JP2020152729A - Ligands that specifically bind to human targets of interest - Google Patents

Abstract

To provide improved human patient diagnosis and therapy through applications of human genetic variation analysis and rationally designed sequence selection.SOLUTION: The invention relates to human targets of interest (TOIs), anti-TOI ligands, kits, compositions and methods.SELECTED DRAWING: None

Description

The techniques described herein relate, among other things, to ligands for treating the disease, such as antibodies or traps, methods, compositions, kits and uses.

Individual humans have been recognized to differ in their sequence, and in recent years several individuals, such as James Watson and Craig Venter, have been sequenced in their genomes. Comparison of the genome sequences of individuals revealed that there are differences in their sequences in both the coding and non-coding parts of the genome. Some of these variations in humans are important and contribute to phenotypic differences between individuals. In extreme cases, these are expected to result in genetic diseases. The 1000 Genomes Project aims to catalog sequences within the human genome, including sequencing the genomes of very large samples of individuals from diverse, industry-recognized human populations.

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The present invention provides improved diagnosis and therapy of human patients through the application of analysis of human genetic variations and selection of reasonably designed sequences. Importantly, the present invention enables tailor-made medicine for the genotype or phenotype of individual human patients.

Analysis of the vast number of naturally occurring genomes of human TOI sequences by the present inventor reveals significant variations across diverse human populations, tailor-made medical and targeted for individual human patients and targets. Provides the ability to associate with a diagnostic approach. Thus, the technical application of these discoveries, according to the invention, contributes to better treatment, prevention and diagnosis in humans and benefits patients by enabling personalized medicine and therapy. This results in better prescribing, less disposal of the drug and improved chances of efficacy, as well as better diagnosis in the patient.

Moreover, the inventor surprisingly found that some rarer natural forms exist in humans much less frequently than common forms, but in large and ethnically diverse human populations. It was presented and usually understood that there are numerous human examples for the presented ethnic groups. Therefore, we anticipate that targeting such rarer types will result in effective treatment, prevention or diagnosis across many human ethnic populations, thereby expanding the usefulness of the invention. I understood that it would be even more useful for patients in such populations.

Accordingly, the inventor of the present invention is of the present invention in terms of guiding the selection of anti-TOI ligands to be administered to human patients for the treatment and / or prevention of diseases and conditions mediated by or related to TOI. Understood that there are various industrial and medical applications. In this manner, the patient receives tailor-made drugs and ligands for their needs as determined by the patient's genetic or phenotypic composition. Along with that, the present invention provides patient genotyping and / or phenotyping associated with such treatments, thereby allowing the drug to be appropriately adapted to the patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment (eg, poor efficacy and / or side effects) with drugs or ligands that are not suitable for the patient, and avoids drug prescribing mistakes and waste. ..

To achieve this object, the present invention provides:

In the first configuration,
A method of treating or preventing a disease or condition mediated by a Target of Interest (TOI) that exists as a different polymorphic variant in humans.
The TOI in a human has a cumulative human allelic frequency of less than 50%, comprising the step of administering an anti-TOI ligand to the human to target the TOI variant in the human and treat or prevent the disease or condition. Encoded by a nucleotide sequence and / or the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%.
b. Prior to step (a), the human is genotyped or genotyped positive for the nucleotide sequence, or phenotyped positive for the TOI variant. ,Method.

In the second configuration
A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
The TOI in a human has a cumulative human allelic frequency of less than 50%, comprising the step of administering an anti-TOI ligand to the human to target the TOI variant in the human and treat or prevent the disease or condition. Encoded by a nucleotide sequence and / or the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%.
b. The method by which the ligand was determined or determined to be capable of binding to said TOI variant prior to step (a).

In the third configuration
A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
The TOI in humans has a cumulative human allelic frequency greater than 50%, comprising the step of administering an anti-TOI ligand to humans to target the TOI variant in humans and treat or prevent said disease or condition. Or / or a variant encoded by a nucleotide sequence having a total human genotype frequency greater than 50%.
b. Prior to step (a), was the human genotyped negative for variant nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%? , Or genotyped, or phenotyped negative for TOI variants encoded by nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total genotype frequency of less than 50%. The method.

In the fourth configuration,
An anti-human TOI ligand for use in methods of treating and / or preventing TOI-mediated diseases or conditions in humans, the TOI is present in humans as different genotypic variants, said the human genome. A ligand comprising a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%, the method comprising administering the ligand to a human.

In the fifth configuration
Less than 50% cumulative human allelic frequency and / or 50 for use in methods involving the step of targeting said TOI in humans with a ligand and treating and / or preventing TOI-mediated diseases or conditions. A ligand that binds to a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a total human genotype frequency of less than%, wherein the method comprises the step of administering the ligand to a human.

In the sixth configuration
Pharmaceutical compositions or kits for treating or preventing TOI-mediated conditions or diseases.

In the seventh configuration,
A method for generating an anti-human TOI antibody binding site, a nucleotide having a step of obtaining multiple anti-TOI antibody binding sites and a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. Amino acid variations to the TOI containing the sequence-encoded amino acid sequence, or from the corresponding sequence encoded by the nucleotide sequence encoding the TOI with the highest cumulative human allele frequency and / or the highest total human genotype frequency. A method comprising screening an antibody binding site for binding to the peptide comprising, and isolating the antibody binding site to which it binds in the screening step.

In the eighth configuration
A method of producing an anti-human TOI antibody in a non-human vertebrate (eg, mouse or rat), a nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. A TOI containing an amino acid sequence encoded by, or its containing amino acid variation from a corresponding sequence encoded by a nucleotide sequence encoding a TOI having the highest cumulative human allele frequency and / or the highest total human genotype frequency. Isolate an antibody that binds to a TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a step of immunizing with a peptide and a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% A method comprising the steps of producing a TOI-binding fragment or derivative of an isolated antibody, optionally.

In the ninth configuration
A kit for determining the TOI genotype of humans, specific for selected TOI nucleotide sequences or RNA transcripts thereof having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50%. Containing nucleic acids containing nucleotide sequences that hybridize with; and / or at least 10 of TOI nucleotide sequences in which the nucleic acid has a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. A kit comprising or an antisense sequence thereof containing a nucleotide sequence comprising contiguous nucleotides.

In the tenth configuration
Drugs for treating and / or preventing TOI-mediated diseases or conditions in humans whose genome contains a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. Use of an anti-TOI ligand that binds to a human TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%.

In the eleventh configuration
Less than 50% cumulative human allelic frequency and / or less than 50% total human genotype frequency in the manufacture of drugs to target and treat and / or prevent TOI-mediated diseases or conditions in humans. Use of an anti-TOI ligand that binds to said human TOI, including the amino acid sequence encoded by the TOI nucleotide sequence having.

In the twelfth configuration
A method of targeting TOI to treat and / or prevent TOI-mediated diseases or conditions in humans with a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. A method comprising administering an anti-TOI ligand to a human comprising a selected TOI nucleotide sequence having, thereby targeting the TOI encoded by the nucleotide sequence.

In the thirteenth configuration
A method of TOI genotyping a human nucleic acid sample to identify the presence of a TOI nucleotide sequence in the sample with a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50%. A method, including steps.

In the 14th configuration
A method for determining the TOI of a human protein sample, in which the TOI amino acid is encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. A method comprising identifying the presence of a sequence.

In one example, the TOI is a human TOI selected from the group consisting of PCSK9, VEGF-A and IL6 receptors. In one example, the TOI is human IL4Ra, PDGF-B, PDGFR-B or Ang-2.

The fifteenth configuration is
(i) The ligand (eg, antibody or fragment)
(a) A variable domain encoded by a human V region nucleotide sequence, wherein the V nucleotide sequence is a variable domain derived from recombination of the human VH, D and JH gene segments or the human VL and JL gene segments; or
(b) Containing the constant region domain encoded by the C region gene segment
The first gene segment of the gene segment of (a) or the C region gene segment of (b) contains a first single nucleotide polymorphism (SNP) encoding a first amino acid polymorphism;
(ii) The human genome contains the first SNP, or the human is an antibody variable domain comprising (a') the first amino acid polymorphism or (b') the first amino acid polymorphism. Provided are ligands, methods, uses, kits or compositions of the invention that express the antibody constant domain containing a type.

The sixteenth configuration is an antibody in which the ligand contains a human antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally, if the variable domain is the VH domain, the human D gene segment). Or contains or consists of fragments; the human genome comprises the human V gene segment and / or the human is of the human V gene segment and the human J gene segment (and optionally the human D gene segment). Provided are the ligands, methods, uses, kits or compositions of the invention that express antibodies comprising recombinantly derived antibody variable domains.

The sixteenth configuration comprises a human heavy chain constant domain in which a ligand (eg, containing or consisting of an antibody or fragment or an Fc-fused human PCSK9 receptor) is encoded by a first constant region nucleotide sequence; human. The ligand, method, use of the invention, wherein the genome of the first constant region nucleotide sequence comprises the same heavy chain constant region nucleotide sequence and / or human expresses an antibody comprising said human constant region nucleotide sequence. Kits or compositions are provided.

In the seventeenth configuration, the ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) corresponds to the Asp corresponding to position 204 of SEQ ID NO: 42 or position 206 of SEQ ID NO: 42. Contains a human gamma-1 heavy chain constant region containing Leu, and the human genome contains such an Asp or a gamma-1 heavy chain constant region nucleotide sequence encoding Leu, or a human has such Asp or Provided are ligands, methods, uses, kits or compositions of the invention that express an antibody comprising the human gamma-1 constant region containing Leu.

In the eighteenth configuration, the ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) corresponds to Pro corresponding to position 72 of SEQ ID NO: 44, position 75 of SEQ ID NO: 44. Human gamma-2 heavy chain determination containing an amino acid selected from the group consisting of Asn, Phe corresponding to position 76 of SEQ ID NO: 44, Val corresponding to position 161 of SEQ ID NO: 44, and Ala corresponding to position 257 of SEQ ID NO: 44. Containing a normal region; the human genome contains a gamma-2 heavy chain constant region nucleotide sequence encoding such a selected amino acid, or a human gamma-2 constant containing such a selected amino acid. Provided are ligands, methods, uses, kits or compositions of the invention that express an antibody comprising a region.

In the nineteenth configuration, the ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) corresponds to Val or 87 of SEQ ID NO: 50, which corresponds to position 84 of SEQ ID NO: 50. Contains a human kappa light chain constant region containing Cys; the human genome contains a kappa light chain constant region nucleotide sequence encoding such Val or Cys, or a human kappa containing such Val or Cys. Provided are ligands, methods, uses, kits or compositions of the invention that express an antibody comprising a light chain constant region.

The twentieth composition comprises or consists of an antibody or fragment in which the ligand contains the VH domain from the recombination of the human VH gene segment, the human D gene segment and the human JH gene segment, and the VH gene segment is (i). ) The IGHV1-18 * 01 and the human genome contain the human IGHV1-18 * 01 nucleotide sequence, or the human expresses an antibody containing a recombinantly derived variable domain of the human IGHV1-18 * 01; (ii) The IVGH1-46 * 01 and the human genome contain the human IGHV1-46 * 01 nucleotide sequence, or the human expresses an antibody containing a recombinantly derived variable domain of the human IGHV1-46 * 01. Provided are the ligands, methods, uses, kits or compositions of the invention selected from the group consisting of.

The 21st configuration is that the ligand contains or consists of an antibody or fragment containing the VL domain from the recombination of the human VL gene segment and the human JL gene segment, and the VL gene segment is (i) IGKV4-1 *. 01 and the human genome contain the human IGKV4-1 * 01 nucleotide sequence, or does the human express an antibody containing a recombinantly derived variable domain of human IGKV4-1 * 01; (ii) IGLV2-14 * 01 and the human genome contain the human IGLV2-14 * 01 nucleotide sequence, or does the human express an antibody containing a recombinantly derived variable domain of human IGLV2-14 * 01; or (iii) IGKV1 The ligand of the invention, wherein the -13 * 02 and the human genome contain the human IGKV1-13 * 02 nucleotide sequence, or the human expresses an antibody comprising a variable domain derived from the recombination of human IGKV1-13 * 02. , Methods, uses, kits or compositions.

The 22nd configuration is a method of treating or reducing the risk of IL4Ra-mediated disease or condition in humans in need thereof, wherein the human is given I75V, E400A, C431R, S503P, Q576R in SEQ ID NO: 67. And a method comprising the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of S752A. As further explained below, these amino acid variations are found in the naturally occurring IL-4Ra variants in humans found in many populations. The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

The 23rd configuration is a ligand (eg, an antibody or antibody fragment) that treats or reduces the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method of which is said to said human. The ligand provides a ligand that specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. .. The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

The twelfth configuration is a method of targeting IL4Ra in humans, wherein the human IL4RA comprises a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Provided is a method comprising administering a ligand that specifically binds to a protein (eg, an antibody or antibody fragment). The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In some cases, humans have or are at risk for an IL4Ra-mediated disease or condition. In some examples, this method treats or reduces the risk of IL4Ra-mediated diseases or conditions in humans.

The 25th configuration is a ligand (eg, antibody or antibody fragment) for targeting IL4Ra in humans, the method comprising administering the ligand to said human, the ligand being SEQ ID NO: 67. Provides a ligand that specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in. The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In some cases, humans have or are at risk for an IL4Ra-mediated disease or condition. In some examples, this method treats or reduces the risk of IL4Ra-mediated diseases or conditions in humans.

In any of the embodiments of the 21st to 25th configurations, (i) the antibody or fragment comprises a VH domain derived from a human VH segment, a human D gene segment and a human JH segment recombination, and the human VH segment is sequenced. The human encodes Framework 1 of SEQ ID NO: 40 and contains the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; (ii) The human comprises a nucleotide sequence encoding the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In addition, or instead, in any of the embodiments of configurations 21-25, (i) the antibody or fragment is at position 189 Leu set forth in SEQ ID NO: 73 or position 289 set forth in SEQ ID NO: 73. The human gamma-4 heavy chain constant region containing Arg, said human containing the IGHG4 * 01 human heavy chain constant region gene segment, or the human being at Leu at position 189 or SEQ ID NO: 73 set forth in SEQ ID NO: 73. Human gamma containing Arg at position 289 shown-4 expresses an antibody comprising a heavy chain constant region; (ii) said human was selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Contains a nucleotide sequence encoding the IL4RA protein containing said mutation.

The 26th configuration is a method of treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof, wherein the human is given 136I, 216S, 241T, 395K, 848T, 858H. , 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y) ), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X Includes amino acids selected from the group consisting of (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Including the step of administering a ligand that specifically binds to the human Nav1.7 protein (eg, an amino acid or an amino acid fragment); (i) the ligand is shown in Leu at position 189 or SEQ ID NO: 73 shown in SEQ ID NO: 73. Contains a human gamma-4 heavy chain constant region containing Arg at position 289, said human containing (i) IGHG4 * 01 human heavy chain constant region gene segment, or human at position 189 shown in SEQ ID NO: 73. Expressing an antibody containing Leu or the Arg at position 289 set forth in SEQ ID NO: 73; (ii) the human expressed 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P. , 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K , 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (here In X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, Methods are described herein comprising a nucleotide sequence encoding the Nav1.7 protein, comprising said amino acid selected from the group consisting of 1161W and 1919G. The invention also provides a corresponding ligand, eg, an antibody or antibody fragment, for use in such a manner.

In some embodiments, the amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is pain. A sexual disorder or condition. In some embodiments, the amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the disease or condition is a painful disease or condition. In some embodiments, the amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y). Amino acids other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R) Yes), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) ) And 1689X (where X is an amino acid other than W); optionally said the disease or condition is a painful disease or condition.

In some embodiments, the constant region gene segment contained in the human is a germline gene segment. In some embodiments, the method further comprises the step of selecting a human containing the nucleotide sequence of (ii) prior to the step of administration.

In some embodiments, humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is). , 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is other than E) 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) , 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), Nav1.7 protein containing the amino acid selected from the group consisting of 422D, 490N, 943L, 1002L, 1161W and 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Amino acid), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, a nucleotide encoding a Nav1.7 protein containing the amino acid selected from the group. It was determined to contain the sequence.

In some embodiments, this method is performed by humans (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K. , 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S) Where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R), Amino acids other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is W) (Amino acids other than), 422D, 490N, 943L, 1002L, 1161W and 1919G, a nucleotide sequence encoding a Nav1.7 protein containing the amino acid selected from the group, and / or (b) 136I, 216S, 241T. , 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than R) , 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is other than I) 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) From the group consisting of 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The selected mutation further comprises the step of determining to include the Nav1.7 protein containing said amino acid, optionally the step of determining is performed prior to administration of the antibody to humans. In some embodiments, the step of determining the biological sample from humans is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I. , 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (here X is an amino acid other than R) Is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than K) , 422D, 490N, 943L, 1002L, 1161W and 1919G. Includes the step of assaying for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group. In some embodiments, the step of assaying the biological sample is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) In X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is W) (Amino acids other than), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is other than W) Contains at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 422D, 490N, 943L, 1002L, 1161W and 1919G. , Or the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the contiguous nucleotide, wherein the sequence of contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P. , 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K , 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R) Is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G, a nucleotide sequence encoding the amino acid selected from the group. Includes, thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (here And X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) , 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than W), From 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G When at least one nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group is present, the steps of forming a complex and; detecting the presence or absence of the complex. In the process of detecting the presence of the complex, humans can use 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X ( Where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) Is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Amino acids), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G, comprising the step of determining to include the Nav1.7 protein containing said amino acid selected from the group. In some embodiments, does the assaying step include nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification? And / or the assaying steps are performed in a multiplex fashion. In some embodiments, the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

In some embodiments, the human is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X). Is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an E) Amino acids other than 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) ), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) , 422D, 490N, 943L, 1002L, 1161W and 1919G have been shown to be heterozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of, optionally, said human. It is further shown that it comprises the nucleotide sequence of SEQ ID NO: 76, or said humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I. , 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (here X is an amino acid other than R) Is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than K) , 422D, 490N, 943L, 1002L, 1161W and 1919G are shown to be homozygous for the nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group.

In some embodiments, it is further determined that the human is or is substantially tolerant to the treatment of pain or pruritus. In some embodiments, the human has been or has been treated for pain or anti-pruritus, or has reduced responsiveness to treatment for pain or pruritus. In some embodiments, the disease or condition is a painful or pruritic disease or condition. In some embodiments, the disease or condition is channel disorder or is associated with channel disorder; or is associated with primary erythromelalgia (PE), paroxysmal severe pain (PEPD) and channel disorder. Selected from the group consisting of painlessness (CIP). In some embodiments, the human has been diagnosed with a painful or pruritic disease or condition.

In some embodiments, the ligand fragment treats or reduces the risk of the human in a painful or pruritic disease or condition.

In some embodiments, the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rs13402180 and rs12478318.

The 27th configuration is a method of treating or reducing the risk of a VEGF-A-mediated disease or condition in a human, wherein the human has rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, An anti-VEGF-A ligand that specifically binds to human VEGF-A expressed by a VEGF-A nucleotide sequence containing an SNP selected from the group consisting of rs833068, rs833069, rs3025000 and rs1570360 (eg, anti-VEGF-A trap, A method comprising the step of administering an antibody or antibody fragment), wherein the human comprises a VEGF-A nucleotide sequence containing the selected SNP, is described herein. The invention also provides a corresponding ligand, eg, an antibody or antibody fragment, for use in such a manner.

The 28th configuration is a method of treating or reducing the risk of a VEGF-A-mediated disease or condition in a human, the anti-VEGF-A that specifically binds to the human VEGF-A. Including the step of administering a ligand (eg, an anti-VEGF-A trap, antibody or antibody fragment), humans have (i) an ARMS2 nucleotide sequence containing G at position rs10490924 of SNP; (ii) at position rs1061170 of SNP. A CFH nucleotide sequence containing T at the position of T or rs3766404; or (iii) a VEGFR2 nucleotide sequence containing rs4576072 or rs6828477 of SNP, and the ligand is Asp corresponding to position 204 of SEQ ID NO: 42 and position 206 of SEQ ID NO: 42. Contains a human gamma-1 heavy chain constant region comprising an amino acid selected from the group consisting of Leu corresponding to, said human containing the IGHG1 * 01 human heavy chain constant region gene segment, or the human being selected said. Methods for expressing antibodies containing human gamma-1 heavy chain constant regions containing amino acids are described herein. These SNPs are associated with an improved response after anti-VEGF-A treatment of eye diseases or conditions. The invention also provides a corresponding ligand, eg, an antibody or antibody fragment, for use in such a manner.

The 29th configuration is a method of treating or reducing the risk of a VEGF-A-mediated disease or condition in a human, comprising administering to the human an anti-human VEGF-A ligand. (i) PDGF-B nucleotide sequence containing an SNP selected from the group consisting of rs142404523 (ie, C corresponding to position -776) and C at position rs1800818 (ie, C corresponding to position -735); or ( ii) A method comprising a PDGFR-B nucleotide sequence containing an SNP selected from the group consisting of rs246395 (ie, G corresponding to position 2601) and rs74943037 (ie, T corresponding to position 1391) is described herein. Will be done. The invention also provides a corresponding ligand, eg, an antibody or antibody fragment, for use in such a manner.

In an example of any of the 27-29 configurations, the ligand comprises an antibody constant region (eg, antibody Fc region). Optionally, the ligand comprises a human gamma-1 heavy chain constant region comprising an amino acid selected from the group consisting of Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, said human. However, IGHG1 * 01 human heavy chain constant region gene segment is contained, or human expresses an antibody containing a human gamma-1 heavy chain constant region containing the selected amino acid.

In an example of any of the 27-29 configurations, the disease or condition is an eye condition or cancer; or angiogenesis or neovascularization.

In an example of any of the configurations 27-29, the method further comprises the step of antagonizing PDGF-B in said human.

In an example of any of the 27-29 configurations, the method further comprises the step of antagonizing angiopoietin-2 (Ang2) in said human.

In a thirtieth configuration, the present invention provides:

A method of treating or reducing the risk of a PD-L1 mediated disease or condition in humans, the PD-L1 comprising variations selected from the variations listed in Table 21 (Table 22). A method comprising administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by a nucleotide sequence.

For example, a method of treating or reducing the risk of a PD-L1-mediated disease or condition in a human, the PD comprising a variation selected from the variations listed in Table 21 (Table 22). The human is selected, comprising the step of administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by the -L1 nucleotide sequence. A method comprising a PD-L1 nucleotide sequence comprising the above variation is provided.

For example, a method of treating or reducing the risk of cancer in a human being expressed in said human by a PD-L1 nucleotide sequence comprising a variation selected from the variations listed in Table 21. Includes the step of administering an anti-PD-L1 ligand that specifically binds to a human PD-L1 (eg, an anti-PD-L1 trap, antibody or antibody fragment), wherein the human comprises the PD- A method is provided that comprises an L1 nucleotide sequence.

For example, a PD-L1 nucleotide sequence that comprises a method of treating or reducing the risk of an autoimmune disease or condition in a human, wherein the human comprises a variation selected from the variations listed in Table 21. Includes the step of administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by the human, said the variation selected by the human. A method is provided that comprises a PD-L1 nucleotide sequence comprising.

For example, a PD-L1 nucleotide sequence that comprises a method of treating or reducing the risk of an inflammatory disease or condition in a human, wherein the human comprises a variation selected from the variations listed in Table 21. Including the step of administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by the human, said the variation selected by the human. A method is provided that comprises a PD-L1 nucleotide sequence that comprises.

In a thirteenth configuration, the invention provides an anti-human PD-L1 ligand (eg, antibody, antibody fragment or human PD-L1 trap) for use in the method of thirtieth configuration.

In a thirty-second configuration, the invention is by targeting an immune cell TOI (eg, PD-1) present in human as multiple variants that differ in one or more amino acid polymorphisms in human. A method of cancer immunotherapy in the art, comprising the step of administering a ligand (eg, an antibody or antibody fragment) to a human, the ligand comprising a first and second protein domain, the first domain being the first. It specifically binds to a TOI variant containing one amino acid polymorphism, the second domain comprises the second polymorphism, and humans (i) the TOI containing the first amino acid polymorphism; and (ii). ) Expressing the protein domain containing the second polymorphism, the ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73. , The human contains the IGHG4 * 01 human heavy chain constant region gene segment, or the human contains the Leu at position 189 shown in SEQ ID NO: 73 or the Arg at position 289 shown in SEQ ID NO: 73. Expressing an antibody comprising a chain constant region; a second domain provides a method of being contained within said gamma-4 heavy chain constant region of a ligand.

This configuration also provides the following aspects:

A method of cancer immunotherapy in humans by targeting PD-1 in humans, which comprises the step of administering an antibody or antibody fragment to a human, the antibody or antibody fragment being detailed in Table 23 (Table 24). Specific binding to PD-1 encoded by a PD-1 nucleotide sequence containing an SNP selected from the group consisting of the variations described, the antibody or antibody fragment is Leu or Leu at position 189 shown in SEQ ID NO: 73. Contains a human gamma-4 heavy chain constant region containing Arg at position 289 set forth in SEQ ID NO: 73, wherein the human contains (i) IGHG4 * 01 human heavy chain constant region gene segment, or the human contains SEQ ID NO: 73. Expressing an antibody containing the human gamma-4 heavy chain constant region containing Leu at position 189 shown in or Arg at position 289 shown in SEQ ID NO: 73; (ii) PD-1 nucleotide sequence containing the selected SNP. Including, method.

A method of cancer immunotherapy in humans by targeting PD-1 in humans, which comprises the step of administering an antibody or antibody fragment to a human, the antibody or antibody fragment being detailed in Table 23 (Table 24). Encoded by a PD-1 nucleotide sequence containing an SNP (first polymorph) selected from the group consisting of the variations described (eg, selected from the group consisting of rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982) An antibody or antibody fragment that specifically binds to PD-1 comprises a human gamma-4 heavy chain constant region comprising Leu at position 189 set forth in SEQ ID NO: 73 or Arg at position 289 set forth in SEQ ID NO: 73. Human gamma-4 in which humans contain (i) IGHG4 * 01 human heavy chain constant region gene segment, or humans contain Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73. A method of expressing an antibody comprising a heavy chain constant region; (ii) comprising a PD-1 nucleotide sequence comprising the selected SNP.

A method of treating cancer in humans, which comprises the step of administering an antibody or antibody fragment to human, wherein the antibody or antibody fragment is PD-L1 or PD-L2 to PD-1 containing the first amino acid polymorphism. The antibody or antibody fragment comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73. i) IGHG4 * 01 Human gamma-4 heavy chain constant region containing the human heavy chain constant region gene segment, or human containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73. (Ii) contains a PD-1 nucleotide sequence encoding PD-1 containing the first amino acid polymorphism, or humans include PD-1 containing the first amino acid polymorphism. The method of expressing.

In alternatives, instead of "methods of cancer immunotherapy" or "methods of treating cancer," the present invention presents "methods of treating or reducing the risk of an autoimmune disease or condition" or "inflammatory." Instead, it provides a method of treating or reducing the risk of a disease or condition, and TOI polymorphisms are associated with an autoimmune disease or condition or an inflammatory disease or condition.

The invention also provides ligands (eg, traps, antibodies or antibody fragments) for use in any of the methods.

In a thirty-third configuration, the invention treats or treats in humans a TOI-mediated disease or condition (eg, anemia) that is present in human as multiple variants that differ in one or more amino acid polymorphisms. A method of reducing risk, comprising the step of administering a ligand (eg, an antibody or antibody fragment) to a human, the ligand comprising a first and second protein domain, the first domain being the first. It specifically binds to a TOI variant containing an amino acid polymorph, the second domain comprises a second polytype, and humans have (i) a TOI containing the first amino acid polymorph; and (ii) said. Expressing a protein domain containing a second polymorph, the ligand optionally contains a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73. Human gamma-4 containing, said human containing the IGHG4 * 01 human heavy chain constant region gene segment, or human containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73. Expressing an antibody containing a heavy chain constant region; a second domain is contained in said gamma-4 heavy chain constant region of the ligand; TOI is human hemoduverin-BMP6-TMPRSS6-hepcidin-ferropatin-transferrin. Provides a method, selected from the axes.

In a thirty-fourth configuration, the invention is a method of treating or reducing the risk of an IL6R-mediated disease or condition in a human in need thereof, wherein the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 is used in said human. Includes the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to the IL6R protein comprising; the human comprises a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78. , Provide a method. In various embodiments of this configuration, the ligand and human are adapted for stationary and / or variable regions. This configuration of the invention also provides corresponding ligands, uses and kits.

In the 35th configuration, the present invention is an anti-TOI ligand for use in humans in a manner that treats or prevents a target (TOI) -mediated disease or condition that exists as a different polymorphic variant in humans. The method comprises administering the ligand to a human, wherein the ligand has a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% (first SNP). It specifically binds to a TOI variant encoded by a nucleotide sequence containing the SNP; where humans provide a ligand comprising a nucleotide sequence encoding a TOI containing said SNP.

A method of treating or preventing a target (TOI) -mediated disease or condition in humans that is present as a different polymorphic variant in humans, wherein less than 50% of the cumulative human allele frequency and / Alternatively, it comprises the step of administering an anti-TOI ligand that specifically binds to a TOI variant encoded by a nucleotide sequence containing an SNP (first SNP) having a total human genotype frequency of less than 50%; humans include said SNP. A method comprising a nucleotide sequence encoding a TOI comprising.

Less than 50% cumulative human allelic frequency and / or 50% in the manufacture of pharmaceuticals for use in methods of targeting a target target (TOI) in humans and treating or preventing TOI-mediated diseases or conditions. The use of an anti-TOI ligand that specifically binds to a human TOI variant comprising an amino acid sequence encoded by a TOI nucleotide sequence comprising an SNP (first SNP) having a total human genotype frequency of less than, wherein the TOI is. Used as a different polymorphic variant in humans, humans contain a nucleotide sequence encoding a TOI containing said SNP.

In an example of a ligand, method or use, the ligand comprises a heavy chain gamma-1,2,3 or 4 constant region encoded by a nucleotide sequence comprising a second SNP, and human comprises the second SNP. Includes heavy chain gamma-1, 2, 3 or 4 constant regions, respectively.

In examples of ligands, methods or uses, the ligand is a human antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally, if the variable domain is the VH domain, the human D gene segment). The human genome comprises the human V gene segment and / or the human is an antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally the human D gene segment). To express an antibody containing. The present invention further provides regimens, ligands and kits.

An example of either configuration or any TOI is as follows.
(i) The ligand comprises a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a recombinantly derived VH domain of the human JH segment, and the human VH segment is the framework 1 of SEQ ID NO: 40. The human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40.
(ii) The ligand comprises a human VH segment IGHV3-7 * 01, a human D gene segment and a recombination-derived VH domain of a human JH segment, and the human contains the IGHV3-7 * 01 VH gene segment or is human. Expresses the VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment.
(iii) The ligand comprises the human Vκ segment IGKV1-12 * 01 and the recombinantly derived Vκ domain of the human Jκ segment, and the human comprises the IGKV1-12 * 01 Vκ gene segment, or the human contains the human Vκ segment. It expresses the Vκ domain derived from recombination of IGKV1-12 * 01 and human Jκ segment.
(iv) The ligand comprises the Vκ domain from the recombination of the human Vκ segment and the human Jκ segment, and the human Vκ segment encodes (i) CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, said human. Does it contain the Vκ gene segment encoding CDR3 containing Pro at position 7 shown in SEQ ID NO: 36, or does human express the Vκ domain containing CDR3 containing Pro at position 7 shown in SEQ ID NO: 36? Or (ii) the Vκ gene segment encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38 and the human encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38, or Humans express the Vκ domain containing FW3, which contains Ser at position 15 shown in SEQ ID NO: 38.
(v) The ligand contains a human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human is (i) IGHG1 * 01 human. An antibody containing a heavy chain constant region gene segment or containing a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4 is expressed. ..
(vi) The ligands are Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 and Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala at position 257 shown in SEQ ID NO: 6, and whether the human contains (i) IGHG2 * 01 human heavy chain constant region gene segment. , Or human, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Val or the antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown in SEQ ID NO: 6.
The (vii) ligand comprises a human kappa chain constant region containing Val at position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16, and the human is (i) IGKC1 * 01 human kappa chain constant region. An antibody containing a gene segment or containing a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16 is expressed.
(viii) the ligand comprises a human IGLC1 * 01 lambda chain constant region, said human containing (i) a human IGLC1 * 01 lambda chain constant region gene segment, or a human containing a human IGLC1 * 01 lambda chain constant region. Expresses an antibody containing.
(ix) The ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human. An antibody comprising a heavy chain constant region gene segment or containing a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73 is expressed. ..
The (x) ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3 * 01) constant region gene segment, wherein the human is (i) said the first constant region gene. An antibody comprising a segment (eg, IGHG3 * 01) or containing a human gamma-3 heavy chain constant region encoded by the first human IGHG3 (eg, IGHG3 * 01) constant region gene segment is expressed. ..
(xi) The ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human epsilon heavy chain constant region encoded by the first constant region gene segment.
(xii) The ligand comprises the human mu heavy chain constant region encoded by the first human mu heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expresses an antibody comprising the human mu heavy chain constant region encoded by the first constant region gene segment.
(xiii) The ligand comprises the human alpha heavy chain constant region encoded by the first human alpha heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human alpha heavy chain constant region encoded by the first constant region gene segment.
The (xiv) ligand comprises the human delta heavy chain constant region encoded by the first human delta heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expressing an antibody comprising the human delta heavy chain constant region encoded by the first constant region gene segment.
The (xv) ligand comprises the human kappa light chain constant region encoded by the first human kappa light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human kappa light chain constant region encoded by the first constant region gene segment.
The (xvi) ligand comprises the human lambda light chain constant region encoded by the first human lambda light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human lambda light chain constant region encoded by the first constant region gene segment.

In some embodiments, the ligand (eg, antibody or antibody fragment) is administered by inhalation, intravenous or subcutaneous administration, and / or is included in an inhalation or injectable formulation.

In some embodiments, the human gamma-4 heavy chain constant region of the ligand comprises the amino acid sequence of SEQ ID NO: 73 or the ADCC inactivated version thereof.

In some embodiments, the human gamma-4 heavy chain constant region comprises 228P and 235E.

Optionally, in the present invention, the TOI is human TOI: PCSK9, IL6R, IL4Ra, VEGF-A, placental growth factor (PGF), PDGF-B, PDGFR-B, Ang-2, Nav1.7, Nav1.8. , Nav1.9, PD-1, PD-L1, ICOS, BMP6, hemodoverin, ferroportin, TMPRSS6, transferrin, human hemochromatosis protein (HFE) and sclerostin.

For example, the TOI is PCSK9.

For example, the TOI is IL6R.

For example, the TOI is IL4Ra.

For example, the TOI is VEGF-A.

For example, TOI is placental growth factor (PGF).

For example, TOI is PDGF-B.

For example, TOI is PDGFR-B.

For example, the TOI is Ang-2.

For example, the TOI is Nav 1.7.

For example, the TOI is Nav1.8.

For example, the TOI is Nav1.9.

For example, the TOI is PD-1.

For example, the TOI is PD-L1.

For example, TOI is ICOS.

For example, the TOI is BMP6.

For example, TOI is hemodaverin.

For example, TOI is ferroportin.

For example, the TOI is TMPRSS6.

For example, TOI is transferrin.

For example, TOI is a human hemochromatosis protein (HFE).

For example, TOI is sclerostin.

For example, in any aspect, embodiment, embodiment or configuration of the invention, the anti-TOI (eg, PCSK9 or IL4Ra) ligand (eg, trap, antibody or fragment) is a human VH segment (eg, human VH3-23). * 04), which contains the VH domain derived from the recombination of the human D gene segment and the human JH segment, the human VH segment encodes Framework 1 of SEQ ID NO: 40, and the human encodes Framework 1 of SEQ ID NO: 40. Contains the VH gene segment encoding, or humans express the VH domain containing Framework 1 of SEQ ID NO: 40. In an alternative example, the ligand comprises a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a recombinantly derived VH domain of a human JH segment, and the human VH segment comprises the SNP rs56069819. The human comprises a VH gene segment containing the SNP rs56069819.

The SNP rs56069819 is present in humans with an average cumulative frequency of 11% according to the Phase I database of 1000 genes, but AFR (23%), EUR (13%), ASW (27%), LWK (15%), Higher in the YRI (28%), CEU (19%) and GBR (15%) populations, and therefore in certain embodiments, anti-TOI (eg, PCSK9 or IL4Ra) antibodies or fragments have Framework 1 of SEQ ID NO: 40. The human VH segment comprises the SNP rs56069819 and the human contains, or contains a VH domain derived from the recombination of the human VH segment (eg, human VH3-23 * 04), human D gene segment and human JH segment. , AFR, EUR, ASW, LWK, YRI, CEU and GBR pedigrees. For example, humans belong to the AFR lineage. For example, humans belong to the EUR pedigree. For example, humans belong to the ASW lineage. For example, humans belong to the LWK lineage. For example, humans belong to the YRI lineage. For example, humans belong to the CEU lineage. For example, humans belong to the GBR pedigree. In one example, the human selected pedigree is one of these listed pedigrees, and the TOI is encoded by SNPs that are also present in such human populations with a higher than average cumulative frequency according to the 1000 genome. Includes amino acid variations (mutations). For example, a ligand (eg, an antibody or fragment) specifically binds to an IL4R protein containing the mutant S752A, and humans belong to the YRI lineage. In this case, the mutation-encoding SNP, plus the SNP rs56069819, is present in the YRI population with a higher than average cumulative frequency. Therefore, this general aspect of the present invention is particularly useful for tailoring to the genomic profile of members of such populations.

In one example, the ligand specifically binds to the f-type of human PCSK9.

In one example, humans express human PCSK9 selected from the f, e, p, and aj types.

In one example, humans belong to the ASW, LWK, YRI, CEU or GBR pedigree.

In one example, the human belongs to the ASW lineage and the human expresses human PCSK9 selected from the f, e, p and aj types; optionally the ligand is from the recombination of human VH3-23 * 04. Includes VH domain.

In one example, the human belongs to the LWK lineage and the human expresses human PCSK9 selected from the f, e, p and aj types; optionally the ligand is from the recombination of human VH3-23 * 04. Includes VH domain.

In one example, the human belongs to the YRI lineage and the human expresses human PCSK9 selected from the f, e, p and aj types; optionally the ligand is from the recombination of human VH3-23 * 04. Includes VH domain.

In one example, the human belongs to the CEU lineage and the human expresses human PCSK9 selected from the f, e, p and aj types; optionally the ligand is from the recombination of human VH3-23 * 04. Includes VH domain.

In one example, the human belongs to the GBR lineage and the human expresses human PCSK9 selected from the f, e, p and aj types; optionally the ligand is from the recombination of human VH3-23 * 04. Includes VH domain.

In one example, the human genome contains a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29, 32, 34 and 36, and the antibody or fragment is selected from human PCSK9 types f, e, p and aj, respectively. The antibody or fragment specifically binds to the VH domain from the recombination of the human VH gene segment, the human D gene segment and the human JH gene segment, and the VH gene segment contains the nucleotide sequence containing SEQ ID NO: 39 or a nucleotide sequence thereof. Contains complements. Optionally, the VH gene segment is VH3-23 * 04 (SEQ ID NO: 38).

The file of this patent or application contains at least one colored drawing. A copy of this patent or publication of the patent application with colored drawings will be provided by the Patent Office upon request and payment of necessary costs.

It is a figure which shows the in silico modeling of the PCSK9 surface variant residue. FIG. 6 shows the cumulative allele frequency distribution across a database of 1000 genome projects of human VH3-23 alleles containing SNP rs56069819 (such alleles are labeled "C" and are the most frequent alleles (which are: This SNP is not included) is indicated as "A"). In this figure, VH3-23 alleles, including SNP rs56069819, are present at a cumulative frequency of 11% across all human ethnic populations taken overall, but specific specific human ethnic subpopulations (ASW, LWK, YRI). , CEU and GBR) indicate that such alleles are present above the average cumulative frequency. Shown in this figure are human PCSK9 variant types (denoted as "Variants") found in various subpopulations above the average appearance of human VH3-23 alleles, including SNP rs56069819. Diagram showing the framework and CDR coded by VH3-23 * 04 from the IMGT database (available from the World Wide Web at www.IMGT.org). FIG. 3 discloses nucleotide sequences in order of appearance as SEQ ID NOs: 117, 117, 117, 119, 119, 119, 120, 122, 124, 39 and 125, respectively. FIG. 3 discloses the encoded amino acid sequences in order of appearance as SEQ ID NOs: 118, 118, 118, 118, 118, 118, 121, 123, 123, 38 and 126, respectively. See Figure 3A. See Figure 3A. See Figure 3A. See Figure 3A. See Figure 3A. It is a figure which shows the arrangement of VH3-23 * 04. Part of VH3-23 * 04 containing FW1 residue changes in rs56069819 (SEQ ID NO: 38). The portion of the nucleic acid sequence encoding rs56069819 is shown (SEQ ID NO: 39). FW1 encoded by VH3-23 * 04 is shown (SEQ ID NO: 40).

Those skilled in the art are aware that SNPs or other changes translated into amino acid variations can result in variability in the higher-order structure or activity of the human target to be addressed. This has led to great interest in personalized medicine that uses genotyping and knowledge of protein and nucleotide variability to more effectively tailor-make patient care and diagnosis. The present invention provides tailor-made medicines and trials specifically directed to a rarer variant of a human target (TOI).

The present invention utilizes the ability to analyze human genetic variations and select reasonably designed sequences. The technical application of these approaches, according to the invention, benefits patients by contributing to better treatment, prevention and diagnosis in humans and providing options to enable personalized medicine and therapy. Bring. This results in better prescribing, less consumption of the drug and improved chances of drug efficacy, as well as better diagnosis in the patient.

As a data source for genomic sequence variations, one of ordinary skill in the art is expected to be aware of the available databases and resources (including updates thereof) provided by:
1. HapMap (The International HapMap Consortium. 2003; available on the World Wide Web at hapmap.ncbi.nlm.nih.gov/index.html.en). The HapMap Project is an international project aimed at identifying chromosomal regions that contain common genetic variants by comparing the gene sequences of different individuals. The HapMap www site provides tools for identifying chromosomal regions and variants within them, with options for delving into population-level frequency data.
2. 1000 Genomes Project (The 1000 Genomes Project Consortium 2010; available at 1000genomes.org/ on the World Wide Web). This resource provides the complete genomic sequence of at least 2500 unidentified individuals from one of 25 distinct populations.
3. Japanese SNP Database (H.Haga et al., 2002; available on the World Wide Web at snp.ims.u-tokyo.ac.jp/index.html). Based on the study, 190,562 human genetic variants will be identified.

The present invention identifies naturally occurring human genome target sequence variants, including those found to be relatively infrequent or rare variants that are distinguished in a particular human ethnic population and in many individual humans. And cataloging.

Aspects of the invention are the rarer, however, of human individuals who have, or are at risk of, suffering from, or are at risk of, a disease or condition mediated by or associated with a target of interest. Nonetheless, it is based on a rational design of sequence selection that addresses the desirability for tailoring pharmaceuticals and diagnostics to a significant group. In devising this rational design of aspects of the invention, the inventor has developed the widespread dissemination of naturally occurring target variant sequences across a large number of diverse human ethnic populations, plus many individuals in one or more individuals. It took into account the importance of working to analyze populations that are likely to exhibit the genotype and / or phenotype of the variant. As part of this design, the inventor understands the importance of adopting the classification of human ethnic groups recognized in the industry, and in this respect the inventor provides the basis for analysis and design. , Placed in the recognized human ethnic population adopted by the 1000 Genomes Project, because the 1000 Genomes Project is a resource that has been and continues to be widely adopted by the scientific and medical community.

In this way, in this aspect of the invention, the inventor has designed the selection criteria for the following variant sequences, which the inventor has learned, which are useful medical drugs to meet tailor-made requirements in the human population. And a diagnostic agent.

Selection Criteria Three or four of the following.
A naturally occurring human target variant sequence with a cumulative human allele frequency of 35% or less;
Naturally occurring human target variant sequences with a total human genotype frequency of 40% or less;
• Naturally occurring human target variant sequences found in many different human populations (using standard categorization of the 1000 Genomes Project; see Table 4 below); and • many such A naturally occurring human target variant sequence found in many individuals distributed across various ethnic populations.

The inventor's choice is to consider nucleotide variations (ie, non-synonymous variations) that produce amino acid variations in the corresponding TOI type as opposed to silent variations that do not alter amino acid residues in the target protein. Included a selection of.

In certain embodiments, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range of 1-20% or 1-15% or 1-10%. ..

In certain embodiments, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, 1-25%, 1-20%, 1-15%, 1 It ranges from ~ about 15%, 1 ~ 10%, 1 ~ about 10%, or 1 ~ 5%, or 1 ~ about 5%.

In certain embodiments, the naturally occurring human target variant sequences are at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human ethnic populations (of the 1000 Genomes Project). Found in (using standard categorization).

In certain embodiments, naturally occurring human target variant sequences are distributed across many such diverse ethnic populations, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70. , 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 individuals.

In one example, the following criteria apply:
A naturally occurring human target variant sequence with a cumulative human allele frequency of 15% or less;
A naturally occurring human target variant sequence with a total human genotype frequency of 20% or less;
• Naturally occurring human target variant sequences found in at least 5 different human populations (using standard categorization of the 1000 Genomes Project); and • Many distributed across many such diverse populations A naturally occurring human target variant sequence found in an individual.

In some examples, the criteria are applied with reference to one or more human genome sequence databases as described herein. For example, the criteria are those that apply to a database of 1000 genomes.

For example, in any aspect, example, embodiment or configuration of the present invention, a database of 1000 genomes has released 13. For example, the 1000 Genomes database is its latest version as of October 1, 2013.

At the option, further sequence analysis and 3D in silico modeling (see, eg, Figure 1) can also be used as additional selection criteria: for selection, its variant amino acid residues (to the most common type of human TOI). Variants in which (as opposed to) are surface-exposed on the target are desirable, which allows the inventor to use such variants in determining the topography of the target, potentially how and where to target. This is because it is considered to contribute to whether ligand binding occurs.

The following bioinformatics protocols are envisioned to identify human sequences for use in the present invention:
(a) A sequence assembly that identifies the genomic region (“target genomic region”) that contains the target sequence of interest and is used by the 1000 Genomes Project or the International HapMap Project (or another selected optimal human gene database). Compute the genomic coordinates that match the structure.
(b) Identify genomic variants that map to the genomic regions previously identified in (a). Allele frequencies are derived for each superior population, preferably for each variant of the subpopulation for which such data are available. The 1000 Genomes Project VWC tool can be used for this process.
(c) Genome variants from the target genomic region so that they contain only variants classified as either "non-synonymous" single nucleotide polymorphisms (SNPs) or "insertions or deletions" (indels) of the genome. Filter the list. Further filtering is done to include those that are only present in sequences with exons. "Non-synonymous" refers to nucleotide variations that result in amino acid variations (ie, excluding silent mutations).
(d) Each of the variants identified for each of the top populations (eg, "European pedigree", "East Asian pedigree", "West African pedigree", "North and South American", and "South Asian pedigree"). Correlate population frequency data to identify variant top populations that are distinguished by less than two top populations. In addition, all identified variants were correlated with each of the subpopulations (eg, the upper population of "European pedigree" was further "CEU-Utah resident with Scandinavian or Western pedigree", "TSI, Italy". (May be divided into groups such as "Tuscans" and "Britishes from England and Scotland"), create a second score for the rarity of variants within the upper population.
(e) Collect one or more sequences that indicate a distinction to a particular subpopulation for use in the present invention, eg, according to selection criteria as described herein.

Human Population Arbitrarily, ethnic populations are selected from those identified in the 1000 Genomes Project database. In this regard, see Table 4 (Table 5), which details the ethnic groups underlying the 1000 Genomes Project database.

NA Rosenberg et al. (Science December 20, 2002: Vol. 298, No. 5602, 2342-2343) studied the genetic structure of human populations of geographically different pedigrees. A total of 52 populations were sampled, and these are the following populations.
African pedigree
[Mbuti Pygmies, Biaka Pygmies, Sans, and Niger-Congolian speakers (Bantu, Yoruba or Mandinka],
Eurasian pedigree
[European pedigree (Orkney Islands, Adygei, Basques, French, Russians, Italians, Sardinians, Toscanans],
Middle Eastern pedigree (Mozabite, Bedouin, Druze, Palestinian),
Central / South Asian pedigree [Bharuch, Brahuy, Makrani, Sindh, Pattern, Burusho, Hazaras, Uighurs, Kalash],
East Asian pedigree
[Han, Dal, Daur, Hogen, Lahu, Miao, Oroqen, She, Tujia, Tu, Xibo, Yi, Mongolian, Nakhi , Cambodian, Japanese, Yakut], Oceanian pedigree (Miao, Papuan); or North and South American pedigree
[Karitiana, Surui, Colombian, Maya, Pima].

According to the International HapMap Project, Nature, December 18, 2003; 426 (6968): 789-96, the goal of the HapMap project is in DNA samples from populations of pedigree from the regions of Africa, Asia and Europe. It is disclosed to determine the common pattern of DNA sequence variations in the human genome by determining the genotypes of more than one million sequence variants, their frequency and the degree of association between them. Related human populations of geographically different pedigree include the Yoruba, Japanese, Chinese, Scandinavian and Western European populations. More specifically, it is as follows.
Utah population of Scandinavian or Western descent (sample collected in 1980 by Center d'Etude du Polymorphisme Humain (CEPH));
A group of Yoruba descent from Ibadan, Nigeria;
Populations with Japanese pedigree; and populations with Han Chinese pedigree from China.

Citing previous publications, the authors suggest that pedigree geography is a reasonable criterion for sampling human populations.

Suitable samples of the human population used in the present invention are as follows.
(a) European pedigree
(b) Nordic pedigree; Western pedigree; Tuscan pedigree; British pedigree, Finnish pedigree or Iberian pedigree.
(c) More specifically, a group of Utah residents of Scandinavian and / or Western descent; a Tuscan population in Italy; a British population in England and / or Scotland; a Finnish population in Finland; or in Spain. Iberian group.
(a) East Asian pedigree
(b) Japanese pedigree; Chinese or Vietnamese pedigree.
(c) More specifically, the Japanese group in Tokyo, Japan; the Han ethnic group in Beijing, China; the Chinese Dai group in Xishuangbanna; the Kin group in Ho Chi Minh City, Vietnam; or the Chinese group in Denver, Colorado, USA.
(a) West African pedigree
(b) Yoruba pedigree; Luiya pedigree; Gambian pedigree; or Malawi pedigree.
(c) More specifically, the Yoruba group in Ibadan, Nigeria; the Luiya group in Webuye, Kenya; the Gambian group in the western part of Gambia; or the Malawi group in Blantyre, Malawi.
(a) North and South American population
(b) Native American pedigree; African Caribbean pedigree; Mexican pedigree; Puerto Rican pedigree; Colombian pedigree; or Peruvian pedigree.
(c) More specifically, a population of African pedigree in the southwestern United States; a population of African Americans in Jackson, Mississippi; a population of African Caribbean in Barbados; a population of Mexican pedigree in Los Angeles, California; Puerto Rico A population of Puerto Ricans in; a population of Colombians in Medellin, Colombia; or a population of Peruvians in Lima, Peru.
(a) South Asian pedigree
(b) Ahom pedigree; Kayast pedigree; Reddy pedigree; Maratha pedigree; or Punjabis pedigree.
(c) More specifically, the Ahom group in Assam, India; the Kayast group in Calcutta, India; the Reddy group in Hyderabad, India; the Malata group in Bombay, India; or the Punjab group in Lahore, Pakistan. ..

In any configuration of the invention, in one embodiment, each human population is selected from the population represented above as "(a)".

In any configuration of the invention, in another embodiment, each human population is selected from the population represented above as "(b)".

In any configuration of the invention, in another embodiment, each human population is selected from the population represented above as "(c)".

In one embodiment, the ethnic groups are ethnic groups with European pedigree, East Asian pedigree, West African pedigree, North and South American pedigree, and South Asian pedigree. Selected from the group consisting of.

In one embodiment, the ethnic group is an ethnic group with Scandinavian pedigree; or an ethnic group with Western pedigree; or an ethnic group with Toscana pedigree; or an ethnic group with British pedigree; or an Icelandic pedigree. Ethnic groups with; or ethnic groups with Finnish pedigree; or ethnic groups with Iberian pedigree; or ethnic groups with Japanese pedigree; or ethnic groups with Chinese pedigree; or ethnic groups with Vietnamese pedigree; or Ethnic groups with Jorba pedigree; or ethnic groups with Luiya pedigree; or ethnic groups with Gambian pedigree; or ethnic groups with Malawi pedigree; or ethnic groups with Native American pedigree; or African Caribbean Ethnic groups with pedigree; or ethnic groups with Mexican pedigree; or ethnic groups with Puerto Rico pedigree; or ethnic groups with Colombian pedigree; or ethnic groups with Peruvian pedigree; or ethnic groups with Arhome pedigree It is selected from a group consisting of an ethnic group of Kayast pedigree; or an ethnic group of Reddy pedigree; or an ethnic group of Malata pedigree; or an ethnic group of Punjab pedigree.

Anti-Target Ligand The present invention provides a useful anti-target ligand for humans who have or are likely to suffer from a disease or condition mediated by or associated with TOI. For example, the ligand specifically binds to the TOI variant according to the present invention. The ligand can inhibit or antagonize the activity of the target, eg, the ligand neutralizes the target. Those skilled in the art are generally expected to be familiar with the neutralization of ligands such as antibodies or antibody fragments and are suitable ligands for specific binding and / or neutralization of the target in in vitro or in vivo assays. Can be easily tested.

An antibody "fragment" comprises a portion of an intact antibody, preferably the antigen binding and / or variable region of an intact antibody. Examples of antibody fragments include dAb, Fab, Fab', F (ab') 2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules and multispecific antibodies formed from antibody fragments. ..

In certain embodiments, the ligand of the invention is or comprises an antibody or antibody fragment, eg, an antibody or fragment comprising a human variable region (and optionally also a human constant region). Anti-TOI or TOI binding or targeting antibodies and fragments can be prepared according to any known method, eg, immunized with TOI, followed by constant regions and optionally to produce human or humanized antibodies. / Or transgenic mice with humanized variable regions [eg, Kymouse ™ or Velocimouse ™, or Omnimouse ™, Xenomouse ™, HuMab Mouse ™ or MeMo Mouse ™], It can be prepared using rats [eg Omnirat ™], camels, sharks, rabbits, chickens or other non-human animals. In some examples, display techniques such as yeast, phage or ribosome display can be used as would be apparent to those skilled in the art. Standard affinity maturation using display techniques, for example, can be performed in a further step after isolation of antibody reads from transgenic animals, phage display libraries or other libraries. Representative examples of suitable techniques are described in US Patent Application Publication No. 20120093818 (Amgen, Inc.), which is incorporated herein by reference, and is detailed, for example, in paragraphs [0309] to [0346]. The way it is done. Although it is based on PCSK9, the antibody production method can be applied to other TOIs as per the broadest scope of the invention.

Generally, VELOCIMMUNE ™ or other mice or rats can be loaded with an antigen of interest and lymphocytes (eg, B cells) are recovered from antibody-expressing mice. Lymphocytes may be fused with myeloma cell lines to prepare immortal hybridoma cell lines, such hybridoma cell lines to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. Screened and selected. DNA encoding the variable regions of the heavy and light chains may be isolated and linked to the desired isotype constant region of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Instead, antigen-specific chimeric antibodies or DNA encoding the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As described below, antibodies are characterized and selected with respect to desired characteristics including affinity, selectivity, epitopes and the like. The mouse constant region is replaced with the desired human constant region, such as fully human antibodies of the invention, eg, wild or modified IgG1 or IgG4 (eg, SEQ ID NOs: 751, 752, 753 in US Patent Application Publication No. 2011/0065902, these. The sequence of is generated herein by reference for use with the ligands of the invention). Although the selected constant region can be modified according to specific use, the features of high affinity antigen binding and target specificity reside in the variable region.

In one example, the ligands of the invention hybridize to TOI variant sequences under stringent conditions, eg, variants (for the most common TOI sequences, eg, refer to the 1000 Genome Project database). A nucleic acid that hybridizes to a nucleotide sequence containing one or more nucleotides, eg RNA, eg siRNA, or comprises.

The ability, specificity and affinity (Kd, K _off and / or K _on ) to bind to the target can be determined by any conventional method in the art, eg, by surface plasmon resonance (SPR). The term "Kd", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

In one embodiment, surface plasmon resonance (SPR) is performed at 25 ° C. In another embodiment, the SPR is performed at 37 ° C.

In one embodiment, SPR is performed at physiological pH, eg at about pH 7 or pH 7.6 [eg, using Hepes buffered saline (also referred to as HBS-EP) at pH 7.6].

In one embodiment, SPR is performed at physiological salt levels, eg, 150 mM NaCl.

In one embodiment, SPR is performed at a detergent level of 0.05% by volume or less, eg, in the presence of 0.05% P20 [polysorbate 20; eg, Tween-20 ™] and 3 mM EDTA.

In one example, SPR is performed at 25 ° C. or 37 ° C. in pH 7.6 buffer, 150 mM NaCl, 0.05% detergent (eg P20) and 3 mM EDTA. The buffer may contain 10 mM Hepes. In one example, SPR is performed in HBS-EP at 25 ° C or 37 ° C. HBS-EP is available from Teknova Inc (California; Catalog No. H8022).

In one example, the affinity of a ligand (eg, an antibody) can be determined using SPR,
1. Anti-mouse (or other relevant human, rat or non-human vertebrate, compatible antibody constant region species) IgG (eg, Biacore ™ BR-1008-38) biosensor chip (eg, GLM chip) To, for example, by coupling with a primary amine coupling;
2. Expose anti-mouse IgG (or other compatible species of antibody) to the test IgG antibody to capture the test antibody on the chip;
3. Pass the test antigen on the capture surface of the chip with 0 nM (ie buffer alone) in addition to 1024 nM, 256 nM, 64 nM, 16 nM, 4 nM;
4. For example, by using surface plasmon resonance under the SPR conditions discussed above (eg, in physiological buffer, at 25 ° C.) to determine the affinity of test antibody binding to the test antigen. It is determined. SPR can be performed using any standard SPR device, for example by Biacore ™ or using ProteOn XPR36 ™ [Bio-Rad®].

Regeneration of the capture surface can be performed with 10 mM glycine at pH 1.7. This removes the captured antibody and allows the surface to be used for another interaction. The combined data can be applied to the built-in 1: 1 model using standard techniques, for example, using the built-in model in the ProteOn XPR36 ™ analysis software.

In some examples, the ligands of the invention are contained in medical containers such as vials, syringes, IV containers or injection devices (eg, intraocular or intravitreal injection devices). In some examples, the ligand is present in vitro, eg, in a sterile container. In one example, the invention provides a kit comprising the ligands, packaging and instructions of the invention for use in the treatment, prevention or diagnosis of TOI-mediated diseases or conditions in humans. In one example, the instructions indicate that humans should be genotyped for the TOI variant sequences of the invention prior to administering the ligand to humans. In one example, the instructions indicate that the human should be phenotyped for the TOI variant of the invention prior to administering the ligand to the human. In one example, humans have the ethnicity of Chinese (eg, Han Chinese) and the instructions are written in Chinese (eg, Mandarin Chinese). In some examples, the instructions include instructions for administering alirocumab or evolocumab to said human.

The present invention relates to concepts detailed in the following aspects.
1. An anti-TOI ligand for use in humans in methods of treating or preventing a disease or condition mediated by a target target (TOI) that exists as a different polymorphic variant in humans, the method of which is to humans. Including the step of administering the ligand, the ligand was encoded by a nucleotide sequence containing an SNP (first SNP) having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. A ligand that specifically binds to a TOI variant; the human comprises a nucleotide sequence encoding a TOI containing said SNP.
2. A method of treating or preventing a target (TOI) -mediated disease or condition in a human that is present as a different polymorphic variant in the human and has a cumulative human allelic frequency of less than 50% in the human. And / or including the step of administering an anti-TOI ligand that specifically binds to a TOI variant encoded by a nucleotide sequence containing an SNP (first SNP) having a total human genotype frequency of less than 50%; humans A method comprising a nucleotide sequence encoding a TOI comprising the SNP.
3. Less than 50% cumulative human allelic frequency and / or in the manufacture of pharmaceuticals for use in methods of targeting targeted targets (TOIs) in humans and treating or preventing TOI-mediated diseases or conditions. Use of an anti-TOI ligand that specifically binds to a human TOI variant containing an amino acid sequence encoded by a TOI nucleotide sequence containing an SNP (first SNP) with a total human genotype frequency of less than 50%, the TOI Is present as a different polymorphic variant in humans, humans contain nucleotide sequences encoding TOIs containing said SNPs, use.
4. The ligand contains a heavy chain gamma-1,2,3 or 4 constant region encoded by a nucleotide sequence containing a second SNP, and a human has a heavy chain gamma-1,2 containing the second SNP. , 3 or 4 The ligand, method or use according to any one of aspects 1 to 3, comprising 3 or 4 constant regions, respectively.
5. The ligand contains a human antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally, if the variable domain is the VH domain, the human D gene segment); the human genome , And / or a human expresses an antibody comprising an antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally the human D gene segment). The ligand, method or use according to any one of aspects 1 to 4.
6. The ligand, method or method according to any one of aspects 1-5, wherein each SNP encodes a non-synonymous amino acid variation and / or each SNP encodes an amino acid that is surface-exposed on a target. use.
7. The ligand, method or use according to any one of aspects 1-6, wherein the ligand is an antibody or antibody fragment; or the ligand is a trap.
8. The method comprises the step of determining that the human is positive for the TOI variant, optionally the step of determining is the step of determining that the human is positive for the nucleotide variant encoding the TOI variant. The ligand, method or use according to any one of aspects 1 to 7, which comprises.
9. The ligand specifically binds to two or more different TOI variants, each encoded by a nucleotide sequence containing an SNP with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. The ligand, method or use according to any one of aspects 1-8, wherein it is possible.
10. The ligand, method or use according to any one of aspects 1-9, wherein the TOI variant has been determined or is determined to be present in at least two different human ethnic populations.
11. Any of aspects 1-10, wherein the cumulative human allele frequency is a frequency in a database of naturally occurring sequences containing at least 1000 sequences sampled from at least 15 different human populations. The ligand, method or use described in one.
12. A kit for treating or preventing a TOI-mediated condition or disease according to any one of aspects 1 to 11, comprising said ligand, wherein the first SNP comprises at least two types. Found in different ethnic groups; optionally combined with labels or instructions for use in the treatment and / or prevention of said disease or condition in humans; optionally, labels or instructions by regulatory authorities. Includes license number issued; optionally, kit containing injection pen or IV container containing ligand.
13. The ligand contains the human gamma heavy chain CH1 domain encoded by the CH1 nucleotide sequence; the human genome contains the gamma heavy chain constant region nucleotide sequence identical to the CH1 nucleotide sequence, and / or the human is said to be the human gamma. The ligand, method, use or kit according to any one of aspects 1-12, which expresses an antibody comprising the CH1 genome.
14. The ligand contains the human gamma heavy chain CH2 domain encoded by the CH2 nucleotide sequence; the human genome contains the gamma heavy chain constant region nucleotide sequence identical to the CH2 nucleotide sequence, and / or the human is said to be the human gamma. The ligand, method, use or kit according to any one of aspects 1 to 13, which expresses an antibody comprising the CH2 domain.
15. The ligand contains the human gamma heavy chain CH3 domain encoded by the CH3 nucleotide sequence; the human genome contains the gamma heavy chain constant region nucleotide sequence identical to the CH3 nucleotide sequence, and / or the human has said human gamma. The ligand, method, use or kit according to any one of aspects 1 to 14, which expresses an antibody comprising the CH3 domain.
16. The ligand contains the human gamma heavy chain CH4 domain encoded by the CH4 nucleotide sequence; the human genome contains the gamma heavy chain constant region nucleotide sequence identical to the CH4 nucleotide sequence, and / or the human is said to be the human gamma. The ligand, method, use or kit according to any one of aspects 1 to 15, which expresses an antibody comprising the CH4 domain.
17. The ligand contains the human gamma heavy chain Fc region encoded by the Fc nucleotide sequence; the human genome contains the gamma heavy chain constant region nucleotide sequence identical to the Fc nucleotide sequence, and / or the human contains the human gamma. The ligand, method, use or kit according to any one of aspects 1 to 16, which expresses an antibody containing an Fc region.
18. The ligand, method, use or kit according to any one of aspects 13-17, wherein the human is genotyped or genotyped for the heavy chain constant region nucleotide sequence.
19. Described in any one of aspects 13-18, wherein the human is phenotypized or phenotypically positive for the gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc. Ligand, method, use or kit.
20. The ligand, method, use or method according to any one of aspects 1-19, wherein the ligand comprises a human IGHG1 * 01 gamma-1 heavy chain constant region; or a human IGHG2 * 01 gamma-1 heavy chain constant region. kit.
21. The ligand is
a) Contains the human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human has the IGHG1 * 01 human heavy chain constant region gene segment. Contains or does the human express an antibody comprising the human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4;
b) Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6, and SEQ ID NO: 6. Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala at position 257 shown, wherein the human contains the IGHG2 * 01 human heavy chain constant region gene segment, or the human contains the selection. Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or SEQ ID NO: 6. Does it express an antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown; or
c) Contains the human gamma-3 heavy chain constant region encoded by the IGHG3 * 01 constant region gene segment, and the human contains the IGHG3 * 01 constant region gene segment, or the human contains the IGHG3 * 01 constant region gene segment. Does it express an antibody containing the encoded human gamma-3 heavy chain constant region; or
(i) A human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is an IGHG4 * 01 human heavy chain constant region gene segment. , Or humans express antibodies containing the human gamma-4 heavy chain constant region containing Leu at position 189 set forth in SEQ ID NO: 73 or Arg at position 289 set forth in SEQ ID NO: 73; where the second The ligand, method, use or kit according to any one of aspects 1 to 20, wherein the domain is contained in said gamma-4 heavy chain constant region of the ligand.
22. TOI is the following human TOI:
a) PCSK9 [Optionally, disease or condition is lipid disorder, hyperlipoproteinemia, hyperlipidemia, abnormal lipidemia, hypercholesterolemia, cardiac attack, stroke, coronary heart disease, atherosclerosis Select from sclerosis, peripheral vascular disease, lameness (eg, lameness associated with elevated cholesterol), type II diabetes, hypertension and cardiovascular disease or condition],
b) IL6R [Optionally, the disease or condition is inflammatory disease or condition, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingivalitis, graft rejection, allogeneic graft Rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic Select from sex erythematosus and coronary heart disease],
c) IL4Ra [Optionally select disease or condition from asthma, COPD, inflammatory bowel disease, fibrotic condition, allergy, graft rejection, delayed hypersensitivity, contact hypersensitivity, nasal polyps and sinusitis Ru],
d) VEGF-A [Optionally, disease or condition is eye condition, cancer; neovascularization, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD), polypoid choroid Selected from angiopathy (PCV), retinal hemangiomatous growth (RAP), diabetic macular edema (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
e) PDGF-B [Optionally, disease or condition is eye condition, cancer; neovascularization, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD), polypoid choroid Selected from angiopathy (PCV), retinal hemangiomatous growth (RAP), diabetic macular edema (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
f) PDGFR-B [Optionally, disease or condition is eye condition, cancer; neovascularization, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD), polypoid choroid Selected from angiopathy (PCV), retinal hemangiomatous growth (RAP), diabetic macular edema (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
g) Angiopoetin (eg Ang-2) [Optionally, disease or condition is eye condition, cancer; angiogenesis, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD) ), Polypoid choroidal angiogenesis (PCV), retinal hemangiomatous growth (RAP), diabetic macular degeneration (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
h) Nav1.7 [Optionally, the disease or condition is pain, pruritus or channel disorder, such as chronic pain, neurogenic pain, painful diabetic neuropathy (PDN), post-herpes neuropathy (PHN). , Triangular nerve pain (TN), spinal cord injury pain, multiple sclerosis pain, phantom limb pain, post-stroke pain, chronic back pain, osteoarthritis pain, cancer-related pain, HIV-related pain, inflammatory pain, primary Select from extremity erythema (PE), paroxysmal extreme pain (PEPD) or channel disorder-related painlessness (CIP)],
i) PD-L1 [Optionally, the disease or condition is cancer, autoimmune disease or condition, viral infection and inflammatory disease or condition, eg, solid tumor, breast cancer, lung cancer (eg, non-small cell lung cancer), Colon cancer, ovarian cancer, melanoma (eg stage IV melanoma), bladder cancer, liver cancer (eg hepatocellular carcinoma), kidney cancer, salivary adenocarcinoma, gastric cancer (stomach cancer), gastric cancer (eg gastric cancer), glioma, pancreatic cancer, thyroid cancer, thoracic epithelial cancer, head cancer and / or cervical cancer, multiple myeloma, T-cell lymphoma, hodgkin lymphoma, renal cell cancer, offspring Miya cervical cancer, colorectal cancer, bloodline neoplasm, metastatic colorectal cancer, uterine or cervical cancer, leukemia (eg, acute myeloid leukemia), diffuse large B-cell lymphoma, follicular center lymphoma , Prostate cancer, Merkel cell carcinoma, HIV infection, hepatitis C infection or Ebola infection].
j) PD-1 [optionally, the disease or condition is, for example, renal cell carcinoma; hematological neoplasm; stage IV melanoma; non-small cell lung cancer; metastatic gastric cancer; breast cancer; solid tumor; bladder cancer; Select from head and neck cancer, colorectal cancer, glioblastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, cervical cancer, myeloid leukemia (eg, acute myeloid leukemia and chronic myeloid leukemia) It is a cancer to be done],
k) BMP6 [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
l) Hemoduverin [optionally, the disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); anemia associated with cancer, kidney condition or GvHD; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
m) Hepcidin [optionally, the disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron refractory iron deficiency anemia (IRIDA)],
n) Ferropatin [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or anemia associated with GvHD; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
o) TMPRSS6 [Optional disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
p) Transferrin [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic diseased anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron refractory iron deficiency anemia (IRIDA)]; and
q) Human hemochromatosis protein (HFE) [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD Related anemia; selected from hemochromatosis and iron refractory iron deficiency anemia (IRIDA)]
The ligand, method, use or kit according to any one of aspects 1 to 21, selected from the group consisting of.

The present invention relates to concepts detailed in the following clauses.

Clause 1: A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
A step of administering an anti-TOI ligand to a human to target the TOI variant in the human and treat or prevent (eg, at least 40, 50, 60, 70, 80, 90 or 95%) the disease or condition. Including, the TOI in said human is encoded by a nucleotide sequence having a cumulative human allelic frequency of less than 50%, and / or the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%. ,
Prior to step (a), the human was genotyped or genotyped positive for the nucleotide sequence, or phenotyped positive for the TOI variant. Alternatively, a method comprising a step of genotyping a human as positive for the nucleotide sequence or a step of phenotyping a human as positive for the TOI variant prior to step (a).

In any aspect, configuration, embodiment, embodiment, clause or concept described herein, the frequency may be determined using bioinformatics.

In any of the embodiments, configurations, examples, embodiments, clauses or concepts described herein, the frequency may be determined by reference to a database containing at least 1000 or 2000 human sequences.

In any of the embodiments, configurations, examples, embodiments, clauses or concepts described herein, "heterozygous human genotype frequency" is one of the rare variant alleles in the sample or database. It means the appearance and the appearance of one of the other alleles (heterozygous state), eg, the cumulative frequency of all genotypes in humans with genotypes in a database of 1000 genomes.

In any of the embodiments, configurations, examples, embodiments, clauses or concepts described herein, "homozygous human genotype frequency" refers to the two appearances of variant alleles (homozygous states). For example, it means the cumulative frequency of genotypes in the database of 1000 genome projects.

In any of the embodiments, configurations, examples, embodiments, clauses or concepts described herein, "total human genotype frequency" refers to heterozygous human genotype frequency and homozygous human genotype frequency. Means the sum of and.

In any of the embodiments, configurations, examples, embodiments, clauses or concepts described herein, the "cumulative human allele frequency" is in a sample or database, or in a human, eg, a database of the 1000 Genomes Project. Refers to the sum of all occurrences of a variant allele (eg, SNP).

Clause 2: As described in Clause 1, prior to step (a), it is determined or determined that the ligand is capable of binding to said TOI variant, eg, with the affinity (Kd) disclosed below. Method.

In one example, the ligand is (or is determined to be) a neutralizer for TOI. In some cases, decisions are made in humans (eg, in clinical trials). In some examples, the determination is made in non-humans, such as mice, rats, rabbits, pigs, dogs, sheep or non-human primates (eg, cynomolgus monkeys, rhesus monkeys or baboons).

Clause 3: A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
A step of administering an anti-TOI ligand to a human to target the TOI variant in the human and treat or prevent (eg, at least 40, 50, 60, 70, 80, 90 or 95%) the disease or condition. Including, the TOI in said human is encoded by a nucleotide sequence having a cumulative human allelic frequency of less than 50%, and / or the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%. ,
b. Prior to step (a), the method by which the ligand was determined or determined to be capable of binding to said TOI variant, eg, with the affinity (Kd) disclosed below.

In one example, the ligand is (or is determined to be) a neutralizer for TOI. In some cases, decisions are made in humans (eg, in clinical trials). In some examples, the determination is made in non-humans, such as mice, rats, rabbits, pigs, dogs, sheep or non-human primates (eg, cynomolgus monkeys, rhesus monkeys or baboons).

Clause 4: The human genome contains a nucleotide sequence encoding the TOI variant; prior to step (a), the nucleotide sequence contains less than 50% cumulative human allele frequency and / or less than 50% total. The method of clause 3 which is determined or is determined to have a human genotype frequency.

TOI variants are not the most frequent.

Clause 5: Prior to step (a), the human was genotyped or genotyped positive for the variant nucleotide sequence, or prior to step (a), the human was genotyped. , The method of clause 3 or 4, comprising the step of genotyping the variant nucleotide sequence as positive.

Clause 6: Whether the human was phenotypically determined to be positive for the TOI variant prior to step (a), or the human was phenotypized prior to step (a). The method according to any of the above clauses, comprising the step of phenotyping a variant nucleotide sequence as positive.

Clause 7: The method of any of the above clauses, wherein the frequency is 10 or less than 15% (eg, 1-10%).

In certain embodiments, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range of 1-20% or 1-15% or 1-10%. ..

In certain embodiments, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, 1-25%, 1-20%, 1-15%, 1 It ranges from ~ about 15%, 1 ~ 10%, 1 ~ about 10%, or 1 ~ 5%, or 1 ~ about 5%.

Clause 8: Ligand encoded by nucleotide sequences having a cumulative human allelic frequency of less than 50% (eg, 1-10%) and / or a total human genotype frequency of less than 50% (eg, 1-20%), respectively. The method according to any of the above clauses, which is capable of binding to two or more different TOI variants.

In certain embodiments, the cumulative human allele frequency of each TOI variant is 30, 25, 20, 15, 10 or 5% or less, eg, 1-20% or 1-15% or 1-10%. Is the range of.

In certain embodiments, the total human genotype frequency of each TOI variant is 35, 30, 25, 20, 15, 10 or 5% or less, eg, 1-25%, 1-20%, 1-. It ranges from 15%, 1 to about 15%, 1 to 10%, 1 to about 10%, or 1 to 5%, or 1 to about 5%.

Clause 9: A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
A step of administering an anti-TOI ligand to a human to target the TOI variant in the human and treat or prevent (eg, at least 40, 50, 60, 70, 80, 90 or 95%) the disease or condition. Including, the TOI in said human is encoded by a nucleotide sequence having a cumulative human allele frequency greater than 50% (eg, highest frequency) and / or a total human genotype frequency greater than 50% (eg, highest frequency). Variant
b. Prior to step (a), was the human genotyped negative for variant nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%? , Or genotyped; or phenotypically negative for TOI variants encoded by nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total genotype frequency of less than 50%. Ruka;
Alternatively, prior to step (a), the human is genotyped negative for a variant nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotypic frequency of less than 50%; Alternatively, a method comprising phenotyping this human as negative for a TOI variant encoded by a nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. ..

In certain embodiments, in (a), the cumulative human allele frequency is 55, 60, 65, 70, 75, 80, 85 or 90 or higher, but 95, 96, 97, 98, 99 or 100%. Less than (eg, in the range of 51-80%).

In certain embodiments, in (a), the total human genotype frequency is 55, 60, 65, 70, 75, 80, 85 or 90 or higher, but 95, 96, 97, 98, 99 or 100%. Less than (eg, in the range of 51-80%).

In certain embodiments, in (b), the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, 1-20% or 1-15% or 1-10. It is in the range of%.

In certain embodiments, in (b), the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, 1-25%, 1-20%, 1 It ranges from ~ 15%, 1 ~ about 15%, 1 ~ 10%, 1 ~ about 10%, or 1 ~ 5%, or 1 ~ about 5%.

Clause 10: Prior to step (a), a human is phenotyped or phenotyped positive for the most frequent TOI variant, or genotyped for its nucleotide sequence. The method described in Clause 9.

In certain embodiments, prior to step (a), humans are 55, 60, 65, 70, 75, 80, 85 or 90 or higher, but less than 95, 96, 97, 98, 99 or 100%. Is genotyped or genotyped positive for a TOI variant nucleotide sequence having a cumulative human allelic frequency (eg, in the range 51-80%), or phenotyped for that TOI variant Will be done.

In certain embodiments, prior to step (a), humans are 55, 60, 65, 70, 75, 80, 85 or 90 or higher, but less than 95, 96, 97, 98, 99 or 100%. A TOI variant with a total human genotyping frequency (eg, in the range of 51-80%) is genotyped or genotyped positive for a TOI variant nucleotide sequence, or phenotyped for that TOI variant. Will be done.

Clause 11: The method of Clause 9 or 10, wherein, prior to step (a), the ligand was determined or determined to be capable of binding to the most frequent TOI variant.

Clause 12: Prior to step (a), clauses 9 and 10 were determined or determined that the ligand was unable to substantially neutralize or inhibit said TOI variant listed in step (b). Or the method according to 11.

"Substantially incapable of neutralizing or inhibiting" means neutralization or inhibition of less than 50, 25, 10, 5 or 0.5% of inhibition or inhibition of the most frequent TOI variants.

Clause 13: The method of any one of Clauses 9-12, wherein the ligand is capable of binding to the most frequent TOI variant.

Clause 14: To any one of Clauses 9-13, the ligand is capable of binding to two or more different TOI variants, each encoded by a nucleotide sequence having a cumulative human allele frequency greater than 50%. The method described.

In certain embodiments, each TOI variant is 55, 60, 65, 70, 75, 80, 85 or 90 or higher, but less than 95, 96, 97, 98, 99 or 100% (eg, 51- It is encoded by a nucleotide sequence with a cumulative human allele frequency (in the range of 80%).

In certain embodiments, each TOI variant is 55, 60, 65, 70, 75, 80, 85 or 90 or higher, but less than 95, 96, 97, 98, 99 or 100% (eg, 51- It is encoded by a nucleotide sequence having a total human genotype frequency (in the range of 80%).

Clause 15: The variant nucleotide sequence listed in step (a) is at least two different human ethnic populations, eg, at least 2, 3, 4, 5, 6, 7, 8 as shown in Table 4. Or the method according to any of the above clauses, which has been determined or is determined to be present in nine different human ethnic groups.

Clause 16: Any of the above clauses, wherein the human frequency is the frequency in a database of naturally occurring sequences containing at least 1000 sequences sampled from at least 15, 20 or 25 different human ethnic populations. The method described in Crab. In certain embodiments, the database is a database of the 1000 Genomes Project as described herein.

Clause 17: An anti-human TOI ligand for use in methods of treating and / or preventing TOI-mediated diseases or conditions in humans, the TOI is present in humans as a different genotypic variant of said human. The genome comprises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%, the method comprising administering the ligand to a human.

In an alternative example, Clause 17 is an anti-human TOI ligand for use in any one of Clauses 1-16, the method comprising administering the ligand to a human. provide.

In certain embodiments, the cumulative human allele frequency is 30, 25, 20, 15, 10 or 5% or less, eg, in the range of 1-20% or 1-15% or 1-10%. ..

In certain embodiments, the total human genotype frequency is 35, 30, 25, 20, 15, 10 or 5% or less, eg, 1-25%, 1-20%, 1-15%, 1 It ranges from ~ about 15%, 1 ~ 10%, 1 ~ about 10%, or 1 ~ 5%, or 1 ~ about 5%.

Clause 18: The ligand according to Clause 17, wherein the ligand is determined or determined to be capable of binding to the human TOI encoded by the nucleotide sequence.

In an alternative example, Clause 18 is less than 50% cumulative for use in methods involving the use of ligands to target said TOI in humans and treatment and / or prevention of TOI-mediated diseases or conditions. A ligand that binds to a human TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a human allelic frequency and / or a total human genotype frequency of less than 50%, the method of administering the ligand to human. Provide ligands, including.

Clause 19: Encoded by a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50% for use in the method described in any one of Clauses 1-16. A ligand that binds to a human TOI comprising a given amino acid sequence, the method comprising the step of administering the ligand to a human.

Clause 20: Described in any one of Clauses 17-19, wherein the human is genotyped or genotyped positive for said TOI nucleotide sequence having a cumulative human allelic frequency of less than 50%. Ligand.

The ligand according to any one of clauses 17-19, wherein human is genotyped or genotyped positive for said TOI nucleotide sequence having a total human genotyping frequency of less than 50%.

Clause 21: Humans are phenotyped or phenotypically positive for a TOI encoded by a nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. The ligand according to any one of Articles 17 to 20, which is determined.

Clause 22: Humans have been genotyped or genotyped for TOI nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total genotype frequency of less than 50%. Optionally, humans have a TOI nucleotide sequence with a cumulative human allele frequency of less than 50%, and a cumulative human allele frequency greater than 50% (eg, with the highest cumulative human allele frequency) and / Or either genotyped or genotyped to contain a TOI nucleotide sequence having a total human genotyping frequency greater than 50% (eg, having the highest total human genotyping frequency), clauses 17-21. One of the ligands listed.

Clause 23: The human genome has been genotyped or genotyped for TOI nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. The ligand according to any one of Articles 17 to 22, which is determined.

Clause 24: The ligand comprises an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%; such binding is optionally possible. The ligand according to any one of Articles 17 to 23, comprising an antibody binding site that binds to a human TOI, which is determined or determined to be.

Clause 25: The ligand according to Clause 24, wherein the ligand is an antibody or antibody fragment.

Clause 26: The ligand comprises a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% or a nucleotide sequence that specifically hybridizes to its RNA transcript; / Or the ligand comprises a nucleotide sequence containing at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50% or an antisense sequence thereof. The ligand according to any one of Articles 17 to 23, which is.

In certain embodiments, the ligand is at least 10,11,12,13,14,15,20,25 of the nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. , 30, 35, 40, 45, 50 or a nucleotide sequence comprising, 100 consecutive nucleotides, or an antisense sequence thereof.

Clause 27: The human genome contains nucleotide sequences with a cumulative human allelic frequency of less than 50%, and the sequences are found in at least two different ethnic populations {eg, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or found in 20 different human populations [eg, streets of populations in Table 4]}, in any one of clauses 17-26 The ligand described. In one example, the numbers conform to the 1000 Genomes Project database.

The human genome contains a nucleotide sequence with a cumulative human allele frequency of less than 50%, and the sequence is found in at least 20 individuals distributed across at least two different ethnic populations (eg, such. At least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, distributed across many different ethnic groups. The ligand according to any one of Articles 17-26) (found in 120, 130, 140 or 150 individuals). In one example, the numbers conform to the 1000 Genomes Project database.

Clause 28: A pharmaceutical composition or kit for treating or preventing a TOI-mediated condition or disease listed in any of the above clauses, comprising the ligand according to any one of Clauses 17-27; Optionally combined with a label or instruction for use in the treatment and / or prevention of a disease or condition in humans; optionally, the label or instruction is a marketing license number (eg, FDA or EMA license number). ) Includes; optionally, the kit comprises an injection pen or IV container containing a ligand, a composition or kit.

In some examples, the label or instructions address or describe use for humans that includes a TOI variant encoded by the nucleotide sequence listed in Clause 17.

Clause 29: A method of generating an anti-human TOI antibody binding site, the step of obtaining multiple anti-TOI antibody binding sites and a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. To a TOI containing an amino acid sequence encoded by a nucleotide sequence having, or from a corresponding sequence encoded by a nucleotide sequence encoding a TOI having the highest cumulative human allele frequency and / or the highest total human genotype frequency. A method comprising a step of screening an antibody binding site for binding to the peptide comprising the amino acid variation of the above, and a step of isolating the binding site of the antibody binding site in the screening step.

In any embodiment of any of the embodiments described herein, the antibody, fragment or binding site is a recombinant.

In an alternative example, Clause 29 is a method of producing anti-human TOI antibodies in non-human vertebrates (eg, mice or rats) with less than 50% cumulative human allele frequency and / or less than 50% total. A TOI containing an amino acid sequence encoded by a nucleotide sequence having a human genotype frequency, or a corresponding nucleotide sequence encoded by a TOI encoding the highest cumulative human allele frequency and / or the highest total human genotype frequency. Includes the step of immunizing with the peptide containing amino acid variations from the sequence and the amino acid sequence encoded by the TOI nucleotide sequence having less than 50% cumulative human allele frequency and / or less than 50% total human genotype frequency. A method comprising isolating an antibody that binds to TOI and optionally producing a TOI-binding fragment or derivative of the isolated antibody.

For example, in any aspect, configuration, example or embodiment, the term "isolated" relating to a ligand, antibody or protein is expected to (1) usually find the ligand, antibody, protein, etc. of interest. Free of at least several other proteins, (2) essentially free of other proteins from the same source, eg, the same species, (3) expressed by cells from different species, (4) ) Separated from at least about 50% of naturally occurring polynucleotides, lipids, carbohydrates, or other materials, (5) by covalent or non-covalent interactions with non-naturally binding polypeptides ) Means operably coupled, or (6) does not occur naturally. Typically, "isolated" ligands, antibodies, proteins, etc. make up at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA of synthetic origin, cDNA, mRNA or other RNA, or any combination thereof, can encode such isolated ligands, antibody proteins, and the like. Preferably, the isolated ligand, antibody protein, etc. substantially comprises a protein or polypeptide or other contaminant found in its natural environment that is expected to interfere with its treatment, diagnosis, prevention, investigation or other use. Not included in.

For example, an "isolated" antibody is an antibody that has been identified, isolated, and / or recovered from components of its production environment (eg, naturally or by recombination). Preferably, the isolated polypeptide does not contain binding to all other components from its production environment, eg, so that the antibody is isolated to FDA approved prospects or approved criteria. Contaminating components of its production environment, such as recombinant transfection of cells, result in materials that are typically expected to interfere with antibody research, diagnostic or therapeutic applications, such as enzymes, hormones, and other proteins. Such or non-protein-like solutes can be mentioned. In a preferred embodiment, the polypeptide is (1) more than 95% by weight of the antibody, if determined by the Lowry method, for example; (2) more than 99% by weight in some embodiments; (2) by use of a spinning cup seeker. Uniform state by SDS-PAGE under non-reducing or reducing conditions using (3) Coomassie blue or preferably silver staining, to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence. Expected to be purified until Isolated antibodies also include in situ antibodies in recombinant cells, as it is expected that at least one component of the antibody's natural environment will be absent. However, it is usually expected that the isolated polypeptide or antibody will be prepared by at least one purification step.

Immunoconjugates The present invention includes therapeutic moieties (“immunoconjugates”) such as ligands (eg, antibodies) conjugated to cytotoxicities, chemotherapeutic agents, immunosuppressants or radioisotopes. Cytotoxic agents include any agent that is harmful to cells. Examples of cytotoxic and chemotherapeutic agents suitable for forming immunoconjugates are known in the art. See, for example, WO 05/103081.

Bispecific Substances The antibodies of the invention can be monospecific, bispecific, or multispecific. The multispecific mAb may be specific for different epitopes of one target polypeptide, or may contain an antigen-binding domain specific for more than one target polypeptide. See, for example, Tutt et al. (1991) J. Immunol. 147: 60-69. The human anti-TOI (eg, anti-PCSK9) mAb may be linked to or expressed with another functional molecule, eg, another peptide or protein. For example, an antibody or fragment thereof (eg, by chemical coupling, genetic fusion, non-covalent binding or another method) to one or more other molecular entities, eg, another antibody or antibody fragment. Can be functionally linked to to produce a bispecific or multispecific antibody with a second binding specificity.

Exemplary bispecific antibody modalities that can be used in the context of the present invention include the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, including the use of first and second Ig CH3. The domains are different from each other for at least one amino acid, and the difference in at least one amino acid reduces the binding of the bispecific antibody to protein A compared to the bispecific antibody with no amino acid difference. In one embodiment, the first Ig CH3 domain binds to protein A and the second Ig CH3 domain reduces binding of protein A such as H95R modifications (according to exon numbering of IMGT; H435R according to EU numbering). Or contains a disrupting mutation. The second CH3 may further contain a Y96F modification (according to IMGT; Y436F according to the EU). Further modifications that may be found in the second CH3 are D16E, L18M, N44S, K52N, V57M, and V821 (according to IMGT; according to EU; D356E, L358M, N384S, K392N) in the case of IgG1 antibody. , V397M, and V422I); N44S, K52N, and V82I (IMGT; according to EU, N384S, K392N, and V422I); in the case of IgG2 antibody; and Q15R, N44S, K52N, V57M, R69K, E79Q in the case of IgG4 antibody. , And V82I (according to IMGT; Q355R, N3845, K392N, V397M, R409K, E419Q, and V422I according to the EU). Variations on the modalities of the bispecific antibodies described above are considered within the scope of the present invention.

Clause 30: The method of clause 29, comprising obtaining a nucleic acid encoding an antibody, fragment, derivative or binding site and optionally inserting the nucleic acid into an expression vector.

Clause 31: A kit for determining the TOI genotype of humans, the selected TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotyping frequency of less than 50% or RNA transcription thereof. Containing nucleic acids containing nucleotide sequences that specifically hybridize to a substance; and / or at least a TOI nucleotide sequence in which the nucleic acid has a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. Contains or antisense a nucleotide sequence containing 10 contiguous nucleotides (eg, at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100). A kit that is an array.

For example, the nucleic acid hybridizes to the region immediately end of the nucleotide that is a variant compared to the corresponding nucleotide of the TOI nucleotide sequence with the highest cumulative human allele frequency and / or the highest total human genotype frequency. In one example, the nucleic acid hybridizes to two or more such variant nucleotides.

Specific hybridization is under stringent conditions as would be apparent to those of skill in the art, such as 5 × SSC, 5 × Denhardt's reagent, and 0.5% SDS, 65 ° C. ..

Clause 32: A kit for TOI genotyping or phenotyping of humans by the ligand described in any one of Clauses 17-27 or the method described in any one of Clauses 29-31. A kit containing the antibodies, fragments or derivatives produced.

For example, the ligand is specific for an epitope containing an amino acid that is a variant compared to the corresponding amino acid of the TOI encoded by the nucleotide sequence having the highest cumulative human allele frequency and / or the highest total human genotype frequency. Join. In one example, the ligand specifically binds to an epitope containing two or more such variant amino acids. In some examples, specific binding means binding with an affinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less, as determined, for example, by SPR.

The term "epitope" is the region of the antigen to which the antibody binds. Epitopes may also be defined as structural or functional epitopes. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. The epitope can also be a three-dimensional epitope, that is, an epitope composed of non-linear amino acids. In certain embodiments, the epitope may include determinants that are chemically active surface atomic groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments. The form may have specific three-dimensional structural features and / or specific charge features.

Clause 33: Treat and / or prevent TOI-mediated diseases or conditions in humans whose genome contains less than 50% cumulative human allelic frequency and / or less than 50% total human genotype frequency. Of an anti-TOI ligand that binds to a human TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% in the manufacture of a drug for use.

Clause 34: Less than 50% cumulative human allelic frequency and / or less than 50% total human genes in the manufacture of drugs to target TOI and treat and / or prevent TOI-mediated diseases or conditions in humans. Use of an anti-TOI ligand that binds to a human TOI containing an amino acid sequence encoded by a genotypic TOI nucleotide sequence.

Use according to clause 33 or 34, wherein the ligand, human, disease or condition is described in any one of clauses 1-27.

Clause 35: A method of targeting TOI to treat and / or prevent TOI-mediated diseases or conditions in humans, with less than 50% cumulative human allelic frequency and / or less than 50% total human genes. A method comprising administering an anti-TOI ligand to a human containing a selected TOI nucleotide sequence having a genotype frequency, whereby the TOI encoded by the nucleotide sequence is targeted.

Clause 36: The step of targeting a human TOI containing an amino acid sequence with the ligand to treat and / or prevent the disease or condition in the human, wherein the amino acid sequence is less than 50% cumulative human allele. 35. The method of clause 35, comprising a step encoded by a nucleotide sequence having a gene frequency and / or a total human genotype frequency of less than 50%.

Clause 37: A method of TOI genotyping a human nucleic acid sample, in which a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50% is present. A method comprising the step of identifying.

In one example, the method involves obtaining a TOI nucleic acid sample from a human and then performing the step of identification.

Clause 38: A method of TOI typing a human protein sample, encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50% in the sample. A method comprising identifying the presence of a TOI amino acid sequence.

In one example, this method involves obtaining a TOI protein sample from a human and then performing the step of identification.

Clause 39: Described in Clause 37 or 38, comprising obtaining a sample of serum, blood, feces, hair, urine or saliva from a human, thereby obtaining a nucleic acid or protein sample for use in the step of identifying the sequence. the method of.

Clause 40: The method of any one of Clauses 37-39, comprising performing the identification step using the ligand according to any one of Clauses 17-27.

Clause 41: With a ligand capable of binding to a human TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%, Clause 38 or Diagnostic kit containing instructions and instructions for performing the method described in 39.

Clause 42: Nucleic acid probes comprising a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50% or a nucleotide sequence that specifically hybridizes to its RNA transcript, and clause. Diagnostic kit containing instructions and instructions for performing the method described in 38 or 39.

Clause 43: To one of the above clauses, where the TOI is encoded by a nucleotide sequence having a cumulative human allelic frequency of 1-10% and / or a total human genotype frequency of 1-about 15% or 1-15%. The method, ligand, composition, kit or use described.

Clause 44: The TOI is the human TOI selected from Table 5; optionally to treat and / or prevent the corresponding disease or condition detailed in Table 5 (Table 6). , Any of the methods, ligands, compositions, kits or uses described in the above clause.

For example, the TOI is human PCSK9, such as mature, cleaved, autocatalytic, or active PCSK9. In one example, the disease is a cardiovascular disease, such as hyperlipidemia.

The ligands of the present invention are useful, for example, in specific binding assays for genotyping or phenotyping humans, purification by TOI affinity, and screening assays for identifying other antagonists with TOI activity. .. Some of the ligands of the invention are useful for inhibiting the binding of TOI to cognate human receptors or proteins, or for inhibiting TOI-mediated activity.

The present invention includes anti-TOI (eg, PCSK9) antibody ligands with a modified glycosylation pattern. In some applications, modifications to remove unwanted glycosylation sites, or removal of fucose moieties to increase antibody-dependent cellular cytotoxicity (ADCC), for example, may be useful [Shield et al. (2002). See JBC 277: 26733]. In other uses, galactosylation modifications may be made to modify complement-dependent cytotoxicity (CDC).

In one example, the invention is a recombinant human antibody or fragment thereof that specifically binds to a TOI (eg, a rare variant as described herein) or with a ligand of the invention that comprises it. , A pharmaceutical composition comprising a pharmaceutically acceptable carrier. In one embodiment, the invention features a composition that is a combination of an antibody ligand or an antigen-binding fragment of an antibody of the invention and a second therapeutic agent. The second therapeutic agents are anti-inflammatory agents, anti-angiogenic agents, analgesics, diuretics, chemotherapeutic agents, anti-neoplastic agents, vasodilators, vasoconstrictors, statins, beta blockers, nutrients, adjuvants, anti-obesity. It can be either a symptom or an anti-diabetic agent.

"Pharmaceutically acceptable" has been or is expected to be approved by the US federal or state government regulators, or is generally accepted by the United States Pharmacopeia or other commonly accepted animals, including humans. Refers to those listed in the Pharmacopoeia. A "pharmaceutically acceptable carrier, excipient, or adjuvant" can be administered to a subject with a drug, eg, any antibody or antibody chain described herein, without disrupting its pharmacological activity. Refers to a carrier, excipient, or adjuvant that is non-toxic when administered in a dose sufficient to deliver a therapeutic dose of the drug.

In one example, the invention features a method for inhibiting TOI activity using an anti-TOI ligand of the invention (eg, an antibody of the invention or an antigen-binding portion of an antibody), the therapeutic method being therapeutically effective. It comprises the step of administering a pharmaceutical composition comprising an amount of a ligand. The disorder being treated is any disease or condition that is ameliorated, alleviated, inhibited or prevented by the removal, inhibition or reduction of TOI activity.

The phrase "therapeutically effective amount" means the amount administered to produce the desired effect. The exact amount is expected to depend on the purpose of the procedure and can be confirmed by one of ordinary skill in the art using known techniques (see, eg, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). That).

The term "specifically binds" and the like means that a ligand, such as an antibody or antigen-binding fragment thereof, forms a complex with a relatively stable antigen under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 × 10 ^-6 M or less (eg, a smaller KD indicates a tighter binding). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance and the like. However, isolated antibodies that specifically bind to human TOI may be cross-reactive with other antigens, such as TOI molecules from other species. Moreover, a multispecific antibody (eg, bispecific) that binds a human TOI to one or more additional antigens nevertheless "specifically binds" to a TOI as used herein. It is considered an antibody.

Genotyping and Phenotyping Those skilled in the art are expected to be familiar with the techniques that can be used for accurate genotyping and application to the present invention. These include the following.
1 Hybridization-based method
1.1 Dynamic allele-specific hybridization
1.2 Molecular beacon
1.3 SNP microarray
2 Enzyme-based method
2.1 Restriction enzyme fragment length polymorphism
2.2 PCR-based method
2.3 Flap endonuclease
2.4 Primer extension
2.5 5'-nuclease
2.6 Oligonucleotide ligation assay
3 Other post-amplification methods based on the physical properties of DNA
3.1 Single-strand higher-order polymorphism
3.2 Temperature gradient gel electrophoresis
3.3 Modified High Performance Liquid Chromatography
3.4 High-resolution melting of the entire amplicon
3.5 Use of DNA mismatch binding proteins
3.6 SNPlex [SNPlex ™ is a genotyping platform exclusively sold by Applied Biosystems].

Next-generation sequencing techniques such as pyrosequencing are also useful.

In addition, GB2444410A, which is incorporated herein by reference in its entirety, and the genotyping method disclosed herein are also referenced.

Miniaturized assays, such as microarrays with oligonucleotide reagents immobilized on a small surface, allow large-scale mutation analysis and high-throughput genotyping (large-scale identification, mapping, and genotyping of single nucleotide polymorphisms in the human genome). Frequently advocated for (judgment) (Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lipshutz R, Chee M, Lander ES, Science. May 15, 1998 280 (5366): 1077-82). Other high-throughput methods identify alleles by differential hybridization, primer extension, ligation and cleavage of allele-specific probes (Review Accessing genetic variation: genotyping single nucleotide polymorphisms, Syvanen AC, Nat Rev Genet. 2001 Dec; 2 (12): 930-42; Review Techniques patents for SNP genotyping, Twyman RM, Primrose SB, Hybridogenomics. 2003 Jan; 4 (l): 67-79).

An approach for fully automated large-scale SNP analysis is a "uniform" assay, a single-phase assay without a separation step, which allows continuous monitoring during amplification. The TaqMan ™ Assay (Applied Biosystems), originally designed for quantitative real-time PCR, is a uniform, single-step assay that is also used to determine DNA mutation status [eg, , AA Komar (eds.), Single Nucleotide Polymorphisms, Methods in Molecular Biology 578, DOI 10.1007 / 978-1-60327-411-1_19, Humana Press, Springer Science + Business Media, Part of LLC; and Single Nucleotide Polymorphisms, Methods in Molecular Biology ™, Vol. 578, 2009, pp. 293-306, The TaqMan Method for SNP Genotyping, Gong-Qing Shen et al.]. The TaqMan SNP genotyping assay utilizes the 5'-exonuclease activity of AmpliTaq Gold ™ DNA polymerase to cleave a double-labeled probe hybridized to the SNP-containing sequence of ssDNA. Cleavage separates the 5'-fluorophore from the 3'-quencher, causing a detectable fluorescent signal. The use of two allele-specific probes with different fluorophores allows SNP determination in the same in vitro without any post-PCR processing. Genotype is determined by the ratio of the intensities of the two fluorescent probes at the end of amplification. Therefore, rather than leveraging the entire set of real-time PCR data as done in quantitative studies, only endpoint data is used.

High-throughput automated TaqMan SNP genotyping is facilitated by the use of a validated, prefabricated, prefabricated TaqMan® genotyping assay, although Custom TaqMan® assays are also available. [High-through put genotyping with single nucleotide polymorphisms, Ranade K, Chang MS, Ting CT, Pei D, Hsiao CF, Olivier M, Pesich R, Hebert J, Chen YD, Dzau VJ, Curb D, Olshen R, Rich N, Cox DR, Botstein D, Genome Res. July 2001; 11 (7): 1262-8; Assessment of two flexible and compatible SNP genotyping platforms: TaqMan SNP Genotyping Assays and the SNPlex Genotyping System, De la Vega FM, Lazaruk KD , Rhodes MD, Wenz MH, Mutat Res. June 3, 2005; 573 (1-2): 111-35]. The results of the assay can be automatically determined by genotyping software on the real-time thermal cycler (eg Bio-Rad IQ software, Applied Biosystems sequence detection software).

Single nucleotide polymorphisms (SNPs) can be determined using TaqMan ™ real-time PCR assay (Applied Biosystems) and commercially available software that assigns genotypes based on reporter probe signals at the end of amplification. Algorithms for automatic genotyping of SNPs using all of TaqMan real-time data are available (A. Callegaro et al., Nucleic Acids Res. 2006; 34 (7): e56, April 14, 2006 at doi. Published online: 10.1093 / nar / gkl185, PMCID: PMC1440877). This algorithm is unique in that it classifies samples according to the behavior of blanks (without DNA samples) and clusters them with heterozygous samples. This classification method eliminates the need for positive controls and allows accurate genotyping, for example, in the rare case of one allele, even in the absence of a genotype class.

Those skilled in the art are expected to be familiar with techniques that can be used for accurate phenotyping and application to the present invention. These include the use of amino acid sequencing of isolated target proteins and comparison of sequences from different variants (eg, the most common variants). Antibodies that specifically and selectively bind in the region of SNP under stringent conditions can also be used to identify specific variants. In another method, the genotype is determined, eg, the corresponding amino acid sequence (phenotype) is determined by in silico translation.

Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising anti-TOI ligands such as antibodies of the invention or antigen-binding fragments thereof. Administration of the therapeutic composition according to the invention is expected to be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. To. A number of suitable formulations can be found in Formulations known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) -containing vesicles [eg LIPOFECTINT ™], DNA conjugates, anhydrous absorption pastes, etc. Examples include oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of preferablys for parenteral excipients" PDA (1998) J Pharm Sci Technol 52: 238-311.

The dose may be changed according to the age and size of the subject to be administered, the target disease, condition, route of administration and the like. When a ligand of the invention, eg, an antibody, is used to treat a variety of TOI-related conditions and diseases in an adult patient, it is usually about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, It is advantageous to administer the antibodies of the invention intravenously in a single dose of about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted.

Various delivery systems are known and are recombinant cells capable of expressing the ligands or pharmaceutical compositions of the invention, eg, encapsulation in liposomes, microparticles, microcapsules, mutant viruses, receptor mediation. It can be used to administer the ligand provided by endocytosis (see, eg, Wu et al. (1987) J. Biol. Chem. 262: 4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The ligand or composition may be administered by any convenient route, eg, by instillation or bolus injection, by absorption through the lining of the epithelial or mucosal skin (eg, oral mucosa, rectum and intestinal mucosa, etc.). It may be administered with a bioactive substance. Administration may be systemic or topical.

Ligands or pharmaceutical compositions can also be delivered in vesicles, especially liposomes [Langer (1990) Science 249: 1527-1533; Treat et al. (1989), Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein. and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid, pp. 317-327; see ibid in general].

In certain circumstances, the ligand or pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used [Langer, supra; see Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201]. In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition and therefore requires only a systemic dose (eg, Goodson, Medical Applications of Controlled Release, supra, supra). See Volume 2, pp. 115-138, 1984).

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, infusion, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibodies or salts thereof in sterile aqueous or oily media customarily used for injection. Aqueous vehicles for injection include, for example, saline, isotonic solutions containing glucose and other auxiliaries, which are suitable solubilizers such as alcohols (eg ethanol), polyvalent. It may be used in combination with alcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysolvate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hardened castor oil)] and the like. As the oil-based medium, for example, sesame oil, soybean oil and the like are adopted. These may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol and the like. The injection thus prepared is preferably filled in a suitable ampoule. The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen-type delivery devices can be readily used in applications in the delivery of the pharmaceutical compositions of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices typically utilize replaceable cartridges containing pharmaceutical compositions. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. In this way the pen-type delivery device can be reused. For disposable pen-type delivery devices, there are no replaceable cartridges. Instead, the disposable pen-type delivery device is pre-filled with the pharmaceutical composition and held in a storage container within the device. When the pharmaceutical composition in the storage container is empty, discard the entire device.

A large number of reusable pen-type and self-injector delivery devices are applied for subcutaneous delivery of the ligands or pharmaceutical compositions of the present invention. Examples are not limited to them at all, but to name a few, AUTOPEN ™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC ™ Pen (Disetronic Medical Systems, Burghdorf). , Switzerland), HUMALOG MIX 75/25 ™ Pen, HUMALOG ™ Pen, HUMALIN 70/30 ™ Pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN ™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ™ (Novo Nordisk, Copenhagen, Denmark), BD ™ Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT ™, OPTIPEN PRO (Trademark) Trademarks), OPTIPEN STARLET ™, and OPTICLIKT ™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices applied to the subcutaneous delivery of the pharmaceutical compositions of the present invention are, but are not limited to, SOLOSTAR ™ Pens (sanofi-aventis), FLEXPEN ™. ) (Novo Nordisk) and KWIKPEN ™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in unit dose dosage forms suitable for matching the doses of the ligand. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per unit dose dosage form; especially in the form of injectables, the aforementioned antibody is contained in about 5 to about 100 mg. It is preferably contained in about 10 to about 250 mg in other dosage forms.

Illustrative TOI
For example, in any configuration, embodiment, concept, embodiment or configuration of the present invention, the TOI thereof is ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aglycan; AGR2; AICDA; AWl; AIG1; AKAP1; AKAP2; AIYIH; Amyloid-Beta; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; Axl; AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); B1yS; BMP1; BMP2; BMP3B (GDF1O); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (Plectin); BRCA1; (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CB1; CCBP2 (D6 / JAB61); CCL1 (I-309); CCL11 (Eotaxin); CCL13 (MCP-4); CCL15 ( MIP-id); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP-1); MCAF; CCL2O (MIP-3a); CCL21 (MIP-2) ); SLC; Exodus-2; CCL22 (MDC / STC-1); CCL23 (MPIF-1); CCL24 (MPIF-2I Eotaxin-2); CCL25 (TECK); CCL26 (Eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MIP-1a); CCL4 (MIP-1b); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 ( CKR1 / HM145); CCR2 (mcp-1RB / RA); CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBI1); CCR8 (CMKBR8 / TER1 / CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR); CD7, CD164; C D19; CD1C; CD2O; CD200; CD-22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD4O; CD4OL; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD8O; CD81; CD83; CD86; CD96; CD207; CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6 CDK7; CDK9; CDKN1A (p2lWap1 / Cip1); CDKN1B (p27Kip1); CDKNIC; CDKN2A (p16lNK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CELSR3; CER1; CHGA; CHGB; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (Claudin-7); CLN3; CLU (clusterlin); CMKLR1; CMKOR1 (RDC1); CNR1; COL18A1; COL1A1; COL4A3; COL6A1; CR2; (M-CSF); CRLF2; CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (catepsin B); CX3CL1 (SCYDi); CX3CR1 (V28); CXCR6; CXCL1 (GRO1) ); CXCL1O (IP-10); CXCL11 (I-TAC / IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78I LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMSTR ISTRL33 I Bonzo); CYB5; CYC1; CYSLTR1; DAB2IP; DAND5; DES; DKFZp451J0118; DNCL1; DPP4; E2 ECGF1; EDG1; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EphA4; EPHB4; EP O; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; F3 (TF); FADD; FasL; FASN; FCER1A; FCER2; FCGR3A; FCRL4; FGF; FGF1 (aFGF); FGF1O; FGF11; FGF12; FGF12B FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF2O; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF) ); FGF8; FGF9; FGFR3; FIGF (VEGFD); FIL1 (Epsilon); FIL1 (Zeta); FLJ12584; FLJ25530; FLRT1 (Fibronectin); FLT1; FOS; FOSL1 (FRA-l); FY (DARC); GABRP ( GABAa); GAGEB1; GAGEC1; Galectin-3; GALNAC4S-65T; GATA3; GDF5; GFI1; GGT1; GHR; GM-CSF; GNAS1; GNRH1; GPR2 (CCR1O); GPR31; GPR44; GPR81 (FKSG8O); GPR87; GRCC10 (C10); GRP; GSN (gersoline); GSTP1; HAVCR1; HAVCR2; HDAC4; EDAC5; HDAC7A; HDAC9; hepsidin; hemodoberin; HGF; HIF1A; HIP1; histamine and histamine receptor; HLA-A; HLA-DRA HM74; HMOX1; HUMCYT2A; ICEBERG; ICOS; 1D2; IFN-a; IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFN gamma; TNFW1; IGF1; IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IL-1; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; 1L13; IL13RA1; IL13RA2; 1L14; 1L15; IL15RA; IL16; 1L17; IL17B; IL17C; IL17R; 1L18; IL18BP; IL18R1; IL18RAP; 1L19; IL1A; IL1B; IL1F1O; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HY1; IL1R 1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1RL2 IL1RN; 1L2; 1L20; IL2ORA; IL21R; 1L22; 1L22R; 1L22RA2; 1L23; 1L24; 1L25; 1L26; 1L27; 1L28A; 1L2 1L3; 1L30; IL3RA; 1L4; IL4R; 1L5; IL5RA; 1L6; IL6 receptor; IL6ST (glycoprotein 130); 1L7; TL7R; 1L8; IL8RA; IL8RB; IL8RB; 1L9; IL9R; ILK; INHA; INHBA; INSL3; INSL4; IRAK1; IRAK2; ITGA1; ITGA2; 1TGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; MTLG; KLF5 (GC box) BP); KLF6; KLK10; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (keratin 19); KRT2A; KRTHB6 (hair-specific type II keratin); LAG3; LAMA5; LEP (Leptin); LIGHT; Lingo-p75; Lingo-Troy; LPS; LRP5; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK MIB1; Midkine; MIF; MIP-2; MK167 (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-ifi); MTSS1; MUC1 (mutin); MYC; MYD88; NCK2; Can; Nav1.7; Nav1.8; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NROB1 NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4; ODZ1; OPG; OPRD1; OX40L; OX40; P2RX7; PAP; PART1; PATE; PAWR; PCA3; PCNA; PCSK9, PD-1, PD-L1; PDGFA; PDGFB; PECAM1; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; placenta growth factor (PIGF); PIK3CG; PLAU (uPA); PLG; PLXDC1; PPBP (CXCL7); PPID; PR1; PRKCQ; PRKD1; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p2lRac2); RARB; RGS1; RGS13; RGS3; RNF11O (ZNF144); ROBO2; ROR1; S100A2; SCGB1D2 (Lipophyllin B) SCGB2A1 (mammaglobin 2); SCGB2A2 (mammaglobin 1); SCYE1 (endothelial monocyte activating cytokine); SDF2; SERPINA1; SERPINIA3; SERPINB5 (maspin); SERPINE1 (PAT-i); SERPINF1; SHBG; SLA2; SLC2A2 SLC33A1; SLC43A1; SLIT2; SPP1; SPRR1B (Spri); ST6GAL1; STAB1; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCP1O; TDGF1; TEK; TGFA; TGFB1; TGFB1I1; TGFB2; TGF3; TGF1; TGFBR3; TH1L; THBS1 (Thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-i); TIM3; TMP3; Tissue factor; TLR1O; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9 TMPRSS6; TNF; TNF-α; TNFAIP2 (B94); TNFAIP3; TNFRSF11A; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF1O (TRAIL); TNFSF11 (TRAIL); ); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (0X40 ligand); TNFSF5 (CD4O ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSF8 (CD3O ligand); TNFSF9 (4-1BB ligand); TOLLIP; Toll-like receptor; TOP2A (topoisomerase lia); TP53; TPM1; TPM2; TRADD; TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TRAILD; TREM1; TREM2; TRPC6; TSLP; TWEAK; VEGFA; VEGFB; VEGFC; Versican; VHL C5; -4; Wnt7A; XCL1 (phosphotactin); XCL2 (SCM-lb); XCR1 (GPR5 / CCXCR1); YY1; and ZFPM2.

In one example, the TOI is a human TOI selected from Table 5.

In one example, the TOI is an OX40 ligand.

In one example, the TOI is OX40.

In one example, the TOI is PCSK9.

In one example, the TOI is the IL6 receptor (IL-6R).

In one example, the TOI is LIGHT.

In one example, the TOI is VEGF-A.

In one example, the TOI is TNF alpha.

In one example, the TOI is PIGF.

In one example, the TOI is IGF1R.

In one example, the TOI is OPG.

In one example, the TOI is ICOS.

In one example, the TOI is NGF.

In one example, the TOI is BMP6.

In one example, the TOI is ferroportin.

In one example, the TOI is TMPRSS6.

In one example, the TOI is hemodaverin.

In one example, the TOI is a VEGF receptor.

In one example, TOI is a PDGF receptor.

In one example, the TOI is a stem cell factor receptor.

In one example, the TOI is hepcidin.

In one example, the TOI is IL-4 receptor alpha.

In one example, the TOI is sclerostin.

In one example, the TOI is the IL-13 receptor.

In one example, the TOI is CD7.

In one example, the TOI is a delta-like ligand-4 (Dll4).

In one example, the TOI is HGF.

In one example, the TOI is angiopoietin-2 (Ang2).

In one example, the TOI is GDF8.

In one example, the TOI is ERBB3.

In one example, the TOI is the IL-17 receptor.

In one example, the TOI is CD40.

In one example, the TOI is a CD40 ligand.

In one example, the TOI is EGFR.

In some examples, a TOI contains a mutation as described herein for that TOI, or is encoded by a nucleotide sequence containing an SNP as described herein for that TOI. Optionally, in addition, the human comprises the TOI containing the mutation; or the human comprises a nucleotide sequence encoding the TOI containing the mutation.

For example, the TOI is human PCSK9 and contains mutations I474, E670G, N425S or Q619P in SEQ ID NO: 1. Optionally, in addition, the human comprises the PCSK9 containing the mutation; or the human comprises a nucleotide sequence encoding the PCSK9 containing the mutation. In alternatives, for example, where it is stated herein that PCSK9 has a specific "type", eg, type f, c, r, p, e, h, aj or q, in alternatives PCSK9 Is a human PCSK9 containing a mutation selected from R46L, A53V, N425S, A443T, I474V, Q619P and E670G in SEQ ID NO: 1 (including, for example, mutants I474, E670G, N425S or Q619P); for example, PCSK9 , Includes I474V in SEQ ID NO: 1, and optionally humans include such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, this method is to treat or prevent abnormal lipidemia. For example, to lower or maintain a lower cholesterol in humans); for example, PCSK9 comprises E670G in SEQ ID NO: 1, and optionally humans encode such PCSK9 or such PCSK9. Containing nucleotide sequences (eg, this method is to treat or prevent abnormal lipidemia, eg to lower or maintain cholesterol lowering in humans); for example, PCSK9 is SEQ ID NO: 1. Includes Q619P in, and optionally humans include such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, this method is to treat or prevent abnormal lipidemia, eg, cholesterol in humans. (To reduce or maintain a lower cholesterol); for example, PCSK9 comprises N425S in SEQ ID NO: 1, and optionally humans include such PCSK9 or a nucleotide sequence encoding such PCSK9. (For example, this method is to treat or prevent abnormal lipidemia, eg to lower or maintain a lower cholesterol in humans); for example, PCSK9 comprises R46L in SEQ ID NO: 1. , Optionally, the human comprises such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, this method is to treat or prevent abnormal lipidemia, eg, to increase cholesterol in humans). For example, PCSK9 contains A53V in SEQ ID NO: 1, and optionally humans such PCSK9 or such PCs. Includes a nucleotide sequence encoding SK9 (eg, this method is to treat or prevent abnormal lipidemia, eg to increase cholesterol in humans); for example, PCSK9 has A443T in SEQ ID NO: 1. Including, optionally, humans comprise such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, this method is to treat or prevent abnormal lipidemia, eg, increase cholesterol in humans. Because).

For example, the TOI is human IL6R and comprises the mutation D358A or V385I in SEQ ID NO: 78. Optionally, in addition, the human comprises the IL6R containing the mutation; or the human comprises a nucleotide sequence encoding the IL6R containing the mutation.

For example, the TOI is human IL4Ra and contains mutations selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Optionally, in addition, the human comprises the IL4Ra containing the mutation; or the human comprises a nucleotide sequence encoding the IL4Ra containing the mutation.

For example, the TOI is human Nav1.7, 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G, including mutations selected from the group, eg, the mutation is 1161W. Optionally, in addition, the human comprises said Nav1.7 containing the mutation; or the human comprises a nucleotide sequence encoding said Nav1.7 containing said mutation.

For example, the TOI is human VEGF-A and is encoded by a VEGF-A nucleotide sequence containing an SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360. Will be done. Optionally, in addition, the human comprises the VEGF-A containing the mutation; or the human comprises a nucleotide sequence encoding the VEGF-A containing the mutation.

For example, the TOI is human PDGF-B, an SNP selected from the group consisting of rs142404523 (ie, C corresponding to position -776) and C at position rs1800818 (ie, C corresponding to position -735). Encoded by the containing PDGF-B nucleotide sequence. Optionally, in addition, the human comprises the PDGF-B containing the mutation; or the human comprises a nucleotide sequence encoding the PDGF-B containing the mutation.

For example, the TOI is human PDGFR-B, a PDGFR-B nucleotide sequence containing an SNP selected from the group consisting of rs246395 (ie, G corresponding to position 2601) and rs74943037 (ie, T corresponding to position 1391). Coded by. Optionally, in addition, the human comprises the PDGFR-B containing the mutation; or the human comprises a nucleotide sequence encoding the PDGFR-B containing the mutation.

For example, the TOI is human PD-L1 and is encoded by a PD-L1 nucleotide sequence containing an SNP selected from the group of PD-L1 SNPs listed herein. Optionally, in addition, the human comprises the PD-L1 containing the mutation; or the human comprises a nucleotide sequence encoding the PD-L1 containing the mutation.

For example, the TOI is human PD-1 and is encoded by a PD-L1 nucleotide sequence containing an SNP selected from the group of PD-1 SNPs listed herein. Optionally, in addition, the human comprises the PD-1 containing the mutation; or the human comprises a nucleotide sequence encoding the PD-1 containing the mutation.

For example, the TOI is a human angiopoietin (eg Ang-2) and angiopoietin (eg Ang-2) containing an SNP selected from the group of SNPs of the angiopoietin (eg Ang-2) listed herein. Encoded by the nucleotide sequence of. Optionally, in addition, does the human contain the angiopoietin (eg, Ang-2) containing the mutation; or the human comprises a nucleotide encoding the angiopoietin (eg, Ang-2) containing the mutation. Contains an array.

For example, the TOI is a human BMP6 and is encoded by a nucleotide sequence of BMP6 containing an SNP selected from the group of BMP6 SNPs listed herein. Optionally, in addition, the human comprises the BMP6 containing the mutation; or the human comprises a nucleotide sequence encoding the BMP6 containing the mutation.

For example, the TOI is a human hemoduverin and is encoded by a nucleotide sequence of hemoduverin containing an SNP selected from the group of hemoduverin SNPs listed herein. Optionally, in addition, the human comprises the hemodaverin containing the mutation; or the human comprises a nucleotide sequence encoding the hemoduverin containing the mutation.

For example, a TOI is a human hepcidin and is encoded by a nucleotide sequence of hepcidin containing an SNP selected from the group of hepcidin SNPs listed herein. Optionally, in addition, the human comprises the hepcidin containing the mutation; or the human comprises a nucleotide sequence encoding the hepcidin containing the mutation.

For example, the TOI is a human ferroportin, encoded by the nucleotide sequence of a ferroportin containing an SNP selected from the group of ferroportin SNPs listed herein. Optionally, in addition, the human comprises the ferroportin containing the mutation; or the human comprises a nucleotide sequence encoding the ferroportin containing the mutation.

For example, the TOI is human TMPRSS6 and is encoded by the nucleotide sequence of TMPRSS6 containing an SNP selected from the group of TMPRSS6 SNPs listed herein. Optionally, in addition, the human comprises the TMPRSS6 containing the mutation; or the human comprises a nucleotide sequence encoding the TMPRSS6 containing the mutation.

For example, the TOI is human transferrin and is encoded by the nucleotide sequence of transferrin, including SNPs selected from the group of transferrin SNPs listed herein. Optionally, in addition, the human comprises the transferrin containing the mutation; or the human comprises a nucleotide sequence encoding the transferrin containing the mutation.

For example, a TOI is a human hemochromatosis protein (HFE) and is encoded by a nucleotide sequence of an HFE containing an SNP selected from the group of SNPs for HFEs listed herein. Optionally, in addition, the human comprises the HFE containing the mutation; or the human comprises a nucleotide sequence encoding the HFE containing the mutation.

For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are shown below. Unless otherwise specified, or implied by context, the following terms and phrases embrace the meanings set forth below. As the scope of the invention is limited only by the claims, the definitions are provided to aid in the description of certain embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. In the event of any apparent discrepancy between the use of terms in the art and their definitions set forth herein, the definitions set forth herein shall prevail.

For convenience, the text, specification, examples and specific terms used in the appended claims are collected here.

The terms "decreased," "decreased," or "reduced" are all used herein to mean a statistically significant amount of reduction. In some embodiments, "decreased," "reduced," or "decreased" typically means a reduction of at least 10% relative to the reference level (eg, the absence of a given treatment). And, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least About 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or More reductions can be mentioned. As used herein, "reduction" does not include complete reduction compared to reference levels. The reduction can preferably be to a level that is acceptable to a given unimpaired individual. However, for the purpose of lowering or lowering cholesterol levels, for example, a reduction of about 5-10 points can be considered a "decrease" or "reduction".

In certain embodiments of all embodiments of the invention, the term "inhibition" is used. Inhibition refers to a reduction of at least 10% compared to a reference level (eg, the absence of a given treatment), eg, at least about 10%, at least about 20%, at least about 25% compared to a reference level. , At least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% , At least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or even more inhibition reductions can be mentioned. "Complete inhibition" refers to 100% inhibition compared to the reference level.

The terms "increased", "increased", "increased", or "activated" are all used herein to mean a statistically significant amount of increase. In some embodiments, the terms "increased", "increased", "increased", or "activated" are at least 10% increased relative to the reference level, eg, compared to the reference level. At least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%. , Or an increase of up to 100%, including equivalence, or any increase between 10 and 100%, or at least about 2 times, or at least about 3 times, or at least about 4 times the reference level. Or it can mean an increase of at least about 5 times, or at least about 10 times, or any increase between 2 and 10 times or more. In the context of a marker or symptom, "increasing" is a statistically significant increase at such a level.

As used herein, the term "substantially" refers to a qualitative condition that presents all or nearly all degrees or degrees of a feature or property of interest. Those skilled in the art of biology will assume that biological and chemical phenomena have been completed and / or completely progressed, or have achieved or avoided absolute consequences. It is expected that they understand that it is rare. The term "substantially" is therefore used herein to capture the non-completion potential specific to many biological and chemical phenomena. To dispel doubt, "substantially" means at least 90% degree or degree of the desired feature or property, eg, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or it. It may refer to a greater degree or degree.

As used herein, "subject" means human or animal. Animals are usually vertebrates, such as primates, rodents, livestock or hunting animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchuck, ferrets, rabbits and hamsters. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual", "patient" and "subject" are used interchangeably herein. In some embodiments, the subject can be a non-human vertebrate, such as a primate, rodent, mouse, rat, pig, sheep, zebrafish, frog, and the like.

Preferably, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cows, but not limited to these examples. Mammals other than humans can be advantageously used as objects to provide animal models of diseases or conditions, such as cardiovascular conditions. The subject may be male or female.

Subjects are those who have or have been previously diagnosed with or have been identified as having or having one or more complications relating to a condition requiring treatment or such condition. Optionally, the subject may have already been treated for the condition or one or more complications associated with the condition. Alternatively, the subject may also be a condition or subject who has not been previously diagnosed with one or more complications associated with that condition. For example, a subject may be a subject who presents one or more risk factors for a condition or one or more complications of that condition, or may be a subject who does not present a risk factor.

A "subject in need" or "human in need" of a particular condition is at risk of having that condition, eg, an increase in cholesterol levels, being diagnosed with that condition, or developing that condition. Can be a target.

As used herein, the terms "protein" and "polypeptide" refer to a series of amino acid residues that are linked together by a peptide bond between the alpha-amino group and the carboxy group of the adjacent residue. As used herein synonymously. The terms "protein" and "polypeptide" refer to polymers of amino acids, including natural amino acids. When referred to as a "modified polypeptide", it refers to a polypeptide containing modified amino acids (eg, phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Point. The terms "protein" and "polypeptide" are often used for relatively large polypeptides, whereas the term "peptide" is often used for small polypeptides, but the usage of these terms in the industry. Are overlapping. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins with specific sequences. Peptide homologues, peptide orthologs, peptide paralogs, peptide fragments and other equivalents, peptide variants, fragments, and analogs can also be used, as these terms are understood by those skilled in the art.

As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to a ribonucleic acid, any molecule incorporating a unit of deoxyribonucleic acid, preferably a polymeric molecule. The nucleic acid may be either single-stranded or double-stranded. The single-stranded nucleic acid can be a single nucleic acid strand of denatured double-stranded DNA. Instead, the nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid may be DNA. In another embodiment, the nucleic acid may be RNA. Suitable nucleic acid molecules are genomic DNA or DNA containing cDNA. Another suitable nucleic acid molecule is RNA, including mRNA. Nucleic acid analogs can also be used in some embodiments.

As used herein, the term "nucleic acid probe" is an isolated oligonucleotide having a nucleic acid sequence that is capable of hybridizing to a target nucleic acid sequence, eg, specifically hybridizing to a target sequence. Refers to a molecule. In some embodiments, the nucleic acid probe may further comprise a detectable label. In some embodiments, the nucleic acid probe may be attached to a solid surface. In some embodiments, the nucleic acid is about 5 nt to about 100 nt in length.

As used herein, the term "siRNA" refers to a nucleic acid that forms an RNA molecule that contains two distinct strands of RNA that are substantially complementary to each other. Typically, the siRNA is at least about 15-40 nucleotides in length (eg, each complementary sequence of a double-stranded siRNA is about 15-40 nucleotides in length, and the double-stranded siRNA is about about 15-40 nucleotides in length. A length of 15-40 base pairs, preferably about 19-25 nucleotides (eg, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In some embodiments, the siRNA may be blunt-ended. In some embodiments, the siRNA may contain a 3'and / or 5'overhang with a length of about 0, 1, 2, 3, 4, or 5 nucleotides in each strand. The length of the overhang is irrelevant between the two strands, that is, the length of the overhang in one strand does not depend on the length of the overhang in the second strand. The siRNA molecule may also contain a 3'hydroxyl group. In some embodiments, the siRNA may contain a 5'phosphate group. When a target gene, eg, siRNA as a target RNA, is present or expressed in the same cell, the siRNA has the ability to reduce or inhibit the expression of the gene or target RNA. SiRNA-dependent post-transcriptional silencing of gene expression involves cleaving the target RNA molecule at a site guided by the siRNA.

As used herein, "PCSK9" or "precursor protein convertase subbutyricin / kexin type 9" refers to serine proteases involved in the regulation of low-density lipoprotein receptor (LDLR) protein levels (Horton et al.). , 2007; Seidah and Prat, 2007). PCSK9 has been shown to interact directly with the LDLR protein, endocytosis with LDLR, and co-immune-fluorescent with LDLR throughout the endosomal pathway (Lagace et al., 2006). PCSK9 is a prohormone-precursor protein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). The sequences of various species of PCSK9 are known, such as human PCSK9 (NCBI gene number 255738). Nucleotide and polypeptide sequences of numerous PCSK9 isoforms, such as SEQ ID NOs: 1-37, are set forth herein.

PCSK9 exists as both professional and mature. PCSK9 pro-type autocatalytics occur between Gln152 and Ser153 [VFAQ | SIP (SEQ ID NO: 116)] (Naureckiene et al., 2003) and have been shown to be required for their cellular secretion (Seidah et al.). , 2003). The inactive form prior to this cleavage may also be referred to herein as the "inactive," "professional," or "unprocessed" form of PCSK9. The C-terminal fragment produced by an autocatalytic event may also be referred to herein as "mature," "cleaved," "processed," or "active" PCSK9. Examples of pro-type and mature PCSK9 isoforms are shown herein, see, eg, SEQ ID NOs: 1-27.

As used herein, the "catalytic domain" of PCSK9 refers to, for example, the portion of the PCSK9 polypeptide corresponding to positions 153 to 449 of PCSK9 of SEQ ID NO: 1. As used herein, the "C-terminal domain" of PCSK9 refers to, for example, the portion of the PCSK9 polypeptide corresponding to positions 450-692 of PCSK9 of SEQ ID NO: 1.

As used herein, a "PCSK9-mediated" disease or condition is caused by changes in PCSK9, such as changes in expression levels, changes in activity, and / or the presence of variants or mutations in PCSK9. Refers to a disease or condition characterized by it. Non-limiting examples of such diseases or conditions include, for example, lipid disorders, hyperlipoproteinemia, hyperlipidemia; abnormal lipidemia; hypercholesterolemia, heart attack, stroke, coronary heart disease. , Atherosclerosis, peripheral vascular disease, lameness (eg, lameness associated with elevated cholesterol), type II diabetes, hypertension, and cardiovascular disease or condition. Another example is acute coronary syndrome. In some examples, the disease or condition is an inflammatory disease or condition or an autoimmune disease or condition. Methods for identifying and / or diagnosing such diseases and conditions are well known to health care workers with conventional skills.

Subjects who are at risk of or have developed a PCSK9-mediated disease or condition are those who present one or more signs or symptoms of such a disease or condition, or such disease. Or a subject with one or more risk factors for a condition, such as an overweight subject, a subject with elevated cholesterol levels, a disease or condition, eg, one or more known to be susceptible to elevated cholesterol levels. Subjects containing the genetic polymorphisms of, eg, subjects with mutations in the LDLR (encoding the low density lipoprotein receptor) or APOB (encoding apolipoprotein B) or PCSK9 gene, and / or such diseases. Or it may be an object with a family history of the condition.

As used herein, "ligand" refers to a molecule that is capable of binding to a second molecule or receptor, eg, specifically capable of binding. In some embodiments, the ligand can be, for example, an antibody, antibody fragment, antibody moiety, and / or affibody.

The term "variant", as used herein, is used in a polypeptide or nucleic acid (eg, the most common in humans, eg, a database as disclosed herein, eg, a database of 1000 genomic projects). Peptides or nucleic acids that differ from (most frequently) in the deletion or addition of one or more amino acids or nucleic acids, but retain one or more specific functions or biological activities of naturally occurring molecules. Point. Amino acid substitutions include alterations in which the amino acids are replaced by different naturally occurring amino acid residues. Such substitutions may also be classified as "conservative", in which case the amino acid residues contained in the polypeptide have similar characteristics in terms of either polarity, side chain functionality or size. Replaced by another naturally occurring amino acid. Such conservative replacements are well known in the art. The substitutions included in the present invention may also be "non-conservative", in which case the amino acid residues present in the peptide are amino acids with different properties, such as naturally occurring amino acids from different groups. Substituted (eg, substituting charged or hydrophobic amino acids with alanine), or instead, naturally occurring amino acids are replaced with unusual amino acids. In some embodiments, the amino acid substitution is conservative. The term variant also includes when used with respect to a polynucleotide or polypeptide, primary and secondary compared to a reference polynucleotide or polypeptide (eg, compared to a wild-type polynucleotide or polypeptide), respectively. , Or a polynucleotide or polypeptide that may have changed in the tertiary structure.

Variants of PCSK9 are provided elsewhere herein. Variants of PCSK9 can include the types described herein, such as a, f, c, r, p, m, eh, aj, and q. Sequences of these variants are shown herein, see, for example, SEQ ID NOs: 1-27 and Table 1 (Table 1), Table 2 (Table 2) or Table 6 (Table 7).

In some embodiments, a "synthetic variant," "recombinant variant," or "chemically modified" polynucleotide or polypeptide variant isolated or produced using methods well known in the art. Can be used. The "modified variant" may include conservative or non-conservative amino acid changes as described below. Changes in polynucleotides can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects of use include insertion and substitution of amino acids and other molecules that are not normally present in the insertion variant, deletion variant, or peptide sequence underlying the variant, eg, but not limited to, human proteins. Includes variants substituted with amino acid substitutions, including the insertion of ornithine, which is not normally present. The term "conservative substitution" as used to describe a polypeptide refers to a change in the amino acid composition of the polypeptide that does not substantially alter the activity of the polypeptide. For example, conservative substitution refers to substituting an amino acid residue with a different amino acid residue having similar chemical properties. Conservative amino acid substitutions include replacement of leucine with isoleucine or valine, replacement of aspartic acid with glutamic acid, or replacement of threonine with serine.

"Conservative amino acid substitution" occurs when one amino acid is replaced by another amino acid with similar structural and / or chemical properties, eg replacement of leucine with isoleucine or valine, replacement of aspartic acid with glutamic acid. , Or by replacing threonine with serine. Therefore, a "conservative substitution" of a particular amino acid sequence is a polypeptide so that the very important amino acid substitution does not reduce the activity of the peptide [ie, the ability of the peptide to penetrate the blood-brain barrier (BBB)]. Refers to substituting an amino acid that is not important for the activity of the amino acid, or substituting an amino acid with another amino acid having similar properties (eg, acidic, basic, positive or negatively charged, polar or non-polar, etc.) .. A table of conservative substitutions showing functionally similar amino acids is well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions with each other: 1) alanine (A), serine (S), leucine (T); 2) aspartic acid (D), glutamic acid (E). 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionin (M), Valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W). [See also Creighton, Proteins, W. H. Freeman and Company (1984), which is incorporated by reference in its entirety. ] In some embodiments, individual substitutions, deletions or additions that modify, add or delete a single amino acid or a small percentage of amino acids are also "conservative" if the change does not reduce the activity of the peptide. It can be regarded as "replacement". Insertions or deletions are typically in the range of about 1-5 amino acids. The selection of conservative amino acids may be based on the position of the amino acid to be substituted in the peptide, eg, the amino acid is either outside the peptide and exposed to a solvent, or inside and exposed to a solvent. It may be selected based on whether or not it is present.

In an alternative embodiment, the location of an existing amino acid, i.e. its exposure to a solvent (ie, the amino acid is exposed to a solvent as compared to an internally localized amino acid that is not exposed to a solvent, or a peptide or polypeptide. Amino acids that are expected to replace existing amino acids can be selected based on (if present on the outer surface of). The choice of such conservative amino acid substitutions is well known in the art, for example, Dordo et al., J. MoI Biol, 1999, 217, 721-739 and Taylor et al., J. Theor. Biol. 119 (1986); 205. As disclosed in ~ 218 and S. French and B. Robson, J. MoI. Evol. 19 (1983) 171. Thus, suitable conservative amino acid substitutions can be selected for amino acids outside the protein or peptide (ie, amino acids exposed to the solvent), eg, but not limited to, the following substitutions: Y at F. Substitution, substitution of T with S or K, substitution of P with A, substitution of E with D or Q, substitution of N with D or G, substitution of R with K, G with N or A Substitution, T at S or K, D at N or E, I at L or V, F at Y, S at T or A, R at K Substitutions, substitutions of G with N or A, substitutions of K with R, and substitutions of A with S, K or P can be used.

In an alternative embodiment, the inclusion of conservative amino acid substitutions suitable for amino acids inside the protein or peptide can also be selected, eg, to amino acids inside the protein or peptide (ie, the amino acids are not exposed to the solvent). Suitable conservative substitutions can be used, eg, but not limited to, the following conservative substitutions: Y at F, T at A or S, I at L or V. Substitution, W at Y, M at L, N at D, G at A, T at A or S, D at N, I at L or Substitutions with V, F with Y or L, S with A or T and A with S, G, T or V can be used. In some embodiments, non-conservative amino acid substitutions are also included in the term variant.

Whether the "antibody", as used herein, is derived from any species that naturally produces the antibody, or is produced by recombinant DNA technology; serum, B cells, hybridomas, immunoglobulins. IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof, whether isolated from Tohma, yeast or bacteria (Fab, F (ab') ₂ , but not limited to, Fab, F (ab') ₂ , Refers to Fv, disulfide-bound Fv, scFv, single-domain antibody, closed higher-order multispecific antibody, disulfide-bound scfv, diabody, etc.). Antibodies may be humanized using conventional techniques.

An "antigen", as described herein, is a molecule that binds to a binding site on a drug that is an antibody. Typically, the antigen is capable of binding to a ligand that is an antibody to trigger an antibody reaction in vivo. The antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term "antigen determinant" refers to an epitope on an antigen that is recognized by an antigen-binding molecule, more specifically by the antigen-binding site of said molecule.

As used herein, the term "antibody fragment" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence that specifically binds to a given antigen. The antibody fragment may include the antibody or a polypeptide containing the antigen binding domain of the antibody. In some embodiments, the antibody fragment may comprise a monoclonal antibody or a polypeptide comprising the antigen binding domain of the monoclonal antibody. For example, an antibody may include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, the antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody fragment" refers to an antigen-binding fragment of an antibody {eg, a single chain antibody, Fab and sFab fragment, F (ab') 2, Fd fragment, Fv fragment, scFv, and domain antibody (dAb) fragment [eg, its. See de Wildt et al., Eur J. Immunol. 1996; 26 (3): 629-39], which is incorporated herein by reference in its entirety]}, plus complete antibodies. Antibodies may have structural characteristics of IgA, IgG, IgE, IgD, IgM (plus their subtypes and combinations). Antibodies may be formed from any source, including mouse, rabbit, pig, rat, and primates (human and non-human primates) as well as primated antibodies. The antibody also includes a midibody, a humanized antibody, a chimeric antibody and the like.

As used herein, an "antibody variable domain" is the light chain of an antibody molecule that contains the amino acid sequences of the complementarity determining regions (CDRs; ie, CDR1, CDR2, and CDR3), and the framework regions (FR). And part of the heavy chain. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. According to the method used in the present invention, the positions of amino acids assigned to CDRs and FRs are determined according to Kabat [Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991)] or It can be defined according to the IMGT nomenclature.

The D domain or region refers to the diversity domain or region of the antibody chain. The J domain or region refers to a ligated domain or region of an antibody chain.

An antibody "gene segment", such as a VH gene segment, D gene segment, or JH gene segment, refers to an oligonucleotide having a nucleic acid sequence encoding that portion of the antibody, eg, a VH gene segment encodes the VH domain of a polypeptide. It is an oligonucleotide containing a nucleic acid sequence to be used.

The VH and VL regions are also hypervariable, called "complementarity determining regions" ("CDRs"), interspersed with more highly conserved regions called "framework regions" ("FR"). It can be subdivided into regions having sex. Framework areas and degrees of CDR are precisely defined (IMGT or Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 , And Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917; these are incorporated herein by reference in their entirety). Each VH and VL is typically composed of 3 CDRs and 4 FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The terms "antigen-binding fragment" or "antigen-binding domain" are used interchangeably herein to refer to one or more fragments of a full-length antibody that retains the ability to specifically bind to a target of interest. Used for. Examples of binding fragments included in the term "antigen-binding fragment" of a full-length antibody are (i) a monovalent fragment consisting of VL, VH, CL and CH1 domains, Fab fragment; (ii) hinge region. F (ab') 2 fragments, which are divalent fragments containing two Fab fragments linked by disulfide bridges in; (iii) Fd fragments consisting of VH and CH1 domains; (iv) consisting of a single arm of antibody. VL and VH domain Fv fragments, (v) dAb fragments consisting of VH or VL domains [Ward et al. (1989) Nature 341: 544-546; in whole incorporated herein by reference]; and (vi) singularity. Included are isolated complementarity determining regions (CDRs) that retain such antigen-binding functionality.

The term "antibody binding site", as used herein, refers to a polypeptide or domain that contains one or more CDRs of an antibody and is capable of binding to an antigen. For example, the polypeptide comprises CDR3 (eg, HCDR3). For example, the polypeptide comprises CDRs 1 and 2 (eg, HCDR 1 and 2) or CDRs 1-3 (eg, HCDR 1-3) of the variable domain of the antibody. In some examples, the antibody binding site is provided by a single variable domain (eg, VH or VL domain). In another example, the binding site comprises a VH / VL pair or two or more such pairs.

The term "specific binding", as used herein, is a chemical interaction between two molecules, compounds, cells and / or particles, wherein the first entity is non-targeted. Refers to a chemical interaction that binds to a second target entity with greater specificity and affinity than when it binds to a third entity. For example, in diagnostic tests, ligand specific binding can identify two variants of the PCSK9 protein as described herein. In some embodiments, the specific binding is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold or greater than the affinity for the third non-target entity, the first. May refer to the affinity of an entity for a second target entity. In the context of oligonucleotide chains that interact through hybridization, the specific binding can be "specific hybridization."

In addition, as described herein, recombinant human (humanized) antibodies can be further optimized to reduce possible immunogenicity while maintaining functional activity for therapy in humans. .. In this regard, functional activity means a polypeptide capable of presenting one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activity includes, for example, the ability to bind to a target molecule.

The term "immunization" generally refers to the step (s) of administering one or more antigens to an animal so that the antibody can be generated in the animal, and immunization refers to the animal. Includes injecting the antigen (s). Immunization may include one or more doses of the antigen (s). Suitable methods are prime-boost and RIMMS procedures as known to those of skill in the art.

As used herein, "affibody" refers to a relatively small synthetic protein molecule that has a high binding affinity for a target protein (eg, PCSK9 or a variant thereof). Affibody is composed of three helix bundle domains derived from the IgG-binding domain of staphylococcal protein A. The protein domain consists of a sequence of 58 amino acids, 13 of which are randomized amino acids, resulting in a variety of affibody variants. Although the affibody molecule is significantly smaller than the antibody (the affibody weighs about 6 kDa, the antibody generally weighs about 150 kDa), its binding site is the surface area of the antibody. It acts like an antibody because it is almost equivalent to the binding site.

"VH3-23 * 04", as used herein, refers to a human VH domain variant comprising the polypeptide sequence of SEQ ID NO: 38. In contrast to the reference sequence, VH3-23 * 04 has a valine residue instead of a leucine residue as a result of the presence of rs56069819SNP in the nucleic acid sequence encoding the VH domain (FIGS. 3 and 4; L24V, Numbering including signal sequences; see valine at position 5 shown in Figure 4). "Rs56069819", as used herein, depends on the strand of DNA from adenosine to cytosine (or being read) in the VH gene segment resulting in a VH domain encoding VH3-23 * 04. Refers to a mutation or variant (from thymine to guanine). rs56069819 is represented in Figure 4 and SEQ ID NO: 39, which illustrates the T → G mutation (note that the dbSNP invasion for RS5606819 represents another strand containing the A → C mutation. ). A further description of VH3-23 * 04 can be found, for example, in US Patent Application Publication No. 2013/0071405, which is incorporated herein by reference in its entirety.

"Determining" or "determining" as used herein refers to confirming, for example, by quantitative or qualitative analysis. As used herein, "determined" may refer to confirmation based on previously or simultaneously obtained information.

In some aspects of all embodiments of the invention, the choice can be made based on instructions described herein by entering relevant data such as a panel of ethnicity or SNP data. It may include automation of software programs and the like realized by.

As used herein, "assaying" is assessing, evaluating, quantifying, measuring or characterizing an analyte, eg, measuring the level of an analyte in a sample, identifying the analyte. , Or to detect the presence or absence of an analyte in a sample. In some embodiments, assaying refers to detecting the presence or absence of an analyte of interest. In some embodiments, assaying refers to quantifying the amount of analyte, eg, providing a measure of concentration or degree of abundance of the analyte. In some embodiments, assaying refers to counting the number of molecules of the analyte present in the sample and / or specimen, eg, determining the copy number of the analyte.

"Multiplex", as used herein, is used in two or more, typically three or more different target sequences, eg, in two or more isoforms of PCSK9, or in PCSK9 and additional targets. Refers to performing the method or process simultaneously and in the same reaction vessel. Multiplex analysis typically involves 10-50; 10-100; 10-1000, 10-5000, 10-100,000 reactions in a multiplex fashion, such as a multi-wall, array, or multi-channel reaction. Includes analysis of.

Analysis or multiplex analysis is also often automated using robotics and software typically run by a computer, robotic handling of samples, automatic or robotic selection of positive or negative results, and targeting. For example, an assay for the presence or absence of a nucleic acid polymorphism or protein variant may be included.

The term "biological sample" or "test sample" as used herein refers to a sample taken or isolated from an organism, such as a sample from a subject. Exemplary biological samples include, but are not limited to, biological fluid samples; serum; plasma; urine; saliva; hair, epithelial cells, skin, tumor biopsies and / or tissue samples and the like. The term also includes a mixture of the above-mentioned samples. The term "test sample" or "biological sample" also includes untreated or pretreated (or pre-processed) biological samples. For nucleic acid analysis, the biological sample is typically expected to contain at least one cell containing the nucleic acid.

Test samples can be obtained by removing a sample of cells from the subject, but using previously isolated cells (eg, isolated at a previous time point and isolated by the same or another person). It can also be achieved by doing. In addition, the test sample may be freshly collected or may be a pre-collected, refrigerated, frozen, or otherwise stored sample.

In some embodiments, the test sample may be an untreated test sample. The phrase "untreated test sample" as used herein refers to a test sample that has not been pretreated with any preceding sample except for dilution and / or suspension in solution. Exemplary methods for treating test samples include, but are not limited to, centrifugation, filtration, ultrasonic crushing, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, eg, frozen tissue. Frozen samples may be thawed prior to adoption of the methods, assays and systems described herein. After thawing, the frozen sample may be centrifuged before being subjected to the methods, assays and systems described herein. In some embodiments, the test sample is a test sample that has been clarified, for example, by centrifugation and collection of a supernatant containing the clarified test sample. In some embodiments, the test sample may be a pre-processed test sample, resulting from a procedure selected from the group consisting of, for example, centrifugation, filtration, melting, purification, and any combination thereof. It may be a supernatant or a filtrate. In some embodiments, the test sample may be treated with chemical and / or biological reagents. Chemical and / or biological reagents can be employed to protect and / or maintain the stability of the sample containing biomolecules (eg, nucleic acids and proteins) therein during processing. One exemplary reagent is a protease inhibitor, which is commonly used to protect or maintain protein stability during processing. One of ordinary skill in the art is familiar with the methods and processes suitable for pre-processing biological samples required to determine the level of expression products as described herein.

"Genotyping", as used herein, determines the composition of specific alleles of a cell and / or subject at one or more positions in the genome, eg, the nucleic acid sequence at that position. Refers to the process of determining by. Genotyping refers to nucleic acid analysis and / or analysis at the nucleic acid level. "Phenotyping", as used herein, is the process of determining the identity and / or composition of a cell and / or expression product of interest, eg, by determining the polypeptide sequence of the expression product. Point to. Phenotyping refers to protein analysis and / or analysis at the protein level.

The term "nucleic acid amplification" as used herein refers to the production of additional copies of a nucleic acid sequence, typically a polymerase chain reaction (PCR) or ligase chain reaction (LCR) technique well known in the art. (Dieffenbach, CW and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY). Other methods for amplification are also considered in aspects of the invention.

The term "allele-specific amplification" refers to a polymorphic region (eg, a single nucleotide) of a gene in which at least one of the primers (eg, an allele-specific primer) has a polymorphism located at or near the 3'end of the primer. Refers to a reaction (eg, PCR reaction) selected from (polymorphism). Mismatched primers are not expected to initiate amplification, whereas matching primers are expected to initiate amplification. The appearance of amplification products is an indicator of the presence of polymorphisms.

"Sequencing", as used herein, determines the exact order of nucleotide bases in a chain of DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) or the exact order of amino acid residues or peptides in a protein. Point. Nucleic acid sequencing can be performed using Sanger sequencing or next-generation high-throughput sequencing.

"Next-generation sequencing," as used herein, is possible with conventional sequencing methods (eg, sequencing by Sanger) by performing and reading thousands to millions of sequencing reactions in parallel. Refers to an oligonucleotide sequencing technique capable of sequencing oligonucleotides at speeds above Non-limiting examples of next-generation sequencing methods / platforms include massively parallel signature sequencing (Lynx Therapeutics); 454 Pyrosequence (454 Life Sciences / Roche Diagnostics); solid phase reversible dye-terminator sequencing ( Solexa / Illumina): SOLiD Technology (Applied Biosystems); Ion Semiconductor Sequencing (ION Torrent); DNA Nanoball Sequencing (Complete Genomics); and Pacific Biosciences, Intelligen Bio-systems, Oxford Nanopore Technologies, And technologies available from Helicos Biosciences. Next-generation sequencing techniques and related sequencing primer constraints and design parameters are well known in the art (eg, Shendure et al., "Next-generation DNA sequencing", Nature, 2008, Vol. 26, No. 10, 1135-1145; Mardis, "The impact of next-generation sequencing technology on genetics", Trends in Genetics, 2007, Vol. 24, No. 3, pp. 133-141; Su et al., "Next-generation sequencing and its applications in molecular diagnostics" Expert Rev Mol Diagn, 2011, 11 (3): 333-43; Zhang et al., "The impact of next-generation sequencing on genetics", J Genet Genomics, 2011, 38 (3): 95-109; (Nyren, P. et al., Anal Biochem 208: 17175 (1993); Bentley, DR Curr Opin Genet Dev 16: 545-52 (2006); Strausberg, RL et al., Drug Disc Today 13: 569-77 (2008); US Patent No. 7,282,337; US Patent 7,279,563; US Patent 7,226,720; US Patent 7,220,549; US Patent 7,169,560; US Patent 6,818,395; US Patent 6,911,345; US Patent Application Publication No. 2006/0252077; 2007/0070349 And see 20070070349; these are incorporated herein by reference in their entirety).

"Nucleic acid hybridization" as used herein refers to the pairing of complementary RNA and DNA strands, as well as the pairing of complementary DNA single strands. In some embodiments, nucleic acid hybridization may refer to a method of determining nucleic acid sequences and / or identity by hybridizing a nucleic acid sample with a probe, such as Northern or Southern blot analysis or microarray analysis.

The terms "treat," "treat," "treat," or "alleviate," as used herein, undo or alleviate the progression or severity of a disease or disorder-related condition. Refers to a therapeutic procedure aimed at alleviating, inhibiting, slowing down, or stopping. The term "treating" includes reducing or alleviating at least one adverse action or symptom of a condition, disease or disorder. In general, treatment is "effective" if one or more symptoms or clinical markers are reduced. Instead, if the progression of the disease slows or stops, the procedure is "effective." That is, "treatment" includes not only improvement of symptoms or markers, but also interruption of progression or worsening of symptoms, or at least delay compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes, whether detectable or undetectable, alleviated one or more symptoms, reduced the degree of disease, and stabilized (but not limited). Disease conditions (which do not worsen), slowing or slowing of disease progression, alleviation or temporary alleviation of the condition, remission (either partial or complete), and / or reduced mortality. The term "treatment" of a disease also includes providing relief of symptoms or side effects (including temporary palliative treatment) of the disease. Complete cure is not considered for the effectiveness of the procedure. Similarly, the method comprises healing in certain embodiments.

The term "pharmaceutical composition" as used herein refers to an active substance in combination with a pharmaceutically acceptable carrier, such as a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein in humans and animals without causing excessive toxicity, irritation, allergic responses, or other problems or complications, within the bounds of certain medical judgment. It is adopted as referring to a compound, material, composition, and / or dosage form corresponding to a suitable benefit / risk ratio suitable for use in contact with the tissue of.

The term "administering", as used herein, delivers a compound as disclosed herein to a subject by a method or route that results in at least partial delivery of the agent to the desired site. Point to that. Pharmaceutical compositions containing the compounds disclosed herein can be administered by any suitable route that results in effective treatment in the subject.

Multiple compositions can be administered separately or simultaneously. Separate administration separates the two compositions at different times, eg at least 10, 20, 30, or 10-60, or 1, 2, 3, 4, 5, 6, 7, 8, 9 , Refers to administration 10 to 12 hours apart. The composition can also be administered 24 hours apart, or even longer. Instead, the two or more compositions can be administered simultaneously, eg, less than 10 minutes or less than 5 minutes apart. In some embodiments, the co-administered compositions can be administered as a mixture with a similar or different sustained release mechanism for each of the components, or without such a mechanism.

"Contacting", as used herein, refers to any suitable means for delivering or exposing a drug to at least one complex, enzyme, or cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery methods well known to those of skill in the art.

As used herein, "obtaining" refers to any method of obtaining, securing, obtaining, or owning a sample, for example. Obtaining a biological sample from a subject is the physical removal of the sample from the subject with or without active participation of the subject (eg, blood sampling or collection of a hair or saliva sample); receiving the sample from the subject ( For example, the subject may include collecting the saliva or hair sample itself and providing it, for example, in a container provided for that purpose); or obtaining the sample from a storage facility, medical facility, or medical provider. .. Obtaining from a human or subject refers to, for example, an active step of collecting blood or collecting a tissue or cell sample.

"Cholesterol level" as used herein refers to one or more levels of total cholesterol, LDL cholesterol, HDL cholesterol, and / or triglycerides. Cholesterol levels can be the levels of cholesterol in the subject's blood.

With respect to cholesterol levels, "maintaining" as used herein refers to preventing deterioration (eg, increase) in levels. In some embodiments, maintaining a particular level refers to a process that does not increase cholesterol levels above 10% over time. Maintaining may also mean maintaining the level achieved so far. For example, if a human is undergoing statin treatment, the cholesterol levels achieved using statin treatment can be maintained.

In some embodiments, subjects treated according to the methods described herein have previously lowered cholesterol levels. "Lower by then", as used herein, indicates that the subject has experienced a decrease in cholesterol levels at a previous time point. The reduction may be due to administration of the pharmaceutical composition (eg, administration of a composition as described herein or another composition, eg, statin), or another cause, eg, dietary changes. And / or may be due to exercise.

The existing treatment for high cholesterol levels is the administration of statins. As described herein, "statins" (also known as HMG-CoA reductase inhibitors) are inhibitors of the enzyme HMG-coA reductase, which mediates cholesterol production in the liver. Statins prevent the binding of HMG-CoA to enzymes by competitively binding to HMG-CoA reductase, thereby inhibiting the activity of reductase (eg, mevalonate production). Non-limiting examples of statins include atorvastatin [LIPITOR ™], fulvastatin [LESCOL ™], lovastatin [MEVACOR ™, ALTOCOR ™], pitavastatin [LIVALO ™], prastatin [ PRAVACHOL ™], rosuvastatin [CRESTOR ™], and simvastatin [ZOCOR ™] can be mentioned. Statins can be administered in combination with other agents, for example in combination with ezetimaive and simvastatin.

Some subjects are resistant to statin treatment or become resistant to statin treatment. "Resistant to statin treatment" or "reduced responsiveness to statin treatment", when used herein, is statistically significantly lower in the subject compared to reference levels for administration of statin. Refers to presenting a response. The reference level can be, for example, the average response in the case of a population of subjects, or the level of an individual subject at an earlier date. Responses to statin treatment are readily measured by those of skill in the art, such as cholesterol levels, changes in cholesterol levels, and / or measurement of HMG-CoA reductase activity.

As used herein, the term "detectable label" refers to a molecule or moiety that is present or absent or detectable, eg, measurable and / or determinable. The detectable label may include, for example, a light absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods for detecting them, and methods for incorporating them into reagents (eg, antibodies and nucleic acid probes) are well known in the art.

In some embodiments, the detectable label is spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical or chemical means, such as fluorescence, chemiluminescence, or chemiluminescence, or any other suitable. Examples include labels that can be detected by means. The detectable label used in the methods described herein is a primary label (in this case, the label comprises a directly detectable portion or a portion producing a directly detectable portion) or a secondary label. (In this case, the detectable label may bind to another moiety that produces a detectable signal, such as a common moiety in immunological labeling using secondary and tertiary antibodies). .. The detectable label may be linked to the reagent by covalent or non-covalent means. Alternatively, the detectable label may be linked, for example, by directly labeling a molecule that achieves binding to the reagent by a ligand-receptor binding pair configuration or other such specific recognition molecule. .. Detectable labels include, but are not limited to, radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detectable label can be a fluorescent compound. When a fluorescent label is exposed to light of a suitable wavelength, its presence can be detected by fluorescence. In some embodiments, the detectable labels are, but are not limited to, fluorescein, phycoerythrin, phycyanine, o-phthaldehyde, fluoresamine, Cy3 ™, Cy5 ™, allophicyanine. , Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5 ™, green fluorescent protein, lordamine, fluorescein isothiocyanate (FITC) and Oregon Green ™, lordamine and derivatives [eg , Texas Red and tetrarhodimine isothiocynate (TRITC)], biotin, phycoerythrin, AMCA, CyDyes ™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F). ), 6-carboxy-2', 4', 7', 4,7-hexachlorofiuorescein (HEX), 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein (dimethoxyfiuorescein) (JOE or J), N, N, N', N'-tetramethyl-6carboxylodamine (TAMRA or T), 6-carboxy-X-lodamine (ROX or R), 5-carboxylodamine-6G (R6G5 or G5), 6-Carboxyrodamine-6G (R6G6 or G6), and Rhodamine 110; Cyanine dyes such as Cy3, Cy5 and Cy7 dyes; Kumarins such as Umberiferon; Benzimide dyes such as Hext 33258; Phenantridin Dyes such as Texas Red; Etidium Dyes; Acrydin Dyes; Carbazole Dyes; Phenoxazine Dyes; Porphyrin Dyes; Cyanine Dyes such as Cy3, Cy5; Cyanine Dyes such as Cy3, Cy5; .. In some embodiments, the detectable label can be a radiolabel including, but not limited to, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³² P, and ³³ P. In some embodiments, the detectable label can be an enzyme, including, but not limited to, horseradish peroxidase and alkaline phosphatase. Enzyme labeling can generate, for example, chemiluminescent, color, or fluorescent signals. Enzymes considered for use as detectable labels include, but are not limited to, malate dehydrogenase, glucose phosphatase, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate. Acid isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase can be mentioned. In some embodiments, the detectable label is, but is not limited to, lucigenin, luminol, luciferin, isolminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. It is a chemiluminescent label containing. In some embodiments, the detectable label can be a spectroscopic colorimetric label comprising colloidal gold or beads of colored glass or plastic (eg, polystyrene, polypropylene, and latex), without limitation.

In some embodiments, the reagent can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems, such as the biotin-streptavidin system, can also be used. In this system, antibodies that are immunoreactive (ie, specific to) the biomarker of interest are biotinylated. The amount of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available from, for example, DAKO; Carpinteria, CA. Reagents may also be detectably labeled using fluorescent metals such as ¹⁵² Eu, or others from the lanthanide series. These metals can be attached to reagents using metal chelating groups such as diethylenetriamine pentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

"License Number" or "Sales License Number", as used herein, is used by the regulatory agency to market certain pharmaceutical products and / or compositions for sale in the competent jurisdiction and /. Or refers to the number issued by the government agency when deciding that it can be supplied. The “supervisory authority”, as used herein, assesses, for example, the safety and efficacy of pharmaceutical products and / or compositions, and sells such products and / or compositions in a given area. / Refers to one of the government agencies involved in managing market transactions. The Food and Drug Administration (FDA) in the United States and the European Medicines Agency (EPA) in Europe are just two examples of such regulatory agencies. Other non-limiting examples include SDA, MPA, MHPRA, IMA, ANMAT, Hong Kong Department of Health-Drug Office, CDSCO, Medsafe, and KFDA.

"Injection device", as used herein, refers to a device designed to make an injection, injecting a temporary fluid into human tissue, typically subcutaneous tissue. Including the step of connecting. Injection further comprises administering a certain amount of liquid drug to the tissue and removing or removing the injection device from the tissue. In some embodiments, the injection device may be an intravenous device or an IV device, which is the type of injection used when the target tissue is blood in the circulatory system, eg, intravenous blood. It is a device. Common, but non-limiting examples of injection devices are needles and syringes.

As used herein, "buffer" refers to a chemical substance capable of absorbing a predetermined amount of acid or base without being subject to strong fluctuations in pH.

"Packaging" as used herein refers to a means of organizing and / or grouping components into units suitable for distribution and / or use. Packaging includes, for example, boxes, bags, syringes, ampoules, vials, tubes, clamshell packaging, sterile conditions, barriers and / or containers for maintaining labels and the like.

"Instructions", as used herein, are written, printed or charted in direct container of the article, eg, text displayed on a vial containing a pharmaceutically active substance, or the composition of interest. Refers to details about the composition and use of the intended product contained in the kit containing the material. The instructions describe how to treat when administration or practice is considered.

"Solid surface" as used herein refers to an object suitable for binding biomolecules. Non-limiting examples of solid surfaces include particles (including, but not limited to, agarose or latex beads or particles or magnetic particles), beads, nanoparticles, polymers, substrates, slides, coverslips, plates, cultures. Dish, wells, membranes, and / or lattices can be mentioned. Solid surfaces include, but are not limited to, many different materials including, but not limited to, polymers, plastics, resins, polysaccharides, silicon or silica-based materials, carbon, metals, inorganic glass, and films.

The "classification" of a subject, eg, the classification of a subject's pedigree, as used herein, is whether the subject has a biological ancestor of origin in a particular geographic area, and thus historically its. Refers to determining whether a particular genetic variant found in a population that has occupied a region is likely to have it. Classification may include, for example, obtaining information about the subject's family, interviewing the subject or a member of the family regarding their biological family pedigree, and / or performing a genetic test. The classification may be based on the criteria used for the 1000 Genomes Project, which is expected to be familiar to those skilled in the art. In some embodiments, at least the genome of interest comprises a significant number of different alleles common to other humans belonging to a particular lineage (eg, determined by referring to the 1000 Genomes Project database). For example, a subject can be classified as belonging to its pedigree if it contains at least 10, 20, 30, 40, 50 or 100 or more common alleles. Table 4 shows abbreviations for specific pedigree groups.

The term "statistically significant" or "significantly" refers to statistical significance and generally means a difference of 2 standard deviations (2SD) or greater.

All numerical values representing the amounts of the components or the reaction conditions used herein are modified by the term "about" in all examples, except as described in the embodiments of the operation or unless otherwise indicated. It shall be understood that it has been done. The term "about" may mean ± 1% when used in connection with a percentage.

As used herein, the terms "include" or "include" are used with respect to the composition, method, and their respective components essential to the method or composition, but are essential or not essential. Regardless, we do not exclude the inclusion of elements that are not explicitly stated.

The term "consisting of" refers to compositions, methods, and their respective components as described herein, but does not include any element not mentioned in the description of its embodiments.

As used herein, the term "becomes essential" refers to the elements required for a given embodiment. The term allows for the presence of elements that do not significantly affect the novel or functional features underlying the embodiment.

The singular terms "one (a)", "one (an)", and "the" include the plural referent unless explicitly stated otherwise in the context. Similarly, the word "or" is intended to include "and" unless explicitly stated otherwise in the context. Methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, but suitable methods and materials will be described below. The abbreviation "eg (e.g.)" is derived from the Latin word exempli gratia and is used herein to give a non-limiting example. Therefore, the abbreviation "for example (e.g.)" is synonymous with the term "for example (for example)".

Definitions of common terms in cell biology and molecular biology are described in The Merck Manual of Diagnosis and Therapy, 19th Edition, Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. . Porter et al. (Ed.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, Jones & Bartlett Publishing, 2009 (ISBN-10) : 0763766321); Kendrew et al. (Ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences. , Colligan et al.

Unless otherwise specified, the present invention is, for example, incorporated herein by reference in its entirety, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press, Inc. Cold Spring Harbor, NY, USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152 , SL Berger and AR Kimmel, Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan et al., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (edited by Juan S. Bonifacino et al., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; Standard procedures as described in Edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Editors of Jennie P. Mather and David Barnes, Academic Press, First Edition, 1998). Made using.

Other terms are defined herein within the description of various aspects of the invention.

All patents and other publications, including references, published patents, published patent applications, and co-pending patent applications cited throughout this application, are, for example, the techniques described herein. For the purposes of explaining and disclosing the techniques described in such gazettes that may be used in connection with, expressly incorporated herein by reference. These gazettes are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard shall be construed as an approval that the inventors have no right to precede such disclosure for the sake of prior invention or for any other reason. All statements regarding the date or indications regarding the content of these documents are based on the information available to the applicant and do not constitute any approval for the accuracy of the date or content of these documents.

The description of embodiments of the present disclosure is not intended to be exhaustive or limited to the exact form disclosed. Specific embodiments of the present disclosure and examples relating thereto are described herein for exemplary purposes, but within the scope of the present disclosure as would be appreciated by those skilled in the art. Various equivalent modifications are possible. For example, the steps or functions of the method are shown in a given order, but in alternative embodiments the functions may be performed in a different order, or they may be performed at substantially the same time. The teachings of the disclosures presented herein can optionally be applied to other procedures or methods. Further embodiments can be provided by combining the various embodiments described herein. Aspects of the present disclosure may optionally be modified to employ the compositions, functions and concepts of the above references and applications to provide further embodiments of the present disclosure. In addition, by considering the equivalence of biological functions, some changes in protein structure can be made without affecting the biological or chemical action in type or quantity. These and other variations can be made in the present disclosure in view of the detailed description. All such modifications are intended to be included within the appended claims.

A specific element of any of the aforementioned embodiments may be combined with or replaced with an element of another embodiment. Further, while the advantages associated with certain embodiments of the present disclosure are described in the context of these embodiments, other embodiments may also present such advantages and not necessarily all embodiments. It does not necessarily offer such an advantage as the form is within the scope of the disclosure.

It is understood that the particular configurations, embodiments, examples, provisions and embodiments described herein are illustrated by way of illustration and not as limitations of the present invention. The main features of the present invention can be adopted in various embodiments without departing from the scope of the present invention. It is expected that one of ordinary skill in the art will recognize or confirm that, in addition to conventional studies, a large number of procedures equivalent to the specific procedures described herein will be used. To. Such equivalent procedures are considered within the scope of the invention and are within the scope of the claims. All publications and patent applications described herein indicate the level of competence of one of ordinary skill in the art in the field in which the present invention relates. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference. The use of the words "one (a)" or "one (an)" means "one" when used in conjunction with the term "contains" in the claims and / or specification. In some cases, it also agrees with the meaning of "one or more," "at least one," and "one or more." Although the use of the term "or" in the claims is used to refer only to alternatives or to mean "and / or" unless the options are explicitly indicated to be mutually exclusive. , The present disclosure supports the definition of alternatives only and "and / or". Throughout this application, the term "about" indicates that a value embraces variations in error specific to each device, method used to determine the value, or variations that exist between study subjects. Used for.

As used herein and in the claims, the terms "including" [and any form of including, such as "comprise" and "comprises"], "having". [And any form of having, such as "have" and "has"], "including" [and any form of including, such as "includes" and "includes (includes)" "include"], or "containing" [and any form of inclusion, such as "contains" and "contain"], is either inclusive or open-ended. And does not exclude the steps of additional unlisted elements or methods.

Unless the context makes it clear that this is not the case, any part of this disclosure may be read in combination with any other part of this disclosure.

References to sequences by "SEQ ID NO:" herein (eg, within the scope of a patent claim) are made by reference to the sequences listed in the table, as well as references to position numbers within "SEQ ID NOs". (For example, within the scope of patent claims) is made by reference to the sequences described in the tables herein (eg, as in Tables 6, 10 and 17). Thus, for example, "Asp corresponding to position 204 of SEQ ID NO: 42 or Leu corresponding to position 206 of SEQ ID NO: 42" is labeled as position 204 and position 206 of SEQ ID NO: 42 shown in Table 10 herein, respectively. Refers to Asp and Leu.

All of the compositions and / or methods disclosed and claimed herein can be made and carried out in light of the disclosure of the present invention without any extra experimentation. Although the compositions and methods of the present invention have been described with respect to preferred embodiments, the compositions and / or methods, as well as the methods described herein, without departing from the concept, nature and scope of the invention. It is expected to be apparent to those skilled in the art that variations may be applied in steps or in sequence of steps. Similar substitutions and modifications apparent to all such skill in the art are considered to be within the essence, scope and concept of the invention as defined by the appended claims.

The present invention will be described in more detail with the following non-limiting examples.

Some embodiments of the techniques described herein can be defined according to any of the following numbered paragraphs:
1. A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
a. In the step of selecting humans who are positive for the TOI polymorphic variant, the TOI in said humans is encoded by a nucleotide sequence containing an allele with a cumulative human allele frequency of less than 50% and / Or the TOI in said human is encoded by a nucleotide sequence containing an allele with a total human genotype frequency of less than 50%, and;
b. A method comprising administering to a human an anti-TOI ligand to target the TOI in the human and treating or preventing the disease or condition.

In the first alternative, paragraph 1
A method of treating or preventing a target (TOI) -mediated disease or condition in a human that is present as a different polymorphic variant in the human, wherein the anti-TOI ligand (eg, an antibody or antibody fragment) is applied to the human. Cumulative human alleles with less than 50% (eg, less than 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%) of anti-TOI ligands, including the step of administering It specifically binds to a TOI variant encoded by a nucleotide sequence containing a frequency SNP, and / or less than 50% of the TOI variants (eg, 40, 35, 30, 35, 20, 15, 10, 5, Encoded by a nucleotide sequence containing an SNP having a total human genotype frequency of 4, 3, 2, or less than 1%; this human comprises a nucleotide sequence encoding a TOI containing (eg, said) said SNP;
(i) The ligand (eg, antibody or fragment) comprises a VH domain derived from recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment comprises an SNP, and the human comprises the SNP. Includes (eg, said) VH segments; or
(ii) The ligand (eg, antibody or fragment) comprises a Vλ domain derived from recombination of a human Vλ segment and a human Jλ segment, the human Vλ segment comprises an SNP, and the human comprises said SNP (eg, said above. ) Includes Vλ segment; or
(iii) The ligand (eg, antibody or fragment) comprises a human Vκ segment and a recombination-derived Vκ domain of a human Jκ segment, the human Vκ segment comprises an SNP, and the human comprises said SNP (eg, said above. ) Includes Vκ segment; or
(iv) The ligand (eg, antibody or fragment) comprises a heavy chain constant region (eg, gamma-1, 2, 3 or 4 constant region) encoded by a nucleotide sequence containing an SNP, and humans have said SNP. Includes (eg, said) heavy chain constant regions (eg, gamma-1, 2, 3 or 4 constant regions, respectively); or
(v) Does the ligand (eg, antibody or fragment) contain a lambda chain constant encoded by a nucleotide sequence containing an SNP and the human comprises the lambda chain constant region containing said SNP (eg, said);
(vi) Provided a method in which a ligand (eg, an antibody or fragment) comprises a kappa chain constant encoded by a nucleotide sequence comprising an SNP, and human comprises the kappa chain constant region comprising said SNP (eg, said). To do.

In the second alternative, paragraph 1
A method of treating or preventing a disease or condition mediated by a target target (TOI) that exists as a different polymorphic variant in humans in humans, the anti-TOI ligand that specifically binds to the human TOI variant. Including the step of administering (eg, an antibody or antibody fragment), the TOI variant is less than 50% (eg, 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or 1%). Containing a single amino acid variation with a cumulative human allelic frequency of less than (ie) (ie, a single amino acid at positions where there is a difference between TOI variants in humans) and / or this amino acid variant is less than 50% (eg, less than 50%). , 40, 35, 30, 35, 20, 15, 10, 5, 4, 3, 2, or less than 1%) total human genotype frequency; humans have a TOI containing the single amino acid variation. Express protein;
(i) Does the ligand (eg, antibody or fragment) contain a VH domain containing the same single amino acid variation and humans express the VH domain containing said single amino acid variation;
(ii) Does the ligand (eg, antibody or fragment) contain a Vλ domain containing a single amino acid variation and humans express a Vλ domain containing the same single amino acid variation;
(iii) Does the ligand (eg, antibody or fragment) contain a Vκ domain containing a single amino acid variation and humans express a Vκ domain containing the same single amino acid variation;
(iv) The ligand (eg, antibody or fragment) contains a single amino acid variation in a heavy chain constant region (eg, gamma-1, 2, 3 or 4 constant region, eg, this constant region is Fc, CH1, Heavy chain constant regions (eg, gamma-1,2,3 or 4 constant regions) containing (eg, CH2 or CH3) and containing the same single amino acid variation in humans, eg, these constant regions are Fc, CH1, Whether to express (CH2 or CH3); or
(v) Does the ligand (eg, antibody or fragment) contain a lambda chain constant region containing a single amino acid variation and humans express a lambda chain constant region containing the same single amino acid variation;
(vi) Provided is a method by which a ligand (eg, an antibody or fragment) comprises a kappa chain constant region containing a single amino acid variation and human expresses a kappa chain constant region containing the same single amino acid variation. ..

The term "contains a single amino acid variation" includes the possibility that additional amino acid variations may be present in the TOI sequence.

In one example, the frequency herein follows a database of 1000 genomes, such as the Phase I database, or, for example, Ensembl on October 1, 2013 or October 1, 2014 (on the World Wide Web, at ensembl.org). Available).

In one example, each SNP encodes a non-synonymous amino acid variation, i.e. the SNP is not a silent mutation.

Optionally, the method comprises selecting the human containing the nucleotide sequence prior to administration.

In the third alternative where the TOI is human PCSK9, paragraph 1
A method of lowering or maintaining previously lowered cholesterol levels in humans in need thereof, such as mutations in said humans as defined herein in SEQ ID NO: 1. , I474V or E670G), including the step of administering an antibody or antibody fragment that specifically binds to the precursor protein convertase subbutyricin / kexin type 9 (PCSK9) containing a C-terminal domain, wherein the antibody is a human VH segment, human D The human VH segment encodes valine at the amino acid corresponding to position 5 of SEQ ID NO: 40, which comprises the VH domain derived from the recombination of the gene segment and the human JH segment, and the human is (i) the framework of SEQ ID NO: 40. Provided is a method comprising a VH gene segment encoding 1 and (ii) a nucleotide sequence encoding the precursor protein convertase subbutyricin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutation in SEQ ID NO: 1. ..

In the fourth alternative where the TOI is human PCSK9, paragraph 1
A method of lowering or maintaining previously lowered cholesterol levels in humans in need thereof, such as mutations in said humans as defined herein in SEQ ID NO: 1. , I474V or E670G), including the step of administering an antibody or antibody fragment that specifically binds to the precursor protein convertase subbutyricin / kexin type 9 (PCSK9) containing a C-terminal domain, wherein the antibody comprises a human VL segment and a human JL. A human VL segment comprising a recombinantly derived VL domain of the segment is Vλ or Vκ disclosed herein, and the human is (i) said in the VL gene segment and (ii) said suddenly in SEQ ID NO: 1. Provided is a method comprising a nucleotide sequence encoding the precursor protein convertase subbutyricin / kexin type 9 (PCSK9), which comprises a C-terminal domain containing a mutation.

In the fifth alternative where the TOI is human PCSK9, paragraph 1
A method of lowering or maintaining previously lowered cholesterol levels in humans in need thereof, such as mutations in said humans as defined herein in SEQ ID NO: 1. , I474V or E670G), including the step of administering an antibody or antibody fragment that specifically binds to the precursor protein convertase subbutyricin / kexin type 9 (PCSK9) containing the C-terminal domain, the antibody is disclosed herein. A precursor protein comprising a C domain encoded by a human CH, Cλ or Cκ gene segment, wherein the human comprises (i) the C gene segment and (ii) the C-terminal domain containing the mutation in SEQ ID NO: 1. Provided is a method comprising a nucleotide sequence encoding the convertase subbutyricin / kexin type 9 (PCSK9).

The following paragraph relates to one of the alternatives to paragraph 1.
2. The method of paragraph 1, wherein prior to step (a), the ligand is determined or determined to be capable of specifically binding to said TOI variant.
3. Includes a step of determining that a human is positive for a TOI polymorphic variant, optionally including a step of determining that a human is positive for a nucleotide variant encoding said TOI variant. , Paragraph 1 or 2.
4. The method of paragraph 3, wherein the determining step comprises assaying a biological sample obtained from the human for a nucleotide polymorphism encoding the TOI polymorph variant.
5. The method of paragraph 3 or 4, wherein the determining step comprises assaying the biological sample obtained from the human for the protein corresponding to the TOI polymorph variant.
6. The method described in any of the paragraphs above, wherein the frequency is less than 15%.
7. The method according to any of the paragraphs above, wherein the frequency is less than 10%.
8. The ligand is specific for two or more different TOI variants, each encoded by a nucleotide sequence containing an allele with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. The method according to any of the above paragraphs, which can be combined.
9. A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
Negative for variant nucleotide sequences containing alleles with less than 50% cumulative human allele frequency and / or less than 50% total human genotype frequency; or less than 50% cumulative human allele frequency and / Or with the step of selecting humans who are negative for the TOI variant encoded by a nucleotide sequence containing an allele with a total human genotype frequency of less than 50%;
b. The step of administering an anti-TOI ligand to a human to target the TOI variant in the human and treat or prevent the disease or condition, wherein the TOI in the human has a cumulative human allele frequency greater than 50% and / Or a method comprising a step, which is a variant encoded by a nucleotide sequence comprising an allele having a total human genotype frequency greater than 50%.
10. The steps, including the step of determining that a human is positive for a TOI polymorphic variant, are optionally determined by whether the human is phenotypically determined to be positive for the most frequent TOI variant. 9. The method of paragraph 9, comprising phenotyping or genotyping the nucleotide sequence thereof.
11. The method of paragraph 10, wherein the determination step comprises assaying for a nucleotide sequence to determine the presence of the allele.
12. The method according to paragraph 11, wherein the assaying step comprises nucleic acid amplification.
13. The method of paragraph 11 or 12, wherein the step of assaying comprises hybridization, sequencing, or next generation sequencing.
14. The method according to any of paragraphs 11-13, further comprising the step of obtaining a biological sample from a human.
15. The method of any one of paragraphs 9-14, wherein the ligand is determined or is capable of specifically binding to the most frequent TOI variant.
16. The method of any one of paragraphs 9-15, wherein the ligand is determined or determined to be unable to substantially neutralize or inhibit the TOI variant.
17. The method of any one of paragraphs 9-16, wherein the ligand is capable of specifically binding to the most frequent TOI variant.
18. Ligands are capable of specifically binding to two or more different TOI variants, each encoded by a nucleotide sequence containing an allele with a cumulative human allele frequency greater than 50%, paragraphs 9-17. The method described in any one of.
19. The method according to any of the paragraphs above, wherein the TOI polymorphic variant has been determined or is determined to be present in at least two different human ethnic populations.
20. The cumulative human allele frequency is described in any of the paragraphs above, which is the frequency in a database of naturally occurring sequences containing at least 1000 sequences sampled from at least 15 different human ethnic populations. the method of.
21. The method according to any one of the preceding paragraphs, wherein the ligand is an antibody, antibody fragment or affibody.
22. A nucleotide sequence in which the ligand specifically hybridizes to a TOI nucleotide sequence or its RNA transcript containing an allele with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. Contains; and / or the ligand comprises or comprises a nucleotide sequence comprising at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. The method according to any one of the above paragraphs, which is an antisense sequence.
23. The allele described in any one of the above paragraphs, wherein the human genome contains an allele with a cumulative human allele frequency of less than 50%, the allele being found in at least two different ethnic populations. Method.
A composition comprising a ligand capable of binding to a target of interest encoded by a nucleotide sequence containing an allele having a cumulative human allele frequency of less than 24.50%, the allele being in at least two different ethnic populations. Found, this composition comprises optionally a pharmaceutically acceptable carrier, and optionally a label or instructions for use in the treatment and / or prevention of said human disease or condition; optionally; And the label or instructions are compositions that include a marketing license number issued by a supervisory authority.
25. A kit for treating or preventing a condition or disease mediated by a target of interest listed in any of the above paragraphs by a nucleotide sequence containing an allele with a cumulative human allele frequency of less than 50%. Containing a ligand capable of specifically binding to the encoded target of interest, this allele has been found in at least two different ethnic populations; optionally, treatment of the disease or condition in the human and / or Combined with a label or instructions for use in prevention; optionally, this label or instructions includes a marketing authorization number issued by the regulatory agency; optionally, this kit contains an injection containing this ligand. A kit containing a pen or IV container.
26. The composition according to paragraph 24 or the kit according to paragraph 25, wherein the regulatory agency is the FDA or EMA.
27. A method of generating anti-human TOI antibody binding sites, the steps of obtaining multiple anti-TOI antibody binding sites and the cumulative human allele frequency of less than 50% and / or the total human genotype frequency of less than 50%. To a TOI containing an amino acid sequence encoded by a nucleotide sequence containing an allele having, or by a nucleotide sequence encoding a TOI containing an allele having the highest cumulative human allele frequency and / or the highest total human genotype frequency A method comprising screening an antibody binding site for binding to the peptide comprising an amino acid variation from the encoded corresponding sequence, and isolating the binding site in the screening step.
28. A method of producing anti-human TOI antibodies in non-human vertebrates by nucleotide sequence containing alleles with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. Amino acid variations from the TOI containing the encoded amino acid sequence, or the corresponding sequence encoded by the nucleotide sequence encoding the TOI containing the allele with the highest cumulative human allele frequency and / or the highest total human genotype frequency. A TOI containing an amino acid sequence encoded by a TOI nucleotide sequence containing an allele containing less than 50% cumulative human allele frequency and / or less than 50% total human genotype frequency with the step of immunizing with that peptide containing A method comprising the step of isolating an antibody that binds to, and optionally producing a TOI binding fragment or derivative of the isolated antibody.
29. The method of paragraph 28, wherein the non-human vertebrate is a mouse or rat.
30. The method of paragraph 29 or 30, comprising the step of obtaining a nucleic acid encoding an antibody, fragment, derivative or binding site and optionally inserting the nucleic acid into an expression vector.
31. A kit for determining the TOI genotype of humans, the selected TOI nucleotide sequence or RNA transcript thereof having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50%. Containing nucleic acids containing nucleotide sequences that specifically hybridize to; and / or at least 10 of TOI nucleotide sequences in which the nucleic acid has a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. A kit comprising or an antisense sequence thereof comprising a nucleotide sequence comprising a contiguous nucleotide.
32. A kit for TOI genotyping or phenotyping humans, a ligand capable of binding to the target target encoded by a nucleotide sequence containing an allele with a cumulative human allele frequency of less than 50%. , Or a kit comprising an antibody, fragment or derivative produced by the method according to any one of paragraphs 28-30.
33. The kit described in paragraph 32, wherein alleles are found in at least two different ethnic populations.
34. To treat and / or prevent TOI-mediated diseases or conditions in humans whose genome contains less than 50% cumulative human allelic frequency and / or less than 50% total human genotype frequency. Anti-TOI that specifically binds to a human TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% in the manufacture of the drug. Use of ligands.
35. TOI-mediated diseases or conditions targeting human TOIs containing amino acid sequences encoded by TOI nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. Use of anti-TOI ligands that specifically bind to the TOI in the manufacture of pharmaceuticals for treatment and / or prevention in humans.
36. A method of targeting a TOI to treat and / or prevent a target (TOI) -mediated disease or condition in humans, with a cumulative human allelic frequency of less than 50% and / or 50%. A step of administering an anti-TOI ligand to a human containing a TOI nucleotide sequence containing an allele selected as having a total human genotype frequency of less than, thereby targeting the TOI encoded by the nucleotide sequence. Method.
37. The ligand comprises the step of targeting a human TOI containing the amino acid sequence to treat and / or prevent the disease or condition in the human, wherein the amino acid sequence has a cumulative human allelic frequency of less than 50%. And / or the method according to paragraph 36, encoded by a nucleotide sequence comprising an allele having a total human genotype frequency of less than 50%.
38. A method of genotyping a target (TOI) of a human nucleic acid sample, in which the cumulative human allele frequency of less than 50% and / or the total human genotyping frequency of less than 50%. A method comprising assaying the presence of a TOI nucleotide sequence comprising an allele having.
39. A method of typing for a target (TOI) of a human protein sample with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% in the sample. A method comprising assaying the presence of a TOI amino acid sequence encoded by a TOI nucleotide sequence comprising an allele.
40. As described in paragraph 38 or 39, comprising obtaining a sample of serum, blood, feces, hair, urine or saliva from a human, thereby obtaining a nucleic acid or protein sample for use in the step of assaying the sequence. Method.
Specific to a ligand capable of targeting a nucleic acid sequence containing an allele having a cumulative human allele frequency of less than 41.50% and / or a total human genotype frequency of less than 50% or a TOI encoded by the nucleic acid sequence. The method according to any one of paragraphs 38-40, comprising performing the identification step using a ligand capable of binding to.
42. Capable of binding to a human TOI containing an amino acid sequence encoded by the target (TOI) nucleotide sequence containing an allele having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% A diagnostic kit that includes a ligand and instructions for performing the method described in any one of paragraphs 38-41.
43. A diagnostic kit in which the ligand is selected from antibodies, antibody fragments, antibody moieties, affibodies, oligonucleotides, modified oligonucleotides, antisense oligonucleotides, siRNAs, and microRNAs.
44. A nucleotide sequence that specifically hybridizes to a target (TOI) nucleotide sequence or RNA transcript thereof that contains an allele with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. A diagnostic kit comprising a nucleic acid probe comprising, and instructions for performing the method according to paragraph 38 or 39.
45. TOI is encoded by a nucleotide sequence having a cumulative human allelic frequency of 1 to 10% and / or a total human genotype frequency of 1 to about 15% or 1 to 15%, described in any of the paragraphs above. Method, ligand, composition, kit or use.
46. The TOI is the human TOI selected from Table 5; optionally to treat and / or prevent the corresponding disease or condition detailed in Table 5 (Table 6). , The method, ligand, composition, kit or use described in any of the above paragraphs.
47. A method of treating or preventing a target (TOI) -mediated disease or condition in humans that exists as a different polymorphic variant in humans and that has been determined to be positive for the TOI polymorphic variant. Including the step of administering an anti-TOI ligand to target the TOI in a human to treat or prevent the disease or condition, the TOI in the human is an allele having a cumulative human allele frequency of less than 50%. A method encoded by a nucleotide sequence comprising and / or a TOI in said human being encoded by an allele containing an allele having a total human genotype frequency of less than 50%.
48. The method of paragraph 47, wherein the anti-TOI ligand is selected from antibodies, antibody moieties, antibody fragments, affibodies, antisense oligonucleotides, siRNAs, and microRNAs.
49. Specific to the TOI described in paragraph 1 for use in the method described in any of the above paragraphs (eg, for use in the method described in any of paragraphs 1-8 and 19-23). Anti-TOI ligand that binds to.
50. The ligand according to paragraph 49, wherein the ligand is an anti-TOI trap, antibody or antibody fragment.
51. The ligand according to paragraph 49 or 50, wherein the ligand is for subcutaneous or intravenous administration to humans or is included in an injectable formulation.
52. An injection device comprising the ligand according to any one of paragraphs 49-51.

Tailorizing additional ligands to the genotype and / or phenotype of human patients As described herein, the invention presents binding site specificity to one or more variant human TOIs (eg, PCSK9). Or intended for ligands (eg, antibodies and fragments) adapted to IL6R). In addition, or instead (and as further illustrated in the non-limiting examples below), an optional aspect of the invention adapts other features of the ligand to the genotype or phenotype of the patient. To provide that. In this regard, for example, the invention includes the ability to adapt variations of amino acid sequences in human patients to one or more ligand sequences or domains outside the binding site. For example, if the ligand comprises or consists of a human TOI binding antibody or anti-human TOI receptor Fc fusion, then this aspect of the invention is in a patient of one or more constant region domains (eg, Fc). Provides a more tailor-made fit to the genotype or phenotype of. In addition, or instead, it is intended that sequence variations at the binding site can be adapted to the patient's genotype or phenotype as well. The inventor presents SNPs in one, more than, or all of the gene segments in which the variable domain and / or constant region domain is induced, for example, the appearance of SNPs in the sequence encoding one or more parts of the ligand. This was done by considering the emergence. The inventor has understood that it would be desirable to adapt the ligand to one or more corresponding variant SNPs found in patients seeking therapeutic and / or prophylactic treatment. Fitting may include designing a ligand specifically for a patient having a known phenotype and / or genotype, or fitting may include variations in the patient's phenotype or genotype and ligand. It may include selecting the ligand by determining that there is an association between the amino acid and / or the variation in the corresponding nucleotide.

An important consideration for the present inventor was the desire to promote the compatibility of the ligand with the patient's body, and in particular the possible patient immune system response to the ligand administered. For example, a human or human patient receiving a humanized antibody drug may increase the immune response to the incoming antibody (the so-called HAHA response), thereby resulting in the immune system of the patient recognizing the drug as outpatient. It has been observed that patients produce anti-drug antibodies. For example, some patients receiving HUMIRA ™ (adalimumab), the most commonly sold antibody drug at present, have an increased HAHA immune response to the drug, which adversely affects treatment. Studies suggest that there is a possibility. JAMA. 2011; 305 (14): 1460 ~ 1468. doi: 10.1001 / jama.2011.406: See "Development of Antidrug Antibodies Against Adalimumab and Association With Disease Activity and Treatment Failure During Long-term Follow-up", GM Bartelds et al. I want to be. The authors concluded that the results of this study showed that the development of anti-drug antibodies was associated with the negative outcome of adalimumab treatment in human RA patients. In addition to discontinuing treatment more frequently and earlier in patients with anti-adalimumab antibody than in patients without anti-adalimumab antibody, they also have higher disease activity during treatment and are very rarely in remission. It is reported that it has also been reached. In addition, two-thirds of anti-adalimumab antibody-positive patients reportedly developed these antibodies during the first 28 weeks of treatment, and the presence of anti-adalimumab antibody substantially resulted in serum adalimumab levels. The data showed that it had an impact.

Therefore, this HAHA theme is an important concern and has been considered by management authorities. For example, the Committee for Medicinal Products for Human Use (EMA) has published the "Guideline on immunogenicity assessment of monoclonal antibodies intended for in vivo clinical use" (on the Worldwide Web, ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/ 06 / WC500128688.pdf; EMA / CHMP / BMWP / 86289/2010, available in the Addendum to EMEA / CHMP / BMWP / 14327/2006), which came into effect on December 1, 2012. As such, identifying and assessing the risk of the appearance and action of anti-drug antibodies is common practice for researchers.

Aspects of the invention design anti-human TOI agents to treat and / or prevent human TOI-related diseases and conditions by more strictly tailoring the ligand itself (as well as its specificity) to the patient. And help address these considerations when administering.

The inventor also considers the desire to tailor-make variations in the ligand constant region (eg, in the case of antibodies or Fc-containing ligands) and expects that the constant region administered to the patient then interacts with the constant region in the patient. It also takes into account that it will be combined with various components that are produced, such as the patient's Fc receptors. Good Fc / Fc receptor interactions are important for drug reuse (via FcRn) to provide a useful half-life in vivo, or for use in cell killing, eg, cancer indicators. sell. Therefore, in this scheme, it is possible to more closely tailor the effector function of the stationary region (eg, Fc) to the patient and promote efficacy. For example, more effective drugs are desirable for better patient treatment and may offer the potential for administration and / or reduction in frequency of administration.

Thus, in an example of this embodiment, the invention provides (set as a clause):

1. (i) The ligand (eg, antibody or fragment)
(c) A variable domain encoded by a human V region nucleotide sequence, wherein the V nucleotide sequence is a variable domain derived from recombination of the human VH, D and JH gene segments or the human VL and JL gene segments; or
(d) Containing a constant region domain encoded by a C region gene segment, the first gene segment of the gene segment of (a) or the C region gene segment of (b) encodes a first amino acid polymorphism. Contains the first single nucleotide polymorphism (SNP) to
(ii) The human genome contains the first SNP, or the human is an antibody variable domain comprising (a') the first amino acid polymorphism or (b') the first amino acid polymorphism. A ligand, method, use, kit or composition of the invention that expresses the antibody constant domain containing a type.

2. The ligand, method, according to Clause 1, wherein the human blood, as determined by an in vitro binding assay, is substantially free of antibodies that specifically bind to the domain containing the first amino acid polymorphism. Use, kit or composition.

3. The ligand, method, use, kit or composition according to Clause 2, wherein the SPR is used to perform the assay.

In an alternative example, ELISA is used.

4. Any of Clauses 1 to 3 where the human genome includes the first gene segment [when (a) applies] or the C region gene segment [when (b) applies]. The ligand, method, use, kit or composition described in one.

5. The first or second segment of the segment of (a), or the C region gene segment of (b), comprises a second SNP encoding a second amino acid polymorphism; said human. The genome contains the second SNP, or the human has (a ") an antibody variable domain containing the second amino acid polymorphism or (b") the first and second amino acid polymorphisms. The ligand, method, use, kit or composition according to any one of Articles 1 to 4, which expresses an antibody constant region domain containing an amino acid.

6. The ligand, method, use, kit or composition according to Clause 5, wherein the human expresses an antibody variable domain comprising the first and second amino acid polymorphisms.

7. The ligand, method, use, kit or composition according to Clause 5 or 6, wherein the first and second SNPs of the genome are contained in the same antibody gene segment.

For example, the first and second SNPs of the genome are included in the IGHG1 * 01 gene segment, and the first segment in (a) is the IGHG1 * 01 gene segment.

For example, the first and second SNPs of the genome are included in the IGHG2 * 01 gene segment, and the first segment in (a) is the IGHG2 * 01 gene segment.

8. The ligand, method, use, kit or composition according to any one of clauses 1 to 7, wherein each SNP is an SNP of a variable region gene segment.

9. Each SNP is an SNP of a constant region gene segment, for example, each SNP is an SNP of a gamma-1 constant region gene segment, or an SNP of a gamma-2 constant region gene segment, or a gamma-3 constant region gene segment. The ligand, method, use, kit or composition according to any one of clauses 1 to 7, which is an SNP or an SNP of a gamma-4 constant region gene segment.

10. As described in Clause 9, the first SNP is the SNP of the CH1, CH2, CH3 or CH4 gene segment and / or the second SNP is the SNP of the CH1, CH2, CH3 or CH4 gene segment. A ligand, method, use, kit or composition.

11. Ligands, methods, uses, kits according to any one of clauses 1-8, wherein each SNP is a variable domain SNP, eg, a VH domain SNP, or a Vκ domain SNP, or a Vλ SNP. Or the composition.

12. The ligand, method, use, kit or composition according to any one of Articles 1 to 11, wherein the constant region domain of (b) is contained in the antibody Fc region.

13. One or less ligand (eg, antibody or fragment), as disclosed herein, with a KD of 1 nM or less (eg, 100 or 10 pM or less), eg, as determined by SPR. The ligand, method, use, kit or composition according to any one of clauses 1-12, which has been determined to specifically bind to multiple human TOI variants.

14. The ligands, methods, uses, kits or compositions of the invention (eg, as described in any one of clauses 1-13), wherein the ligands are the human V gene segment and the human J gene segment (and any). Optionally, if the variable domain is a VH domain, it comprises or consists of an antibody or fragment comprising a human antibody variable domain derived from a recombination of the human D gene segment); the human genome comprises the human V gene segment. Includes and / or humans express antibodies comprising antibody variable domains derived from the recombination of said human V gene segment and human J gene segment (and optionally human D gene segment), ligands, methods, uses, kits. Or the composition.

In one example, the V gene segment is either WO2013041844, a database of 1000 genomes and / or the V gene segment disclosed at www.imgt.org, the disclosure of which (including disclosure of sequences) is in the present invention. Explicitly incorporated herein by reference for use.

15. The ligand (eg, containing or consisting of an antibody or fragment or Fc-fused human TOI receptor) comprises a human heavy chain constant domain encoded by a first constant region nucleotide sequence; the human genome, Any of the inventions (eg, clauses 1-14), wherein the first constant region nucleotide sequence comprises the same heavy chain constant region nucleotide sequence and / or a human expresses an antibody comprising the human constant region. Ligands, methods, uses, kits or compositions (listed in one).

16. The ligand (eg, containing or consisting of an antibody or fragment or Fc-fused human TOI receptor) comprises the human gamma heavy chain CH1 domain encoded by the CH1 nucleotide sequence; the human genome contains the CH1 nucleotide sequence. The ligand of the invention (eg, as described in any one of Clauses 1-15), which comprises the same gamma heavy chain constant region nucleotide sequence in and / or in which a human expresses an antibody comprising said human gamma CH1 domain. , Method, use, kit or composition.

17. The ligand (eg, containing or consisting of an antibody or fragment or Fc-fused human TOI receptor) comprises a human gamma heavy chain CH2 domain encoded by a CH2 nucleotide sequence; the human genome contains said CH2 nucleotide sequence. The ligand of the invention (eg, as described in any one of Clauses 1-16), which comprises the same gamma heavy chain constant region nucleotide sequence in and / or in which a human expresses an antibody comprising said human gamma CH2 domain. , Method, use, kit or composition.

18. The ligand (eg, containing or consisting of an antibody or fragment or Fc fusion human TOI receptor) comprises a human gamma heavy chain CH3 domain encoded by a CH3 nucleotide sequence; the human genome contains the CH3 nucleotide sequence. The ligand of the invention (eg, as described in any one of Clauses 1-17), which comprises the same gamma heavy chain constant region nucleotide sequence in and / or in which a human expresses an antibody comprising said human gamma CH3 domain. , Method, use, kit or composition.

19. The ligand (eg, containing or consisting of an antibody or fragment or Fc fusion human TOI receptor) comprises a human gamma heavy chain CH4 domain encoded by a CH4 nucleotide sequence; the human genome contains the CH4 nucleotide sequence. The ligand of the present invention (eg, as described in any one of Clauses 1-18), which comprises the same gamma heavy chain constant region nucleotide sequence in and / or in which a human expresses an antibody comprising said human gamma CH4 domain. , Method, use, kit or composition.

20. The ligand (eg, containing or consisting of an antibody or fragment or an Fc-fused human TOI receptor) comprises a human gamma heavy chain Fc region encoded by an Fc nucleotide sequence; the human genome contains the Fc nucleotide sequence. The ligand of the present invention (eg, as described in any one of Clauses 1-19), which comprises the same gamma heavy chain constant region nucleotide sequence in and / or in which a human expresses an antibody comprising said human gamma Fc region. , Method, use, kit or composition.

21. The ligand, method, use, kit or composition according to any one of Articles 16 to 20, wherein the human gamma heavy chain is a human gamma-1 heavy chain.

22. The ligand, method, use, kit or composition according to any one of Articles 16 to 20, wherein the human gamma heavy chain is a human gamma-2 heavy chain.

23. The ligand, method, use, kit or composition according to any one of Articles 16 to 20, wherein the ligand comprises a human IGHG1 * 01 gamma-1 heavy chain constant region.

24. The ligand, method, use, kit or composition according to any one of Articles 16 to 20, wherein the ligand comprises a human IGHG2 * 01 gamma-1 heavy chain constant region.

25. The ligand, method, use, kit or kit according to any one of clauses 15-24, wherein the human is genotyped or genotyped for the heavy chain constant region nucleotide sequence. Composition.

26. The ligand, method, use, kit or composition according to Clause 23, wherein the human is genotyped or genotyped positive for the human IGHG1 * 01 nucleotide sequence.

27. The ligand, method, use, kit or composition according to Clause 24, wherein the human is genotyped or genotyped for the human IGHG2 * 01 nucleotide sequence.

28. Described in any one of Clauses 16-24, wherein the human is phenotypized or phenotypically positive for the gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc. Ligand, method, use, kit or composition.

29. (i) Whether humans are phenotypized or phenotypically positive for the human IGHG1 * 01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc, in accordance with Clause 23. Or (ii) the ligand, method, according to Clause 28, wherein the human was phenotypically determined to be positive for the human IGHG2 * 01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc in accordance with Clause 24. Use, kit or composition.

30. Humans are positive for the gamma heavy chain constant region nucleotide sequence, eg, positive for the gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc nucleotide sequence; the human IGHG1 * 01 gamma heavy chain constant region, Positive for CH1, CH2, CH3, CH4 or Fc nucleotide sequence; or genotyping for the human IGHG2 * 01 gamma heavy chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence. The method or use described in any one of Clauses 16-24 and 26-29.

31. Humans are positive for said gamma heavy chain constant domain, eg, said gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc; said human IGHG1 * 01 gamma heavy chain constant domain, CH1, CH2, Clauses 16-24 and 26-30, including the step of phenotyping positive for CH3, CH4 or Fc; or positive for said human IGHG2 * 01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc. The method or use described in any one of.

Examples of tailor-made ligands We have analyzed the variability and distribution of amino acids among large representative human samples. Table 9 shows the results of analysis of antibody gene segment examples.

In the first example, the inventor individually or in combination with the rarer IGH-gamma-1 SNPs 204D (observed cumulative frequency 0.296) and 206L (observed cumulative frequency 0.283). I confirmed the possibility of working on it. These residues are part of the CH3 domain and as such they form part of the antibody Fc region. Therefore, these CH3 variations and patient compatibility are particularly beneficial for the reasons discussed above. Therefore, this example provides aspects detailed in the following clauses.

32. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) comprises Asp corresponding to position 204 of SEQ ID NO: 42 or Leu corresponding to position 206 of SEQ ID NO: 42. Human gamma-1 heavy chain constant region containing, the human genome contains a gamma-1 heavy chain constant region nucleotide sequence encoding such Asp or Leu, or human gamma containing such Asp or Leu -1 A ligand, method, use, kit or composition of the invention (eg, as described in any one of Clauses 1-31) that expresses an antibody comprising a constant region.

A person skilled in the art will determine the human genome sequence, for example by using a sample containing genomic DNA and / or RNA, and the sampled sequence and the sequence of the human allele (eg, IMGT, 100 Genome or the present specification). Expected to be familiar with techniques for sequencing and comparing using bioinformatics or other computer tools for comparison with (as shown in other databases as disclosed in the book). To. In some examples, the sample is a blood or saliva or cheek swab sample.

33. Ligands, methods, uses, kits according to Clause 32, wherein the ligand comprises a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42. Or the composition.

34. The human genome contains a gamma-1 heavy chain constant region nucleotide sequence encoding such Asp and Leu, or an antibody in which a human contains a human gamma-1 constant region containing such Asp and Leu. The ligand, method, use, kit or composition that is expressed, as described in Clause 32 or 33.

35. The ligand according to clause 32, 33 or 34, wherein the ligand comprises the human IGHG1 * 01 gamma-1 heavy chain constant region, eg, the Fc, CH1, CH2 and / or CH3 domains encoded by human IGHG1 * 01. , Method, use, kit or composition.

36. One of clauses 32 to 35, wherein the human genome contains the human IGHG1 * 01 nucleotide sequence, or the human expresses an antibody comprising the human constant domain encoded by the human IGHG1 * 01 nucleotide sequence. The ligand, method, use, kit or composition described in.

37. The ligand, method, use, kit or composition according to any one of Articles 32 to 36, wherein the ligand comprises a hinge region encoded by human IGHG1 * 01.

38. The ligand, method, use, kit or composition according to any one of Articles 32 to 37, wherein the ligand comprises or comprises an antibody comprising a heavy chain comprising SEQ ID NO: 61.

39. The ligand, method, use, kit or composition according to any one of Articles 32 to 38, wherein the human belongs to the European lineage.

As shown in Table 9 (Table 10), 204D and 206L are found in such humans.

40. The ligand, method, use, kit or composition according to any one of Articles 32 to 39, wherein the human is genotyped or genotyped positive for said Asp and / or Leu. object.

41. The ligand, method, use, kit or composition according to any one of Articles 32 to 40, wherein the human is genotyped or genotyped positive for human IGHG1 * 01.

42. Ligands, methods, uses, kits or compositions according to any one of clauses 32 to 41, wherein the human is phenotypized or phenotypically positive for CH3 of human IGHG1 * 01. object.

43. Does the genome contain codons encoding said Asp and / or Leu; contains human IGHG1 * 01; or comprises the step of selecting said human containing CH3 of human IGHG1 * 01, clauses 32-42. The method or use described in any one.

44. The method according to any one of clauses 32 to 43, comprising the step of selecting the human whose phenotype comprises the Asp and / or Leu; human IGHG1 * 01 region; or CH3 of human IGHG1 * 01. Or use.

44a. The ligand, method, use, kit or composition according to any one of Articles 32 to 44, wherein human expresses an antibody comprising a human gamma-1 constant region, such as Asp and Leu.

In the second example, the inventor confirmed the possibility of working on the SNP of IGH-gamma-2. This included consideration of variations in the Fc region, and in this respect the inventor focused on positions 161 and 257 in the Fc region. Therefore, this example provides aspects detailed in the following clauses.

45. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) is Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, sequence. Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Phe corresponding to position 76 of number 44, Val corresponding to position 161 of SEQ ID NO: 44 and Ala corresponding to position 257 of SEQ ID NO: 44. An antibody in which the human genome contains a gamma-2 heavy chain constant region nucleotide sequence encoding such a selected amino acid, or a human contains a human gamma-2 constant region containing such a selected amino acid. The ligand, method, use, kit or composition of the invention (eg, described in any one of clauses 1-31) that expresses.

46. The ligands are (i) Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, and (ii) SEQ ID NO: optionally. Human gamma containing Val corresponding to position 161 of 44 and / or Ala corresponding to position 257 of SEQ ID NO: 44-2 Heavy chain constant region; gamma in which the human genome encodes the amino acid of such (i). -The ligands, methods, uses, kits of Clause 45, which contain a double chain constant region nucleotide sequence or in which a human expresses an antibody comprising a human gamma-2 constant region containing such an amino acid (i). Or the composition.

This example focuses on the CH1 variation.

47. The ligands are (i) Val corresponding to position 161 of SEQ ID NO: 44 and Ala corresponding to position 257 of SEQ ID NO: 44, and optionally (ii) Pro corresponding to position 72 of SEQ ID NO: 44, SEQ ID NO: Contains a human gamma-2 heavy chain constant region containing amino acids selected from the group consisting of Asn corresponding to position 75 of 44 and Phe corresponding to position 76 of SEQ ID NO: 44; the human genome is such (i). 45 or 46, which comprises a gamma-2 heavy chain constant region nucleotide sequence encoding an amino acid of, or human expresses an antibody comprising a human gamma-2 constant region comprising such an amino acid (i). Ligands, methods, uses, kits or compositions.

This example focuses on Fc variations.

48. The ligand is in any one of clauses 45-47, including the human IGHG2 * 01 gamma-2 heavy chain constant region, eg, the Fc, CH1, CH2 and / or CH3 domains encoded by human IGHG2 * 01. The ligand, method, use, kit or composition described.

49. Any one of Clauses 45-48, wherein the human genome contains the human IGHG2 * 01 nucleotide sequence, or the human expresses an antibody comprising the human constant domain encoded by the human IGHG2 * 01 nucleotide sequence. The ligand, method, use, kit or composition described in.

50. The ligand, method, use, kit or composition according to any one of Articles 45-49, wherein the ligand comprises a hinge region encoded by human IGHG2 * 01.

51. The ligand, method, use, kit or composition according to any one of Articles 45-50, wherein the ligand comprises or comprises an antibody comprising a heavy chain comprising SEQ ID NO: 63 or 65.

52. The ligand, method, use, kit or composition according to any one of Articles 45-51, wherein the human belongs to a European, African-American, or European-American lineage.

53. Either Articles 45-52, where humans are genotyped or genotyped positive for one, more than, or all of the above Pro, Asn, Phe, Val and Ala. One of the ligands, methods, uses, kits or compositions described.

54. The ligand, method, use, kit or composition according to any one of Articles 45-53, wherein the human is genotyped or genotyped positive for human IGHG2 * 01.

55. Ligands, methods, uses, kits or compositions according to any one of clauses 45-54, wherein the human is phenotypized or phenotypized for CH1 of human IGHG2 * 01. object.

56. Ligands, methods, uses, kits or compositions according to any one of Articles 45-55, wherein the human is phenotypized or phenotypized for CH2 of human IGHG2 * 01. object.

57. Ligands, methods, uses, kits or compositions according to any one of clauses 45-56, wherein the human is phenotypized or phenotypically positive for CH3 of human IGHG2 * 01. object.

58. Does the genome contain codons encoding one, more than, or all of the Pro, Asn, Phe, Val and Ala; contains human IGHG2 * 01; or CH1 of human IGHG2 * 01, The method or use according to any one of clauses 45-57, comprising the step of selecting said human comprising CH2 and / or CH3.

59. The phenotype comprising one, more than one, or all of the Pro, Asn, Phe, Val and Ala; human IGHG2 * 01 region; or CH1, CH2 and / or CH3 of human IGHG2 * 01. The method or use according to any one of clauses 45-58, including the step of selecting a human.

60. The ligand, method, use, kit according to any one of clauses 45-59, wherein human expresses an antibody comprising a human gamma-2 constant region, including such Pro, Asn, Phe, Val and Ala. Or the composition.

In the third example, the inventor worked on a variation of the human kappa constant region. Therefore, aspects of the invention also provide:

61. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) comprises Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50. Contains the human kappa light chain constant region; the human genome contains the kappa light chain constant region nucleotide sequence encoding such Val or Cys, or the human contains the human kappa light chain constant region containing such Val or Cys. A ligand, method, use, kit or composition of the invention (eg, as described in any one of Clauses 1-60) that expresses an antibody comprising.

62. The ligand, method, use, kit or composition according to Clause 61, wherein the ligand comprises a human kappa light chain constant region containing Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50. object.

63. A clause in which the human genome contains a kappa light chain constant region nucleotide sequence encoding such Val and Cys, or a human expresses an antibody comprising a human kappa constant region containing such Val and Cys. 61 or 62. Ligand, method, use, kit or composition.

64. The ligand, method, use, kit or composition according to any one of Articles 61 to 63, wherein the antibody or fragment comprises the human IGKC * 01 kappa light chain constant region.

65. The ligand, method, use, kit or composition according to any one of Articles 61-64, wherein the ligand comprises or comprises an antibody comprising a light chain comprising SEQ ID NO: 62 or 66.

66. The ligand comprises or consists of an antibody comprising a human Vκ gene segment and a recombinantly derived light chain variable domain of the human Jκ gene segment, and the Jκ gene segment is IGKJ2 * 01 (SEQ ID NO: 57). The ligand, method, use, kit or composition according to any one of clauses 61-65.

67. The ligand, method, use, kit or composition according to any one of Articles 61-66, wherein the human is phenotypized or phenotypically positive for the Val and / or Cys. object.

68. The ligand, method, use, kit or composition according to any one of Articles 61-67, wherein the human is genotyped or genotyped positive for human IGKC * 01.

69. The ligand, method, use, kit or composition according to any one of Articles 61-68, wherein the human is phenotypized or phenotypized for the human IGKC * 01 domain. ..

70. The method or use according to any one of clauses 61-69, wherein the genome comprises a codon encoding said Val and / or Cys; or comprises the step of selecting said said human containing human IGKC * 01. ..

71. Whether the phenotype comprises such Val and / or Cys; or the method or use according to any one of clauses 61-70, which comprises the step of selecting said human containing the human IGKC * 01 domain. ..

72. The ligand, method according to any one of clauses 61-71, wherein human expresses an antibody comprising a human kappa constant domain such as Val and Cys, eg, a human IGKC * 01 constant domain. , Use, kit or composition.

In the fourth example, the inventor worked on a variation of the human lambda constant region. Therefore, this example provides aspects detailed in the following clauses.

73. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) comprises a human IGLC2 * 01 light chain constant region; the human genome contains the human IGLC2 * 01 nucleotide sequence. Ligands, methods, uses, kits or compositions of the invention (eg, described in any one of Clauses 1-60) that contain or human express an antibody comprising the human light chain IGLC2 * 01 constant region. object.

74. The ligand, method, use, kit or composition according to Clause 73, wherein the antibody comprises a light chain comprising SEQ ID NO: 64.

75. The ligand, method, use, kit or composition according to Clause 73 or 74, wherein the human is genotyped or genotyped positive for human IGLC2 * 01.

76. Ligands, methods, uses, kits or compositions according to any one of Articles 73-75, wherein the human is phenotypized or phenotypically positive for the human IGLC2 * 01 domain. ..

77. The method or use according to any one of clauses 73-76, comprising the step of selecting said human whose genome contains human IGLC2 * 01.

78. The method or use according to any one of clauses 73-77, comprising the step of selecting said human whose phenotype comprises a human IGLC2 * 01 domain.

79. The ligand, method, use, kit or composition according to any one of clauses 73-78, wherein the human expresses an antibody comprising the human lambda IGLC2 * 01 constant domain.

In the fifth example, the inventor worked on variations of the human heavy chain variable region. Therefore, this example provides aspects detailed in the following clauses.

80. The ligand contains or consists of an antibody or fragment containing the VH domain from the recombination of the human VH gene segment, human D gene segment and human JH gene segment, and the VH gene segment is selected from the following group. (I) The IGHV1-18 * 01 and the human genome contain the human IGHV1-18 * 01 nucleotide sequence, or the human expresses an antibody containing a variable domain derived from the recombination of the human IGHV1-18 * 01. Or (ii) the IGHV1-46 * 01 and the human genome contain the human IGHV1-46 * 01 nucleotide sequence, or the human antibody contains a variable domain derived from the recombination of the human IGHV1-46 * 01. A ligand, method, use, kit or composition of the invention (eg, as described in any one of clauses 1-79) that expresses.

81. The ligand, method, use, kit or kit according to Clause 80, wherein the antibody or fragment comprises one more or all of the CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human IGHG2 * 01. Composition.

82. The ligand, method, use, kit or composition according to Clause 80 or 81, wherein the antibody or fragment comprises a heavy chain comprising SEQ ID NO: 63 or 65.

83. Humans are genotyped or genotyped positive for the selected VH gene segment, positive for human IGHV1-18 * 01 or IVGH1-46 * 01, clauses 80-82. The ligand, method, use, kit or composition according to any one.

84. Any of clauses 80-83, including the step of genotyping a human as positive for the selected VH gene segment, eg, positive for human IGHV1-18 * 01 or IVGH1-46 * 01. The method or use described.

In the sixth example, the inventor worked on variations of the human light chain variable region. Therefore, this example provides aspects detailed in the following clauses.

85. The ligand contains or consists of an antibody or fragment containing the VL domain from the recombination of the human VL gene segment and the human JL gene segment, and the VL gene segment is selected from the group consisting of: (i) Does the IGKV4-1 * 01 and the human genome contain the human IGKV4-1 * 01 nucleotide sequence, or does the human express an antibody containing a variable domain derived from the recombination of human IGKV4-1 * 01; (ii) ) Does the IGLV2-14 * 01 and the human genome contain the human IGLV2-14 * 01 nucleotide sequence, or does the human express an antibody containing a variable domain derived from the recombination of human IGLV2-14 * 01; (iii) The IGKV1-13 * 02 and the human genome contain the human IGKV1-13 * 02 nucleotide sequence, or humans express antibodies containing the recombinantly derived variable domain of human IGKV1-13 * 02. The ligand, method, use, kit or composition of the invention (eg, as described in any one of clauses 1-84).

86. The ligand, method, use, kit or composition according to Clause 85, wherein the antibody comprises a light chain comprising SEQ ID NO: 62, 64 or 66.

87. The antibody or fragment comprises a light chain variable domain derived from recombination of the human Vκ gene segment and the human Jκ gene segment, and the Jκ gene segment is IGKJ2 * 01 (SEQ ID NO: 57); (i) or (iii). ) Applies to the ligand, method, use, kit or composition according to Clause 85 or 86.

88. Whether humans have been genotyped or genotyped positive for the selected VL gene segment, eg, human IGKV4-1 * 01, IGLV2-14 * 01 or IGKV1-13 * 02 The ligand, method, use, kit or composition according to any one of clauses 85-87.

89. The step of genotyping a human as positive for the selected VL gene segment, eg, a human being positive for human IGKV4-1 * 01, IGLV2-14 * 01 or IGKV1-13 * 02. And the method or use described in Clause 88, including the step of genotyping.

90. A clause that binds to said human TOI with a dissociation constant (Kd) of 1 nM or less (eg, 100, 10 or 1 pM or less) if the ligand (eg, antibody or fragment) is determined by SPR. The ligand, method, use, kit or composition according to any one of 1 to 89.

In a specific embodiment, the ligand, antibody or fragment of the invention comprises an Fc region, which is 234D, 234E, 234N, 234Q, 234T when numbered by the EU index as described in Kabat. , 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F , 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 33
From 1N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W Contains at least one unnatural amino acid residue selected from the group. Optionally, the Fc region may contain additional and / or alternative unnatural amino acid residues known to those of skill in the art (eg, US Pat. No. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publication WO 01/58957; WO 02. / 06919; WO04 / 016750; WO04 / 029207; see WO04 / 035752 and WO05 / 040217).

In one example, the ligand, antibody or fragment binds one or more human binding sites specifically to TOI (eg, PCSK9, LDL receptor, IL4Ra, IL6R, PD-L1, PD-1, PDGF-B). , PDGFR-B, angiopoietin, BMP6, hemoduverin, hepcidin, ferroportin, transferrin, HFE, TMPRSS6, VEGFA, Nav1.8 or Nav1.7). In certain embodiments, the binding site comprises or consists of a human antibody variable domain or human receptor for TOI (eg, a human receptor binding site for VEGFA). Furthermore, the ligand, antibody or fragment may optionally be completely human (eg, human Fc region containing or not containing human constant region). In some examples, the ligand is afuri vercept, alirocumab, salilumab or dupilumab. A fully human ligand can be used with a human patient if the V and / or C regions are used in the context of the invention tailored to the human genotype and / or phenotype as described herein. Maximize the suitability of.

Some embodiments of the techniques described herein can be defined according to any of the following numbered paragraphs:
1. A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
The TOI in a human has a cumulative human allelic frequency of less than 50%, comprising the step of administering an anti-TOI ligand to the human to target the TOI variant in the human and treat or prevent the disease or condition. Encoded by a nucleotide sequence and / or the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%.
b. Prior to step (a), the human is genotyped or genotyped positive for the nucleotide sequence, or phenotyped positive for the TOI variant. ,Method.
2. The method of paragraph 1, wherein the ligand is determined or determined to be capable of binding to the TOI variant prior to step (a).
3. A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
The TOI in a human has a cumulative human allelic frequency of less than 50%, comprising the step of administering an anti-TOI ligand to the human to target the TOI variant in the human and treat or prevent the disease or condition. Encoded by a nucleotide sequence and / or the TOI in said human is encoded by a nucleotide sequence having a total human genotype frequency of less than 50%.
b. The method by which the ligand was determined or determined to be capable of binding to said TOI variant prior to step (a).
4. The human genome contains a nucleotide sequence encoding the TOI variant; prior to step (a), the nucleotide sequence contains less than 50% cumulative human allele frequency and / or less than 50% total human. The method according to paragraph 3, wherein it is determined or is determined to have a genotype frequency.
5. The method of paragraph 3 or 4, wherein prior to step (a), the human has been genotyped or genotyped positive for the variant nucleotide sequence.
6. The method according to any of the paragraphs above, wherein the human is phenotypically or phenotypically tested positive for the TOI variant prior to step (a).
7. The method according to any of the paragraphs above, wherein the frequency is less than 10 or 15%.
8. The ligand is capable of binding to two or more different TOI variants, each encoded by a nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. , The method described in any of the above paragraphs.
9. A method of treating or preventing a disease or condition mediated by a target (TOI) that exists as a different polymorphic variant in humans in humans.
The TOI in humans has a cumulative human allelic frequency greater than 50%, comprising the step of administering an anti-TOI ligand to humans to target the TOI variant in humans and treat or prevent said disease or condition. Or / or a variant encoded by a nucleotide sequence having a total human genotype frequency greater than 50%.
b. Prior to step (a), was the human genotyped negative for variant nucleotide sequences having a cumulative human allelic frequency of less than 50% and / or a total genotype frequency of less than 50%? , Or genotyped; or phenotypically negative for TOI variants encoded by nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total genotype frequency of less than 50%. The method.
10. Prior to step (a), a paragraph in which humans are phenotyped or phenotyped positive for the most frequent TOI variants, or genotyped for their nucleotide sequences. The method described in 9.
11. The method of paragraph 9 or 10, wherein prior to step (a), the ligand was determined or determined to be capable of binding to the most frequent TOI variant.
12. Prior to step (a), paragraphs 9, 10 or are determined that the ligand is, or is determined, unable to substantially neutralize or inhibit said TOI variant listed in step (b). The method described in 11.
13. The method according to any one of paragraphs 9-12, wherein the ligand is capable of binding to the most frequent TOI variant.
14. Described in any one of paragraphs 9-13, wherein the ligand is capable of binding to two or more different TOI variants, each encoded by a nucleotide sequence having a cumulative human allele frequency greater than 50%. the method of.
15. The method according to any of the preceding paragraphs, wherein the variant nucleotide sequence listed in step (a) has been determined or is determined to be present in at least two different human ethnic populations.
16. The method described in any of the paragraphs above, wherein the human frequency is the frequency in a database of naturally occurring sequences containing at least 1000 sequences sampled from at least 15 different human ethnic populations. ..
17. An anti-human TOI ligand for use in methods of treating and / or preventing TOI-mediated diseases or conditions in humans, where TOI is present in humans as a different genotypic variant and is the human genome. Contains a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%, the method comprising administering the ligand to a human.
18. The ligand according to paragraph 17, wherein the ligand is determined or determined to be capable of binding to the human TOI encoded by the nucleotide sequence.
19. Less than 50% cumulative human allelic frequency and / or for use in methods involving the step of targeting TOI in humans with ligands to treat and / or prevent TOI-mediated diseases or conditions. A ligand that binds to a human TOI comprising an amino acid sequence encoded by a TOI nucleotide sequence having a total human genotype frequency of less than 50%, the method comprising the step of administering the ligand to a human.
20. The description in any one of paragraphs 17-19, wherein humans are genotyped or genotyped for said TOI nucleotide sequence having a cumulative human allelic frequency of less than 50%. Ligand.
The ligand according to any one of paragraphs 17-19, wherein human is genotyped or genotyped positive for said TOI nucleotide sequence having a total human genotyping frequency of less than 50%.
21. Humans have been phenotyped or phenotyped positive for a TOI encoded by a nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotypic frequency of less than 50%. The ligand according to any one of paragraphs 17 to 20, which is:
22. Humans have been genotyped or genotyped for TOI nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total genotype frequency of less than 50%; Optionally, the human has a TOI nucleotide sequence with a cumulative human allele frequency of less than 50%, and a TOI nucleotide having a cumulative human allele frequency greater than 50% and / or a total human genotype frequency greater than 50%. The ligand according to any one of paragraphs 17-21, which is genotyped or genotyped to include a sequence.
23. The human genome has been genotyped or genotyped for TOI nucleotide sequences with a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. The ligand according to any one of paragraphs 17-22.
24. The ligand contains an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%; such binding is possible at the option. The ligand according to any one of paragraphs 17-23, comprising an antibody binding site that binds to a human TOI, determined or determined to be present.
25. The ligand according to paragraph 24, wherein the ligand is an antibody or antibody fragment.
26. The ligand comprises a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50% or a nucleotide sequence that specifically hybridizes to its RNA transcript; and / Or the ligand comprises a nucleotide sequence comprising at least 10 contiguous nucleotides of a nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%, or in an antisense sequence thereof. A ligand according to any one of paragraphs 17-23.
27. The human genome comprises a nucleotide sequence having a cumulative human allelic frequency of less than 50%, which sequence is found in at least one of two different ethnic populations, paragraphs 17-26. Ligand.
28. A pharmaceutical composition or kit for treating or preventing a TOI-mediated condition or disease listed in any of the above paragraphs, comprising the ligand according to any one of paragraphs 17-27; optional. Optionally, in combination with a label or instruction for use in the treatment and / or prevention of the disease or condition in the human; optionally, this label or instruction comprises a marketing authorization number (eg, FDA or EMA). Permission number); optionally, this kit comprises an injection pen or IV container containing this ligand, a composition or kit.
29. A method of generating an anti-human TOI antibody binding site, which involves obtaining multiple anti-TOI antibody binding sites and a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. To a TOI containing an amino acid sequence encoded by a nucleotide sequence having, or from a corresponding sequence encoded by a nucleotide sequence encoding a TOI having the highest cumulative human allele frequency and / or the highest total human genotype frequency. A method comprising a step of screening an antibody binding site for binding to the peptide comprising an amino acid variation and a step of isolating the binding site of the antibody binding site in the screening step.
30. A method of producing anti-human TOI antibodies in which non-human vertebrates (eg, mice or rats) have a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. Amino acid variations from a TOI containing an amino acid sequence encoded by a nucleotide sequence, or a corresponding sequence encoded by a nucleotide sequence encoding a TOI having the highest cumulative human allele frequency and / or the highest total human genotype frequency. An antibody that binds to a TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50% with the step of immunizing with that peptide. A method comprising the step of isolating and optionally producing a TOI binding fragment or derivative of the isolated antibody.
31. The method of paragraph 29 or 30, comprising the step of obtaining a nucleic acid encoding an antibody, fragment, derivative or binding site and optionally inserting the nucleic acid into an expression vector.
32. A kit for determining the TOI genotype of humans, the selected TOI nucleotide sequence or RNA transcript thereof having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50%. Containing nucleic acids containing nucleotide sequences that specifically hybridize to; and / or at least 10 of TOI nucleotide sequences in which the nucleic acid has a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%. A kit comprising or an antisense sequence thereof comprising a nucleotide sequence comprising a contiguous nucleotide.
33. A kit for TOI genotyping or phenotyping of humans, produced by the ligand described in any one of paragraphs 17-27 or the method described in any one of paragraphs 29-31. A kit containing the antibody, fragment or derivative of the product.
34. To treat and / or prevent TOI-mediated diseases or conditions in humans whose genome contains less than 50% cumulative human allelic frequency and / or less than 50% total human genotype frequency. Use of anti-TOI ligands that bind to human TOIs, including amino acid sequences encoded by TOI nucleotide sequences with less than 50% cumulative human allele frequency and / or less than 50% total human genotype frequency in the manufacture of pharmaceuticals. ..
35. Less than 50% cumulative human allelic frequency and / or less than 50% total human genotype in the manufacture of drugs to target and treat and / or prevent TOI-mediated diseases or conditions in humans Use of an anti-TOI ligand that binds to a human TOI containing an amino acid sequence encoded by a frequent TOI nucleotide sequence.
36. A method of targeting TOI to treat and / or prevent TOI-mediated diseases or conditions in humans, with less than 50% cumulative human allelic frequency and / or less than 50% total human genotype. A method comprising the step of administering an anti-TOI ligand to a human containing a selected TOI nucleotide sequence having a frequency, whereby the TOI encoded by the nucleotide sequence is targeted.
37. The ligand comprises the step of targeting a human TOI containing an amino acid sequence to treat and / or prevent the disease or condition in the human, the amino acid sequence having a cumulative human allelic frequency of less than 50%. And / or the method of paragraph 36, encoded by a nucleotide sequence having a total human genotype frequency of less than 50%.
38. A method for determining the TOI genotype of a human nucleic acid sample, the presence of a TOI nucleotide sequence in the sample having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50%. A method comprising the step of identifying.
39. A method of TOI typing a human protein sample, encoded by a TOI nucleotide sequence having a cumulative human allele frequency of less than 50% and / or a total human genotyping frequency of less than 50% in the sample. A method comprising identifying the presence of a TOI amino acid sequence.
40. Paragraph 38 or 39, which comprises the step of obtaining a sample of serum, blood, feces, hair, urine or saliva from a human, thereby obtaining a nucleic acid or protein sample for use in the step of identifying the sequence. the method of.
41. The method of any one of paragraphs 38-40, comprising performing the identification step using the ligand according to any one of paragraphs 17-27.
42. Ligand capable of binding to a human TOI containing an amino acid sequence encoded by a TOI nucleotide sequence having a cumulative human allelic frequency of less than 50% and / or a total human genotype frequency of less than 50%, as described in paragraph 38 or 39. Diagnostic kit that includes instructions and instructions for doing the method.
Nucleic acid probes comprising TOI nucleotide sequences with a cumulative human allelic frequency of less than 43.50% and / or a total human genotype frequency of less than 50% or nucleotide sequences that specifically hybridize to RNA transcripts thereof, and paragraph 38 or 39. A diagnostic kit that includes instructions and instructions for performing the methods described in.
44. TOI is encoded by a nucleotide sequence having a cumulative human allelic frequency of 1 to 10% and / or a total human genotype frequency of 1 to about 15% or 1 to 15%, described in any of the paragraphs above. Method, ligand, composition, kit or use.
45. The TOI is the human TOI selected from Table 5; optionally to treat and / or prevent the corresponding disease or condition detailed in Table 5 (Table 6). , The method, ligand, composition, kit or use described in any of the above paragraphs.
46. (i) The ligand (eg, antibody or fragment) is
(e) A variable domain encoded by a human V region nucleotide sequence, wherein the V nucleotide sequence is a variable domain derived from recombination of the human VH, D and JH gene segments or the human VL and JL gene segments; or
(f) Containing a constant region domain encoded by a C region gene segment, the first gene segment of the gene segment of (a) or the C region gene segment of (b) encodes a first amino acid polymorphism. Contains the first single nucleotide polymorphism (SNP) to
(ii) The human genome contains the first SNP, or the human is an antibody variable domain comprising (a') the first amino acid polymorphism or (b') the first amino acid polymorphism. A ligand, method, use, kit or composition according to any of the above paragraphs that expresses the antibody constant domain comprising a type.
47. The ligand, method, according to paragraph 46, wherein the human blood, when determined by an in vitro binding assay, is substantially free of antibodies that specifically bind to the domain containing the first amino acid polymorphism. Use, kit or composition.
48. The ligand, method, use, kit or composition according to paragraph 47, wherein the SPR is used to perform the assay.
49. Any of paragraphs 46-48, wherein the human genome comprises the first gene segment [when (a) applies] or the C region gene segment [when (b) applies]. The ligand, method, use, kit or composition described in one.
50. The first or second segment of said segment of (a), or said C region gene segment of (b), comprises a second SNP encoding a second amino acid polymorphism; said human. The genome contains the second SNP, or the human has (a ") an antibody variable domain containing the second amino acid polymorphism or (b") the first and second amino acid polymorphisms. The ligand, method, use, kit or composition according to any one of paragraphs 46-49, which expresses an antibody constant region domain comprising.
51. The ligand, method, use, kit or composition according to paragraph 50, wherein the human expresses an antibody variable domain comprising the first and second amino acid polymorphisms.
52. The ligand, method, use, kit or composition according to paragraph 50 or 51, wherein the first and second SNPs of the genome are contained in the same antibody gene segment.
53. The ligand, method, use, kit or composition according to any one of paragraphs 46-52, wherein each SNP is an SNP of a variable region gene segment.
54. Each SNP is an SNP of a constant region gene segment, for example, each SNP is an SNP of a gamma-1 constant region gene segment, or an SNP of a gamma-2 constant region gene segment, or a gamma-3 constant region gene segment. The ligand, method, use, kit or composition according to any one of paragraphs 46-52, which is an SNP, or SNP of a gamma-4 constant region gene segment.
55. As described in paragraph 54, where the first SNP is the SNP of the CH1, CH2, CH3 or CH4 gene segment and / or the second SNP is the SNP of the CH1, CH2, CH3 or CH4 gene segment. A ligand, method, use, kit or composition.
56. Ligands, methods, uses, kits according to any one of paragraphs 46-53, wherein each SNP is a variable domain SNP, eg, a VH domain SNP, or a Vκ domain SNP, or a Vλ SNP. Or the composition.
57. The ligand, method, use, kit or composition according to any one of paragraphs 46-56, wherein the constant region domain of (b) is contained in the antibody Fc region.
58. One or less ligand (eg, antibody or fragment), as disclosed herein, with a KD of 1 nM or less (eg, 100 or 10 pM or less), eg, as determined by SPR. The ligand, method, use, kit or composition according to any one of paragraphs 46-57, which has been determined to specifically bind to a plurality of human TOI variants.
59. Lagent contains an antibody or fragment containing a human antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally, the human D gene segment if the variable domain is the VH domain). Or consists of; the human genome comprises said human V gene segment and / or human is derived from recombination of said human V gene segment and human J gene segment (and optionally human D gene segment). The ligand, method, use, kit or composition according to any one of the above paragraphs (eg, according to any one of paragraphs 46-58) that expresses an antibody comprising an antibody variable domain.
60. The ligand (eg, containing or consisting of an antibody or fragment or Fc-fused human TOI receptor) comprises a human heavy chain constant domain encoded by a first constant region nucleotide sequence; the human genome, Described in any of the preceding paragraphs, wherein the first constant region nucleotide sequence comprises the same heavy chain constant region nucleotide sequence and / or a human expresses an antibody comprising said human constant region (eg, paragraph 46). A ligand, method, use, kit or composition (described in any one of 59 to 59).
61. The ligand (eg, containing or consisting of an antibody or fragment or Fc fusion human TOI receptor) comprises a human gamma heavy chain CH1 domain encoded by a CH1 nucleotide sequence; the human genome contains the CH1 nucleotide sequence. The same gamma heavy chain constant region nucleotide sequence is contained in, and / or a human expresses an antibody containing the human gamma CH1 domain according to any one of the above paragraphs (eg, any one of paragraphs 46 to 60). Ligand, method, use, kit or composition (described in).
62. The ligand (eg, containing or consisting of an antibody or fragment or Fc fusion human TOI receptor) comprises a human gamma heavy chain CH2 domain encoded by a CH2 nucleotide sequence; the human genome contains the CH2 nucleotide sequence. The same gamma heavy chain constant region nucleotide sequence is contained in, and / or a human expresses an antibody containing the human gamma CH2 domain according to any one of the above paragraphs (eg, any one of paragraphs 46 to 61). Ligand, method, use, kit or composition (described in).
63. The ligand (eg, containing or consisting of an antibody or fragment or Fc fusion human TOI receptor) comprises a human gamma heavy chain CH3 domain encoded by a CH3 nucleotide sequence; the human genome contains the CH3 nucleotide sequence. The same gamma heavy chain constant region nucleotide sequence is contained in, and / or a human expresses an antibody containing the human gamma CH3 domain according to any one of the above paragraphs (eg, any one of paragraphs 46 to 62). Ligand, method, use, kit or composition (described in).
64. The ligand (eg, containing or consisting of an antibody or fragment or Fc fusion human TOI receptor) comprises a human gamma heavy chain CH4 domain encoded by a CH4 nucleotide sequence; the human genome contains the CH4 nucleotide sequence. The same gamma heavy chain constant region nucleotide sequence is contained in, and / or a human expresses an antibody containing the human gamma CH4 domain according to any one of the above paragraphs (eg, any one of paragraphs 46 to 63). Ligand, method, use, kit or composition (described in).
65. The ligand (eg, containing or consisting of an antibody or fragment or an Fc-fused human TOI receptor) comprises a human gamma heavy chain Fc region encoded by an Fc nucleotide sequence; the human genome contains said Fc nucleotide sequence. The same gamma heavy chain constant region nucleotide sequence is contained in, and / or a human expresses an antibody containing the human gamma Fc region, according to any one of the above paragraphs (eg, any one of paragraphs 46 to 64). Ligand, method, use, kit or composition (described in).
66. The ligand, method, use, kit or composition according to any one of paragraphs 61-65, wherein the human gamma heavy chain is a human gamma-1 heavy chain.
67. The ligand, method, use, kit or composition according to any one of paragraphs 61-65, wherein the human gamma heavy chain is a human gamma-2 heavy chain.
68. The ligand, method, use, kit or composition according to any one of paragraphs 61-65, wherein the ligand comprises a human IGHG1 * 01 gamma-1 heavy chain constant region.
69. The ligand, method, use, kit or composition according to any one of paragraphs 61-65, wherein the ligand comprises a human IGHG2 * 01 gamma-1 heavy chain constant region.
70. The ligand, method, use, kit or kit according to any one of paragraphs 60-69, wherein the human is genotyped or genotyped for the heavy chain constant region nucleotide sequence. Composition.
71. The ligand, method, use, kit or composition according to paragraph 68, wherein the human is genotyped or genotyped positive for the human IGHG1 * 01 nucleotide sequence.
72. The ligand, method, use, kit or composition according to paragraph 69, wherein the human is genotyped or genotyped positive for the human IGHG2 * 01 nucleotide sequence.
73. Described in any one of paragraphs 61-69, wherein the human is phenotypized or phenotypically positive for the gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc. Ligand, method, use, kit or composition.
74. (i) Whether humans are phenotypized or phenotypically positive for the human IGHG1 * 01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc, in accordance with Clause 23. Or (ii) the ligand, method, set forth in paragraph 63, wherein the human was phenotyped positive for the human IGHG2 * 01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc, in accordance with Clause 24. Use, kit or composition.
75. Humans are positive for the gamma heavy chain constant region nucleotide sequence, eg, positive for the gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc nucleotide sequence; the human IGHG1 * 01 gamma heavy chain constant region, Includes the step of genotyping positive for the CH1, CH2, CH3, CH4 or Fc nucleotide sequence; or positive for the human IGHG2 * 01 gamma heavy chain constant region, CH1, CH2, CH3, CH4 or Fc nucleotide sequence. The method or use described in any one of paragraphs 61-69 and 71-74.
76. Humans are positive for said gamma heavy chain constant domain, eg, said gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc; said human IGHG1 * 01 gamma heavy chain constant domain, CH1, CH2, Paragraphs 61-69 and 71-75, including the step of phenotyping positive for CH3, CH4 or Fc; or positive for said human IGHG2 * 01 gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc. The method or use described in any one of.
77. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) comprises Asp corresponding to position 204 of SEQ ID NO: 42 or Leu corresponding to position 206 of SEQ ID NO: 42. Human gamma-1 heavy chain constant region containing, the human genome contains a gamma-1 heavy chain constant region nucleotide sequence encoding such Asp or Leu, or human gamma containing such Asp or Leu -The ligand, method, use, kit or composition according to any one of the above paragraphs (eg, according to any one of Clauses 46-76) that expresses an antibody comprising a constant region.
78. The ligand, method, use, kit according to paragraph 77, wherein the ligand comprises a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42. Or the composition.
79. The human genome contains a gamma-1 heavy chain constant region nucleotide sequence encoding such Asp and Leu, or an antibody in which a human contains a human gamma-1 constant region containing such Asp and Leu. The ligand, method, use, kit or composition expressed, according to paragraph 77 or 78.
80. Ligand, method of paragraph 77, 78 or 79, wherein the ligand comprises the human IGHG1 * 01 gamma-1 heavy chain constant region, eg, the Fc, CH1, CH2 and / or CH3 domains encoded by human IGHG1 * 01. , Use, kit or composition.
81. Any one of paragraphs 77-80, wherein the human genome contains the human IGHG1 * 01 nucleotide sequence, or human expresses an antibody comprising the human constant domain encoded by the human IGHG1 * 01 nucleotide sequence. The ligand, method, use, kit or composition described in.
82. The ligand, method, use, kit or composition according to any one of paragraphs 77-81, wherein the ligand comprises a hinge region encoded by human IGHG1 * 01.
83. The ligand, method, use, kit or composition according to any one of paragraphs 77-82, wherein the ligand comprises or comprises an antibody comprising a heavy chain comprising SEQ ID NO: 61.
84. The ligand, method, use, kit or composition according to any one of paragraphs 77-83, wherein the human belongs to the European lineage.
85. The ligand, method, use, kit or composition according to any one of paragraphs 77-84, wherein the human is genotyped or genotyped positive for said Asp and / or Leu. object.
86. The ligand, method, use, kit or composition according to any one of paragraphs 77-85, wherein the human is genotyped or genotyped positive for human IGHG1 * 01.
87. The ligand, method, use, kit or composition of any one of paragraphs 77-86, wherein the human is phenotypized or phenotypized for CH3 of human IGHG1 * 01. object.
88. Paragraphs 77-87, comprising the step of selecting the human whose genome contains the codon encoding the Asp and / or Leu; the human IGHG1 * 01; or the CH3 of the human IGHG1 * 01. The method or use described in any one.
89. The method of any one of paragraphs 77-88, comprising the step of selecting the human whose phenotype comprises the Asp and / or Leu; human IGHG1 * 01 region; or CH3 of human IGHG1 * 01. Or use.
90. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) is Pro, corresponding to position 72 of SEQ ID NO: 44, Asn, sequence corresponding to position 75 of SEQ ID NO: 44. Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Phe corresponding to position 76 of number 44, Val corresponding to position 161 of SEQ ID NO: 44 and Ala corresponding to position 257 of SEQ ID NO: 44. An antibody in which the human genome contains a gamma-2 heavy chain constant region nucleotide sequence encoding such a selected amino acid, or a human contains a human gamma-2 constant region containing such a selected amino acid. The ligand, method, use, kit or composition of any of the aforementioned paragraphs that expresses.
91. The ligands are (i) Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, and (ii) SEQ ID NO: optionally. Human gamma containing Val corresponding to position 161 of 44 and / or Ala corresponding to position 257 of SEQ ID NO: 44-2 Heavy chain constant region; gamma in which the human genome encodes the amino acid of such (i). -The ligand, method, use, kit or composition of paragraph 90, which comprises a double chain constant region nucleotide sequence or in which a human expresses an antibody comprising a human gamma-2 constant region containing such an amino acid (i). object.
92. The ligands are (i) Val corresponding to position 161 of SEQ ID NO: 44 and Ala corresponding to position 257 of SEQ ID NO: 44, and optionally (ii) Pro corresponding to position 72 of SEQ ID NO: 44, SEQ ID NO: Contains a human gamma-2 heavy chain constant region containing amino acids selected from the group consisting of Asn corresponding to position 75 of 44 and Phe corresponding to position 76 of SEQ ID NO: 44; the human genome is such (i). A ligand according to paragraph 90 or 91, comprising a gamma-2 heavy chain constant region nucleotide sequence encoding an amino acid of, or in which a human expresses an antibody comprising a human gamma-2 constant region comprising the amino acid of (i). , Method, use, kit or composition.
93. The ligand of any one of paragraphs 90-92, wherein the ligand comprises the human IGHG2 * 01 gamma-2 heavy chain constant region, eg, the Fc, CH1, CH2 and / or CH3 domains encoded by human IGHG2 * 01. , Method, use, kit or composition.
94. Any one of paragraphs 90-93, wherein the human genome comprises the human IGHG2 * 01 nucleotide sequence, or human expresses an antibody comprising the human constant domain encoded by the human IGHG2 * 01 nucleotide sequence. Ligand, method, use, kit or composition.
95. The ligand, method, use, kit or composition of any one of paragraphs 90-94, wherein the ligand comprises a hinge region encoded by human IGHG2 * 01.
96. A ligand, method, use, kit or composition of any one of paragraphs 90-95, wherein the ligand comprises or comprises an antibody comprising a heavy chain comprising SEQ ID NO: 63 or 65.
97. A ligand, method, use, kit or composition of any one of paragraphs 90-96, wherein the human belongs to a European, African-American, or European-American lineage.
98. Any of paragraphs 90-97, wherein the human is genotyped or genotyped positive for one, more than, or all of the above Pro, Asn, Phe, Val and Ala. One ligand, method, use, kit or composition.
99. The ligand, method, use, kit or composition of any one of paragraphs 90-98, wherein the human is genotyped or genotyped positive for human IGHG2 * 01.
100. A ligand, method, use, kit or composition of any one of paragraphs 90-99, wherein a human is phenotypized or phenotypized for CH1 of human IGHG2 * 01.
101. The ligand, method, use, kit or composition of any one of paragraphs 90-100, wherein the human is phenotypized or phenotypized for CH2 of human IGHG2 * 01.
102. The ligand, method, use, kit or composition of any one of paragraphs 90-101, wherein the human is phenotypized or phenotypized for CH3 of human IGHG2 * 01.
103. Does the genome contain codons encoding one, more than, or all of said Pro, Asn, Phe, Val and Ala; contains human IGHG2 * 01; or CH1 of human IGHG2 * 01, The method or use according to any one of paragraphs 90-102, comprising the step of selecting the human comprising CH2 and / or CH3.
104. The phenotype comprising one, more than one, or all of said Pro, Asn, Phe, Val and Ala; human IGHG2 * 01 region; or CH1, CH2 and / or CH3 of human IGHG2 * 01. The method or use of any one of paragraphs 90 to 103, including the step of selecting a human.
105. The ligand, method, use, kit according to any one of paragraphs 90-104, wherein human expresses an antibody comprising a human gamma-2 constant region, including such Pro, Asn, Phe, Val and Ala. Or the composition.
106. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) comprises Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50. Contains the human kappa light chain constant region; the human genome contains the kappa light chain constant region nucleotide sequence encoding such Val or Cys, or the human contains the human kappa light chain constant region containing such Val or Cys. The ligand, method, use, kit or composition according to any of the above paragraphs, which expresses an antibody comprising.
107. The ligand, method, use, kit or composition of paragraph 106, wherein the ligand comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50. object.
108. The paragraph in which the human genome contains a kappa light chain constant region nucleotide sequence encoding such Val and Cys, or a human expresses an antibody comprising a human kappa constant region containing such Val and Cys. The ligand, method, use, kit or composition according to 106 or 107.
109. The ligand, method, use, kit or composition according to any one of paragraphs 106-108, wherein the antibody or fragment comprises the human IGKC * 01 kappa light chain constant region.
110. The ligand, method, use, kit or composition according to any one of paragraphs 106-109, wherein the ligand comprises or comprises an antibody comprising a light chain comprising SEQ ID NO: 62 or 66.
111. The ligand comprises or consists of an antibody comprising a human Vκ gene segment and a recombinantly derived light chain variable domain of the human Jκ gene segment, and the Jκ gene segment is IGKJ2 * 01 (SEQ ID NO: 57). The ligand, method, use, kit or composition according to any one of paragraphs 106-110.
112. The ligand, method, use, kit or composition according to any one of paragraphs 106-111, wherein the human is phenotypized or phenotypically positive for the Val and / or Cys. object.
113. The ligand, method, use, kit or composition according to any one of paragraphs 106-112, wherein the human is genotyped or genotyped positive for human IGKC * 01.
114. The ligand, method, use, kit or composition according to any one of paragraphs 106-113, wherein the human is phenotypized or phenotypically positive for the human IGKC * 01 domain. ..
115. The method or use according to any one of paragraphs 106-114, wherein the genome comprises a codon encoding said Val and / or Cys; or comprises the step of selecting said human containing human IGKC * 01. ..
116. The method or use according to any one of paragraphs 106-115, wherein the phenotype comprises such Val and / or Cys; or comprises the step of selecting said human containing the human IGKC * 01 domain. ..
117. The ligand, method according to any one of paragraphs 106-116, wherein human expresses an antibody comprising a human kappa constant domain such as Val and Cys, eg, a human IGKC * 01 constant domain. , Use, kit or composition ,.
118. The ligand (eg, containing or consisting of a TOI receptor fused to an antibody or fragment or Fc) comprises a human IGLC2 * 01 light chain constant region; the human genome contains the human IGLC2 * 01 nucleotide sequence. The ligand, method, use, kit or composition according to any of the above paragraphs, which comprises or human expresses an antibody comprising the human light chain IGLC2 * 01 constant region.
119. The ligand, method, use, kit or composition according to paragraph 118, wherein the antibody comprises a light chain comprising SEQ ID NO: 64.
120. The ligand, method, use, kit or composition according to paragraph 118 or 119, wherein the human is genotyped or genotyped positive for human IGLC2 * 01.
121. Ligands, methods, uses, kits or compositions according to any one of paragraphs 118-120, wherein the human is phenotypized or phenotypized for the human IGLC2 * 01 domain. ..
122. The method or use according to any one of paragraphs 118-212, comprising the step of selecting said human whose genome comprises human IGLC2 * 01.
123. The method or use according to any one of clauses 73-77, comprising the step of selecting said human whose phenotype comprises a human IGLC2 * 01 domain.
124. The ligand, method, use, kit or composition according to any one of paragraphs 108-123, wherein human expresses an antibody comprising a human lambda IGLC2 * 01 constant domain.
125. The ligand contains or consists of an antibody or fragment containing the VH domain from the recombination of the human VH gene segment, human D gene segment and human JH gene segment, and the VH gene segment is selected from the following group. (I) IGHV1-18 * 01 and the human genome contain the human IGHV1-18 * 01 nucleotide sequence, or humans express antibodies containing the recombinantly derived variable domain of human IGHV1-18 * 01. Or (ii) IVGH1-46 * 01 and the human genome contain the human IGHV1-46 * 01 nucleotide sequence, or the human antibody contains a variable domain derived from the recombination of human IGHV1-46 * 01. The ligand, method, use, kit or composition according to any of the above paragraphs, which expresses.
126. The ligand, method, use, kit or kit according to paragraph 125, wherein the antibody or fragment comprises one more or all of the CH1 domain, CH2 domain, CH3 domain, hinge or Fc encoded by human IGHG2 * 01. Composition.
127. The ligand, method, use, kit or composition according to paragraph 125 or 126, wherein the antibody or fragment comprises a heavy chain comprising SEQ ID NO: 63 or 65.
128. Humans are genotyped or genotyped positive for the selected VH gene segment, positive for human IGHV1-18 * 01 or IVGH1-46 * 01, paragraphs 125-127. The ligand, method, use, kit or composition according to any one.
129. In any of paragraphs 125-128, comprising the step of genotyping a human as positive for the selected VH gene segment, eg, positive for human IGHV1-18 * 01 or IGVH1-46 * 01. The method or use described.
130. The ligand contains or consists of an antibody or fragment containing the human VL gene segment and the recombinantly derived VL domain of the human JL gene segment, and the VL gene segment is selected from the group consisting of: (i) Does the IGKV4-1 * 01 and the human genome contain the human IGKV4-1 * 01 nucleotide sequence, or does the human express an antibody containing a variable domain derived from the recombination of human IGKV4-1 * 01; (ii) ) Does the IGLV2-14 * 01 and the human genome contain the human IGLV2-14 * 01 nucleotide sequence, or does the human express an antibody containing a variable domain derived from the recombination of human IGLV2-14 * 01; (iii) The IGKV1-13 * 02 and the human genome contain the human IGKV1-13 * 02 nucleotide sequence, or humans express antibodies containing the recombinantly derived variable domain of human IGKV1-13 * 02. The ligand, method, use, kit or composition described in any of the above paragraphs.
131. The ligand, method, use, kit or composition according to paragraph 130, wherein the antibody comprises a light chain comprising SEQ ID NO: 62, 64 or 66.
132. The antibody or fragment comprises a light chain variable domain derived from recombination of the human Vκ gene segment and the human Jκ gene segment, and the Jκ gene segment is IGKJ2 * 01 (SEQ ID NO: 57); (i) or (iii). ) Applies to the ligand, method, use, kit or composition according to paragraph 130 or 131.
133. Whether humans have been genotyped or genotyped positive for the selected VL gene segment, eg, human IGKV4-1 * 01, IGLV2-14 * 01 or IGKV1-13 * 02 The ligand, method, use, kit or composition according to any one of paragraphs 130-132.
134. A step of genotyping a human as positive for the selected VL gene segment, eg, a human being positive for human IGKV4-1 * 01, IGLV2-14 * 01 or IGKV1-13 * 02. The method or use according to paragraph 133, comprising the step of genotyping, if any.
135. The ligand (eg, antibody or fragment) binds to the human TOI with a dissociation constant (Kd) of 1 nM or less (eg, 100, 10 or 1 pM or less) as determined by SPR. Ligand, method, use, kit or composition described in any of the paragraphs.
136. The ligand, method, use, kit or composition according to any of the above paragraphs, wherein the TOI is human PCSK9 or human IL-6R.

Throughout the text of the invention, eg, in the claims described herein, the position numbers relating to the sequences are the positions detailed in the tables described herein, eg, Table 10 or Table 17. Refers to the positions listed in (Table 18).

(Example 1)
Rare PCSK9 Variant Precursor Protein Convertase Subtilisin Kexin Type 9 (PCSK9) is a serine protease involved in the regulation of low-density lipoprotein receptor (LDLR) protein levels (Horton et al., 2007; Seidah and Prat, 2007). In vitro experiments have shown that the addition of PCSK9 to HepG2 cells lowers the level of cell surface LDLR (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004). Experiments with mice have shown that increased PCSK9 protein levels decrease LDLR protein levels in the liver (Benjannet et al., 2004; Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004). However, PCSK9 knockout mice increased the level of LDLR in the liver (Rashid et al., 2005). In addition, various human PCSK9 mutations have been identified that result in either increased or decreased levels of plasma LDL (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has been shown to interact directly with the LDLR protein, endocytosis with LDLR, and co-immune-fluorescent with LDLR throughout the endosomal pathway (Lagace et al., 2006).

PCSK9 is a prohormone-precursor protein convertase in the subtilisin (S8) family of serine proteases (Seidah et al., 2003). Humans have nine prohormonal-precursor protein convertases that can be split between the S8A and S8B subfamilies (Rawlings et al., 2006). Flynn, PC1 / PC3, PC2, PACE4, PC4, PC5 / PC6 and PC7 / PC8 / LPC / SPC7 are classified in the subfamily S8B. Crystal structures and NMR structures of different domains from mouse furin and PCI reveal subtilisin-like pro- and catalytic domains, as well as the C-terminal P domain immediately relative to the catalytic domain (Henrich et al., 2003; Tangrea). Et al., 2002). Based on the amino acid sequence similarity within this subfamily, it is speculated that all seven members have similar structures (Henrich et al., 2005). SKI-l / SlP and PCSK9 are classified in the subfamily S8A. Sequence comparisons with these proteins also suggest the presence of subtilisin-like pro- and catalytic domains (Sakai et al., 1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins, the amino acid sequence C-terminus to the catalytic domain is more variable, suggesting the presence of the P domain.

Prohormones-precursor protein converting enzymes are expressed as thymogens, which mature through a multi-step process. The function of the pro domain in this process is doubled. This prodomain first acts as a chaperone and is required for proper folding of the catalytic domain (Ikemura et al., 1987). Once the catalytic domain is folded, autocatalytic action occurs between the prodomain and the catalytic domain. After this initial cleavage reaction, the prodomain remains bound to the catalytic domain, where the prodomain then acts as an inhibitor of catalytic activity (Fu et al., 2000). If the conditions are correct, maturation proceeds with a second autocatalytic event at sites within the prodomain (Anderson et al., 1997). This second cleavage event results in the dissociation of the prodomain and catalytic domain, followed by the active protease.

The autocatalytic action of PCSK9 zymogen occurs between Glnl52 and Serl53 (VFAQ | SIP (SEQ ID NO: 116)) (Naureckiene et al., 2003) and has been shown to be required for its secretion from cells (Seidah et al., 2003). No second autocatalytic event was observed at sites within the prodomain of PCSK9. Purified PCSK9 is composed of two species that can be separated by non-reducing SDS-PAGE (17 kd prodomain and 65 kd catalytic plus C-terminal domain). PCSK9 was not isolated without its inhibitory prodomain, and measurements of the catalytic activity of PCSK9 were variable (Naureckiene et al., 2003; Seidah et al., 2003).

In certain embodiments, the PCSK9 polypeptide is a terminal residue, eg, a leader sequence residue, a targeting residue, an amino-terminal methionine residue, a lysine residue, a tag residue and / or Contains fusion protein residues. "PCSK9" was also called FH3, NARC1, HCHOLA3, precursor protein convertase subtilisin / kexin type 9, and neuroapoptotic regulatory convertase 1. The PCSK9 gene encodes a precursor protein convertase protein belonging to the proteinase K subfamily of the secretory subtilase family. The term "PCSK9" means both the precursor protein and the product produced after the autocatalytic action of the precursor protein. When referring only to autocatalytic products (eg, for antigen-binding proteins or ligands that bind to cleaved PCSK9), the proteins are "mature," "cleaved," or "processed." Or it may refer to "active" PCSK9. When referring only to the inactive form, the protein can be referred to as the "inactive," "precursor," or "unprocessed" form of PCSK9. The term PCSK9 also refers to the PCSK9 amino acid sequence, eg, glycosylated PCSK9 sequence, PCSK9 sequence (then the signal sequence is cleaved), PCSK9 sequence (then the prodomain is cleaved from the catalytic domain). Also includes PCSK9 molecules that incorporate post-translational modifications (not separated from the catalytic domain) (eg, FIGS. 1A and 1A of US Patent Application Publication No. 20120093818A1, which is incorporated herein by reference in its entirety). See 1B).

The present invention provides anti-PCSK9 ligands as described herein; and PCSK9-binding or targeting ligands. This ligand has various usefulness. Some ligands are used in affinity purification of PCSK9, specifically in human CSK9 or its ligands, and in screening assays to genotype or phenotype humans, for example in specific binding assays. It is useful for identifying other antagonists with PCSK9 activity. Some ligands of the invention are useful for inhibiting the binding of PCSK9 to LDLR or for inhibiting PCSK9-mediated activity.

Anti-PCSK9 ligands (eg, antibodies and antisense RNA) have been developed on the basis of targeting and neutralizing the so-called "wild" human PCSK9, which is a commonly present form (eg, each of them in its entirety). (See U.S. Patent Application Publication No. 20120093818A1 and U.S. Patent Application Publication No. 20110065902A1), incorporated herein by reference. Although such treatments are useful for human patients carrying this form of human PCSK9, we have targeted a fairly rare but also naturally occurring form of PCSK9 in the human population. It was considered useful to consider the possibility of doing so. In this way, the inventor (at the protein or nucleic acid level) as a useful target for the treatment, prevention and diagnosis of humans mediated by or associated with PCSK9 activity or appropriate for a disease or condition associated thereto. We have reached an insight into the natural existence and distribution of rare human PCSK9 types that may function. This specifically provides tailor-made treatment, prevention and diagnosis in humans lacking the common PCSK9 gene or protein (ie, U.S. Patent Application Publication No. 20120093818A1 and U.S. Patent Application Publication No. 20110065902A1 to produce antibodies. Type a or type a') used in.

Those skilled in the art will appreciate that other changes translating into SNPs or amino acid variations can result in variability in the activity and / or higher-order structure of the human target to be targeted. This has led to increased interest in personalized medicine, where protein and nucleotide variability genotyping and knowledge can be used to more effectively tailor the care and diagnosis of patients. Therefore, the present invention provides tailor-made medicines and tests specifically directed to the more rare PCSK9 polymorphic variant. Such forms or "alleles" (at the nucleotide level), in many cases determined by the present inventor, include numerous changes at the nucleotide and amino acid levels from the corresponding common types of nucleotide and amino acid sequences. That is, there are numerous non-synonymous changes at the nucleotide level that translate into a number of corresponding changes in protein targets in humans.

Moreover, the inventor has surprisingly shown that rare natural forms are shown in a large number of and ethnically diverse human populations, and usually for representative ethnic populations, with many human examples. Nevertheless, we have noticed that it is much less frequent in humans than the common type. Therefore, the inventor has found that targeting such rare types provides effective treatment, prevention or diagnosis across many human ethnic groups, thereby expanding the usefulness of the present invention. ..

Given this perception, we in view that PCSK9 guides the selection of anti-PCSK9 ligands for administration to human patients for the treatment and / or prevention of diseases or conditions mediated or associated. It has been understood that there are significant industrial and medical applications of the present invention. In this manner, the patient receives tailor-made drugs and ligands according to the needs determined by the patient's genetic or phenotypic composition. Along with that, the present invention provides patient genotyping and / or phenotyping associated with such treatments, thereby allowing the drug to be appropriately adapted to the patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment (eg, poor efficacy and / or side effects) with drugs or ligands that are not suitable for the patient, and avoids drug prescribing mistakes and waste. ..

In the development of this idea, the inventor decided to determine a set of human PCSK9 variants based on the following criteria, which would provide useful drugs and diagnoses for tailor-made needs in the human population. It is a standard that the present inventor realizes to provide. The inventor has selected variants that have at least three of the following four criteria:
--PCSK9 variants with cumulative human allele frequencies ranging from 1 to 10%;
--PCSK9 variants with total human genotype frequencies ranging from 1 to about 15%;
--PCSK9 variants found in many different human ethnic populations [using the standard categorization of the 1000 Genomes Project, an acceptable standard in the art; see Table 4 below];
--PCSK9 variants found in many individuals distributed across many such different ethnic groups

Based on these criteria, the inventor identified the variants listed in Table 1 below (excluding type a).

In our selection, as a study, nucleotide variations (ie, non-synonymous variations) that produced amino acid variations in the corresponding PCSK9 types, as opposed to silent variations that did not alter the amino acid residues in the target protein. Included a selection of.

Variant allele nucleotide sequence and therefore
(i) The nucleotide sequence of the allele f is identical to SEQ ID NO: 28, except that the nucleotide sequence of this allele f contains the GTC codon instead of the ATC codon at the position labeled "I474V" in SEQ ID NO: 28. is there.
(ii) The nucleotide sequence of allele c is identical to SEQ ID NO: 28, except that the nucleotide sequence of allele c contains the GGG codon instead of the GAG codon at the position labeled "E670G" in SEQ ID NO: 28. is there.
(iii) The nucleotide sequence of the allele r contains SEQ ID NO: 28 and the GTC codon instead of the ATC codon at the position where the nucleotide sequence of this allele r is labeled "I474V" in SEQ ID NO: 28; SEQ ID NO: 28. It is the same except that the GGG codon is included instead of the GAG codon at the position displayed as "E670G" in.
(iv) The nucleotide sequence of allele p contains SEQ ID NO: 28 and the GTC codon instead of the GCC codon at the position where the nucleotide sequence of this allele p is labeled "A53V" in SEQ ID NO: 28; SEQ ID NO: 28 It is the same except that the GTC codon is included instead of the ATC codon at the position displayed as "I474V".
(v) The nucleotide sequence of the allele m is the same as SEQ ID NO: 28, except that the nucleotide sequence of this allele m contains the ACC codon instead of the GCC codon at the position labeled "A443T" in SEQ ID NO: 28. is there.
(vi) The nucleotide sequence of allele e contains SEQ ID NO: 28 and the AGT codon instead of the AAT codon at the position where the nucleotide sequence of this allele e is labeled "N425S" in SEQ ID NO: 28; SEQ ID NO: 28 It is the same except that the GTC codon is included instead of the ATC codon at the position displayed as "I474V".
(vii) The nucleotide sequence of allele h contains SEQ ID NO: 28 and the ACC codon instead of the GCC codon at the position where the nucleotide sequence of this allele h is labeled "A443T" in SEQ ID NO: 28; It is the same except that the CCG codon is included instead of the CAG codon at the position displayed as "Q619P" in 28.
(viii) The nucleotide sequence of the allele aj contains SEQ ID NO: 28 and the CTT codon instead of the CGT codon at the position where the nucleotide sequence of this allele aj is labeled "R46L" in SEQ ID NO: 28; It is the same except that the GTC codon is included instead of the ATC codon at the position displayed as "I474V" in 28.
(ix) The nucleotide sequence of allele q contains SEQ ID NO: 28 and the GTC codon instead of the GCC codon at the position where the nucleotide sequence of this allele q is labeled "A53V" in SEQ ID NO: 28; It is the same except that it contains the GGG codon instead of the GAG codon at the position labeled "E670G" in 28.

Variant precursor amino acid sequence
(The numbering is as shown in SEQ ID NO: 1 above)
(A) The f-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, except that this f-type amino acid sequence is at position 474 and contains valine. It is the same.
(B) The c-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, except that this c-type amino acid sequence is at position 670 and contains glycine. It is the same.
(C) The r-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, and this r-type amino acid sequence is at position 474, valine, and position 670. It is the same except that it contains glycine.
(D) The p-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, and this p-type amino acid sequence is at position 53, valine, and position 474. It is the same except that it contains valine.
(E) The m-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, except that this m-type amino acid sequence is at position 443 and contains threonine. It is the same.
(F) The e-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, and this e-type amino acid sequence is at position 425, serine, and position 474. It is the same except that it contains valine.
(G) The h-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, and this h-type amino acid sequence is at position 443, threonine, and position 619. It is the same except that it contains proline.
(H) The aj-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, and this aj-type amino acid sequence is at position 46, leucine, and position 474. It is the same except that it contains valine.
(I) The q-type amino acid sequence is the amino acid sequence from amino acid number 31 to (and including) amino acid number 692 of SEQ ID NO: 1, and this q-type amino acid sequence is at position 53, valine, and position 670. It is the same except that it contains glycine.

Variant mature amino acid sequence
(The numbering is as shown in SEQ ID NO: 1 above)
The (A') f-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the f-type amino acid sequence is at position 474 and contains valine.
The (B') c-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the c-type amino acid sequence is at position 670 and contains glycine.
The (C') r-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the r-type amino acid sequence contains valine at position 474 and glycine at position 670.
The (D') p-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the p-type amino acid sequence is at position 474 and contains valine.
The (E') m-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the m-type amino acid sequence is at position 443 and contains threonine.
The (F') e-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the e-type amino acid sequence contains serine at position 425 and valine at position 474.
The (G') h-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the h-type amino acid sequence contains threonine at position 443 and proline at position 619.
The (H') aj-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the aj-type amino acid sequence is at position 474 and contains valine.
The (I') q-type amino acid sequence is the same as that of SEQ ID NO: 2, except that the q-type amino acid sequence is at position 670 and contains glycine.

The mature p is the same as the mature f and aj.

The mature c is the same as the mature q.

Further sequence analysis and 3D in silico modeling (see Figure 1) revealed that the selected variants also met the following selection criteria:-
—— PCSK9 variants in which variant amino acid residues (relative to the common form of human PCSK9) are found in the mature form of the target (ie, outside the prodomain); and
—— PCSK9 variants in which variant amino acid residues (relative to the common form of human PCSK9) are surface-exposed on the target, due to the present inventor determining the topography of the target, and potential. Was determined by how and where ligand binding to the target occurs.

As can be seen from FIG. 1, the identified positions 425, 443, 474, 619 and 670 (found in another selected variant of the invention) are all surface exposed and outside the prodomain. Variants 425 and 443 are surface-exposed on the catalytic domain, while variants 474, 619 and 670 are surface-exposed on the C-terminal domain.

In the first example, the present invention addresses the need to treat humans with the rare, naturally occurring PCSK9 alleles, genotypes and phenotypes (rare types of proteins). In this regard, the present invention provides the following aspects:

In the first aspect: a method of treating and / or preventing a PCSK9-mediated disease or condition in humans whose genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, the ligand to humans. Anti-human PCSK9 ligand for use in methods involving the administration of.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or SEQ ID NO: 29-37. It is selected from the group consisting of 32, 34, 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In other cases, this nucleotide sequence comprises human PCSK9 containing mutants R46L, A53V, N425S, A443T, I474V, Q619P and E670G in SEQ ID NO: 1 (eg, including mutants I474, E670G, N425S or Q619P). For example, PCSK9 contains I474V in SEQ ID NO: 1, and optionally this human contains such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method is used). To treat or prevent dyslipidemia, eg, to lower or maintain lowered cholesterol in humans); for example, PCSK9 contains E670G in SEQ ID NO: 1 and is optionally optional. In this human contains such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method lowers cholesterol in humans to treat or prevent dyslipidemia, for example, Or to maintain lowered cholesterol); for example, PCSK9 contains Q619P in SEQ ID NO: 1, and optionally this human contains such PCSK9 or a nucleotide sequence encoding such PCSK9. (For example, here this method is to treat or prevent dyslipidemia, eg, to lower or maintain lowered cholesterol in humans); for example, PCSK9 is in SEQ ID NO: 1. N425S, and optionally this human contains such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method is used to treat or prevent dyslipidemia, eg, eg. To lower or maintain lowered cholesterol in humans); for example, PCSK9 contains R46L in SEQ ID NO: 1, and optionally this human is such PCSK9 or such. Contains a nucleotide sequence encoding PCSK9 (eg, where this method is to treat or prevent dyslipidemia, eg, to increase cholesterol in humans); for example, PCSK9 is included in SEQ ID NO: 1. A53V is included, and optionally this human contains such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method treats or prevents dyslipidemia). For example, to increase cholesterol in humans); for example, PCSK9 contains A443T in SEQ ID NO: 1, and optionally this human encodes such PCSK9 or such PCSK9. It comprises a nucleotide sequence (eg, here this method is to treat or prevent dyslipidemia, eg, to increase cholesterol in humans).

In the second aspect: Aspect 1 which is determined or determined to be capable of binding to human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q. Ligand.

In an alternative method, this ligand binds to human PCSK9 containing mutants R46L, A53V, N425S, A443T, I474V, Q619P and E670G in SEQ ID NO: 1, including, for example, mutants I474, E670G, N425S or Q619P. Determined or determined to be possible; for example, PCSK9 contains I474V in SEQ ID NO: 1, and optionally this human has such PCSK9 or a nucleotide sequence encoding such PCSK9. Includes (eg, where this method is to treat or prevent dyslipidemia, eg, to lower or maintain lowered cholesterol in humans); for example, PCSK9 has SEQ ID NO: 1. E670G is contained therein, and optionally this human comprises such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method is used herein to treat or prevent dyslipidemia, eg, For example, PCSK9 contains Q619P in SEQ ID NO: 1 and, optionally, this human is such PCSK9 or such PCSK9 or to maintain lowered cholesterol in humans. Contains a nucleotide sequence encoding PCSK9 (eg, where this method is to treat or prevent dyslipidemia, eg, to lower or maintain reduced cholesterol in humans); For example, PCSK9 comprises N425S in SEQ ID NO: 1, and optionally humans include such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method presents with dyslipidemia). To treat or prevent, for example, to lower or maintain lowered cholesterol in humans); for example, PCSK9 contains R46L in SEQ ID NO: 1, and optionally this human Containing such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, here this method is to treat or prevent dyslipidemia, eg, to increase cholesterol in humans); eg, PCSK9 comprises A53V in SEQ ID NO: 1, and optionally this human contains such PCSK9 or a nucleotide sequence encoding such PCSK9 (eg, where this method is used). For the treatment or prevention of dyslipidemia, eg, to increase cholesterol in humans); for example, PCSK9 comprises A443T in SEQ ID NO: 1, and optionally this human is such PCSK9. Or it comprises a nucleotide sequence encoding such PCSK9 (eg, here this method is to treat or prevent dyslipidemia, eg, to increase cholesterol in humans).

In some examples of any embodiment, the ligand is 2, 3, 4 or more human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q, or It binds (or is determined to bind) to the PCSK9 of the alternative method.

In an example of any embodiment, the ligand is PCSK9, eg, human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q, or said PCSK9 in said alternative. Contains a protein domain that specifically binds to.

Terms such as "specifically bind" mean that a ligand, eg, an antibody or antigen-binding fragment thereof, forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 × lO ^-6 M (eg, the smaller the KD, the tighter the binding). Methods of determining which of the two molecules specifically binds are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance and the like. However, isolated antibodies that specifically bind to human PCSK9 may exhibit cross-reactivity with other antigens, such as PCSK9 molecules from other species. In addition, human PCSK9 and multivalent-specific antibodies that bind to one or more additional antigens (eg, bispecific) nevertheless "specifically bind" to PCSK9 when used herein. Is considered an antibody.

In some examples of any embodiment, this ligand mimics the EGA domain of the LDL receptor and is selected from the group consisting of PCSK9, eg, types f, c, r, p, m, e, h, aj and q. Contains or consists of human PCSK9, or a protein that specifically binds to said PCSK9 in the alternative method.

In some examples of any embodiment, the ligand is PCSK9, eg, human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q, or said in the alternative. Compete with PCSK9.

In some examples of any embodiment, the method (prior to administration of the ligand) is that the ligand is selected from the group consisting of types f, c, r, p, m, e, h, aj and q. Includes the step of determining that PCSK9, or the PCSK9 in the alternative method, can be bound.

In some examples of any aspect, binding is determined by SPR. In some examples of any aspect, the binding is determined by ELISA.

In some examples of any aspect, the type is a mature type.

In some examples of any aspect, the type is a precursor type.

In a third aspect: (i) PCSK9 of the alternative method, or (ii) a disease or condition mediated by PCSK9 that binds to human PCSK9 containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27. A ligand for use in a method comprising the step of using a ligand for targeting PCSK9 in humans to treat and / or prevent, the method comprising administering the ligand to humans. Including.

In some examples, the disease or condition is mediated by (i) PCSK9 of the alternative, or (ii) human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.

In some examples, it is selected from the group consisting of SEQ ID NOs: 4-23, 26 and 27, or selected from the group consisting of SEQ ID NOs: 4-14 and 18-27, or SEQ ID NOs: 4-14, 18- Amino acid sequence selected from the group consisting of 23, 26 and 27. These are naturally occurring sequences that do not contain 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the amino acid sequence is SEQ ID NO: 18, 19 or 20 containing 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In one example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 26 and 27, including 670G, which is a marker of the severity of coronary atherosclerosis (Chen). 2005).

In some examples, an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequences that have a naturally occurring combination of differences from SEQ ID NOs: 1-3 (type a) and meet the criteria set above.

In one example, the amino acid sequence is SEQ ID NO: 4.

In one example, the amino acid sequence is SEQ ID NO: 5.

In one example, the amino acid sequence is SEQ ID NO: 6.

In one example, the amino acid sequence is SEQ ID NO: 7.

In one example, the amino acid sequence is SEQ ID NO: 8.

In one example, the amino acid sequence is SEQ ID NO: 9.

In one example, the amino acid sequence is SEQ ID NO: 10.

In one example, the amino acid sequence is SEQ ID NO: 11.

In one example, the amino acid sequence is SEQ ID NO: 12.

In one example, the amino acid sequence is SEQ ID NO: 13.

In one example, the amino acid sequence is SEQ ID NO: 14.

In one example, the amino acid sequence is SEQ ID NO: 15.

In one example, the amino acid sequence is SEQ ID NO: 16.

In one example, the amino acid sequence is SEQ ID NO: 17.

In one example, the amino acid sequence is SEQ ID NO: 18.

In one example, the amino acid sequence is SEQ ID NO: 19.

In one example, the amino acid sequence is SEQ ID NO: 20.

In one example, the amino acid sequence is SEQ ID NO: 21.

In one example, the amino acid sequence is SEQ ID NO: 22.

In one example, the amino acid sequence is SEQ ID NO: 23.

In one example, the amino acid sequence is SEQ ID NO: 24.

In one example, the amino acid sequence is SEQ ID NO: 25.

In one example, the amino acid sequence is SEQ ID NO: 26.

In one example, the amino acid sequence is SEQ ID NO: 27.

In a fourth aspect: the ligand of aspect 3, wherein the human genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37; or a nucleotide sequence encoding PCSK9 in the alternative method.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or SEQ ID NO: 29-37. It is selected from the group consisting of 32, 34, 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In a fifth aspect: a human has been genotyped or genotyped positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence. The ligand of any of the aforementioned embodiments.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or SEQ ID NO: 29-37. It is selected from the group consisting of 32, 34, 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In a sixth aspect: humans are (i) selected from the alternative PCSK9 or (ii) groups consisting of f, c, r, p, m, e, h, aj and q types, or human PCSK9. A ligand of any of the aforementioned embodiments that has been genotyped or genotyped at least for its catalytic or C-terminal domain.

In one example, the form is mature.

In one example, the form is prodromal.

In a seventh aspect: the method is positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence; or the nucleotide sequence encoding PCSK9 of the alternative method. A ligand of any of the aforementioned embodiments, comprising the step of genotyping a human, if any.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or SEQ ID NO: 29-37. It is selected from the group consisting of 32, 34, 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In an eighth aspect: the method is (i) PCSK9 of the alternative, or (ii) human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q. Or a ligand of any of the aforementioned embodiments comprising the step of genotyping a human that is at least catalytic or positive for the C-terminal domain thereof.

In one example, the form is mature.

In one example, the form is prodromal.

In a ninth aspect: humans are heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence; or a nucleotide sequence encoding PCSK9 in the alternative method. Genotyped or genotyped; where, optionally, humans consist of the nucleotide sequence of SEQ ID NO: 28, or at least its catalytic domain or C-terminal domain coding sequence, and SEQ ID NOs: 29-37. Any of the aforementioned genotyped or genotyped containing a nucleotide sequence selected from the group or at least its catalytic domain or C-terminal domain coding sequence; or a nucleotide sequence encoding PCSK9 of the alternative method. A ligand of the embodiment.

"Heterozygous" is defined herein as a nucleotide sequence in which one allele of a human genotype is selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence. A, a'type or containing any PCSK9 (eg, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence). It means that it may be an allele).

In one example, this method (prior to administration of a ligand) makes a human heterozygous for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence. The step of genotyping and optionally also the nucleotide selected from the group consisting of the nucleotide sequence of SEQ ID NO: 28, or at least its catalytic domain or C-terminal domain coding sequence, and SEQ ID NOs: 29-37. Includes the step of genotyping a sequence or at least its catalytic domain or C-terminal domain coding sequence.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or 29-32, 34, Selected from the group consisting of 35 and 37.

These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In a tenth aspect: a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, or at least its catalytic or C-terminal domain, which is the ligand of any one of aspects 1-9. Encoding sequence; or a ligand that is homozygous or genotyped for the nucleotide sequence encoding PCSK9 of the alternative method.

"Homozygous" is defined herein as in human genotype, where each allele is the same nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, or at least its catalytic or C-terminal domain. It means that it contains a code array.

In one example, the method is homozygous for the nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or at least its catalytic domain or C-terminal domain coding sequence; or the nucleotide sequence encoding PCSK9 of the alternative method. Includes the steps of zygosity and genotyping of humans.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or SEQ ID NOs: 29-32. Selected from the group consisting of 34, 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In an eleventh aspect: (i) binding to PCSK9 of the alternative method, or (ii) human PCSK9 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27, and optionally such binding. A ligand of any of the aforementioned embodiments, comprising a determined antibody binding site determined or determined to be possible.

In one example, the method comprises determining that the ligand is capable of binding to said human PCSK9 (prior to administration of the ligand).

In one example, the bond is a specific bond. In one example, the ligand binds (or is determined to bind) to PCSK9 with an affinity (Kd) of 1 mM, 100 nM, 10 nM or 1 nM or less. In certain embodiments, this affinity is less than 10, 100 or 1000 fM.

In some examples, binding or affinity is determined by SPR or ELISA.

In some examples, the disease or condition is mediated by human PCSK9, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-27.

In some examples, it is selected from the group consisting of SEQ ID NOs: 4-23, 26 and 27, or selected from the group consisting of SEQ ID NOs: 4-14 and 18-27, or SEQ ID NOs: 4-14, 18- Amino acid sequence selected from the group consisting of 23, 26 and 27. These are naturally occurring sequences that do not contain 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the amino acid sequence is SEQ ID NO: 18, 19 or 20 containing 425S, which is associated with elevated LDL-C (Pisciotta et al 2006).

In one example, an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 26 and 27, including 670G, which is a marker of the severity of coronary atherosclerosis (Chen). 2005).

In some examples, an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-14 and 18-27; or selected from the group consisting of SEQ ID NOs: 10-14, 18-23, 26 and 27. These are sequences that have a naturally occurring combination of differences from SEQ ID NOs: 1-3 (type a) and meet the criteria set above.

In one example, the amino acid sequence is SEQ ID NO: 4.

In one example, the amino acid sequence is SEQ ID NO: 5.

In one example, the amino acid sequence is SEQ ID NO: 6.

In one example, the amino acid sequence is SEQ ID NO: 7.

In one example, the amino acid sequence is SEQ ID NO: 8.

In one example, the amino acid sequence is SEQ ID NO: 9.

In one example, the amino acid sequence is SEQ ID NO: 10.

In one example, the amino acid sequence is SEQ ID NO: 11.

In one example, the amino acid sequence is SEQ ID NO: 12.

In one example, the amino acid sequence is SEQ ID NO: 13.

In one example, the amino acid sequence is SEQ ID NO: 14.

In one example, the amino acid sequence is SEQ ID NO: 15.

In one example, the amino acid sequence is SEQ ID NO: 16.

In one example, the amino acid sequence is SEQ ID NO: 17.

In one example, the amino acid sequence is SEQ ID NO: 18.

In one example, the amino acid sequence is SEQ ID NO: 19.

In one example, the amino acid sequence is SEQ ID NO: 20.

In one example, the amino acid sequence is SEQ ID NO: 21.

In one example, the amino acid sequence is SEQ ID NO: 22.

In one example, the amino acid sequence is SEQ ID NO: 23.

In one example, the amino acid sequence is SEQ ID NO: 24.

In one example, the amino acid sequence is SEQ ID NO: 25.

In one example, the amino acid sequence is SEQ ID NO: 26.

In one example, the amino acid sequence is SEQ ID NO: 27.

In a twelfth aspect: the ligand of aspect 11, which is an antibody or antibody fragment. For example, an antibody or antibody fragment is a PCSK9 antagonist, eg, neutralizes PCSK9.

Examples of such antibodies include, for example, WO2008 / 057457, WO2008 / 057458, WO2008 / 057459, WO2008 / 063382, WO2008 / 133647, WO2009 / 100297, WO2009 / 100318, WO2011 / 037791, WO2011 / 053759, WO2011 / 053783, WO2008 / 125623, WO2011 / 072263, WO2009 / 055783, WO2010 / 029513, WO2011 / 111007, WO2010 / 077854, the disclosure of which and the sequences of such antibodies are described herein in their entirety by reference for use in the present invention. It is used in the book. One specific example is AMG 145 (Amgen), LY3015014 (Eli Lilly) or alirocumab. Advantageously, this ligand is alirocumab or comprises alirocumab. Alternatively, this ligand is evolocumab or comprises evolocumab.

In some examples, the ligand is SAR236553 / REGN727 (Sanofi Aventis / Regeneron) or a PCSK9-binding derivative thereof.

In some examples, the ligand comprises or consists of a neutralizing antibody that binds to PCSK9, where the antibody binds to PCSK9, reducing the probability that PCSK9 will bind to the LDLR.

A ligand of embodiment 11, which is a PCSK9 antagonist and, for example, neutralizes PCSK9.

In an example of any aspect of the invention, the ligands are evolocumab, lD05-IgG2 (Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat), LY3015014 (Eli Lilly) and alirocumab ( Includes or consists of ligands selected from SAR236553 / REGN727; Sanofi Aventis / Regeneron).

In a thirteenth aspect: a ligand sequence of any one of aspects 1-10, (i) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, or at least its catalytic domain or C-terminal domain coding sequence. Of a contiguous nucleotide that specifically hybridizes to (or hybridizes to the nucleotide sequence encoding PCSK9 of the alternative) or specifically to the antisense sequence or RNA transcript of the sequence. The sequence of contiguous nucleotides containing the sequence hybridizes to at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, or hybridizes to its antisense sequence or RNA transcript, respectively. Do soybeans; and / or (ii) contain at least 10 consecutive nucleotide sequences of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 (or a nucleotide sequence encoding PCSK9 of the alternative method). Or a ligand that is an antisense sequence or RNA version of the contiguous nucleotide, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28.

In some examples, the nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or SEQ ID NO: 29-37. It is selected from the group consisting of 32, 34, 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37 encoding 670G, a marker of the severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, this nucleotide sequence is SEQ ID NO: 29.

In one example, this nucleotide sequence is SEQ ID NO: 30.

In one example, this nucleotide sequence is SEQ ID NO: 31.

In one example, this nucleotide sequence is SEQ ID NO: 32.

In one example, this nucleotide sequence is SEQ ID NO: 33.

In one example, this nucleotide sequence is SEQ ID NO: 34.

In one example, this nucleotide sequence is SEQ ID NO: 35.

In one example, this nucleotide sequence is SEQ ID NO: 36.

In one example, this nucleotide sequence is SEQ ID NO: 37.

In certain embodiments, the ligand comprises at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or 100 contiguous nucleotides of said nucleotide sequence.

In a fourteenth aspect: the disease or condition is hyperlipidemia, hypercholesterolemia (eg, familial hypercholesterolemia), heart attack, stroke, coronary heart disease, atherosclerosis or cardiovascular disease. A ligand of any of the aforementioned embodiments, which is a disease or condition.

The disease or condition is hypercholesterolemia, hyperlipidemia, hypercholesterolemia, dyslipidemia, bile-stagnation liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or cardiovascular disease. A ligand of any of the aforementioned embodiments.

In some examples, the disease or condition is hypercholesterolemia. The term "hypercholesterolemia" as used herein refers to a condition in which cholesterol levels rise above desired levels. In some embodiments, this means elevated serum cholesterol levels. In some embodiments, the desired level considers various "risk factors" known to those of skill in the art (and described or cited in US Patent Application Publication No. 20120093818).

In humans, familial hypercholesterolemia, statin intolerance, statin uncontrolled, or hypercholesterolemia, dyslipidemia, biliary stagnation liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis or A ligand of any of the aforementioned embodiments identified as heterozygous for the risk of developing cardiovascular disease.

In a fifteenth aspect: the ligand of any of the aforementioned embodiments, wherein the disease or condition is associated with an increase in LDL cholesterol.

Cholesterol levels are measured in milligrams (mg) of cholesterol per deciliter of blood in the United States and some other countries. In Canada and most European countries, the millimolar of cholesterol per liter (L) of blood is measured. Below are the ideal and elevated ranges of the general guidelines.

Therefore, the increase in LDL cholesterol is 160 mg / dL or more (4.1 mmol / L or more).

In a sixteenth aspect: the ligand of any of the aforementioned embodiments, wherein the ligand inhibits human PCSK9 binding to the human LDL receptor and is optionally determined or determined to be capable of such inhibition inhibition. ..

In one example, the method involves determining (before administering the ligand) that the ligand is capable of such inhibition.

The determination of inhibition is, for example, in a blood or serum sample, at rtp (room temperature), ph7, 37 ° C., and / or inhibition under physiological conditions of the human body.

In a seventeenth aspect: a ligand of any of the aforementioned embodiments, wherein the human is resistant or substantially resistant to treatment with a statin (eg, abolstatin and / or fluvastatin) for the disease or condition.

In the eighteenth aspect: a ligand of any of the aforementioned aspects,
(i) Are their genomes containing SEQ ID NO: 29, where this human is of ASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU lineage; or
(ii) Are their genomes containing SEQ ID NO: 30, where this human is of ASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU lineage; or
(iii) Are their genomes containing SEQ ID NO: 32, where this human is of ASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU lineage; or
(iv) Are their genomes containing SEQ ID NO: 33, where this human is of LWK, ASW, YRI or CLM lineage; or
(v) Are their genomes containing SEQ ID NO: 34, where this human is of LWK, ASW or YRI lineage; or
(vi) Are their genomes containing SEQ ID NO: 35, where this human is of PUR, TSI, FIN or CEU lineage; or
(vii) Are their genomes containing SEQ ID NO: 36, where this human is of LWK, ASW or YRI lineage; or
(viii) Treat and / or prevent PCSK9-mediated diseases or conditions in humans whose genomes contain SEQ ID NO: 37, where the human is of CHS, ASW, JPT, PUR or CHB lineage. Because of the ligand.

In the nineteenth aspect: a ligand of any of the aforementioned aspects,
(i) The f-type of PCSK9 is expressed, and here this human is
Are you of ASW, YRI, GBR, TSI, CLM, LWK, MXL, JPT, PUR, IBS, FIN or CEU pedigree; or
(ii) The c-type of PCSK9 is expressed, and here this human is
Are you of ASW, YRI, GBR, TSI, CLM, CHB, LWK, CHS, JPT, PUR, FIN or CEU pedigree; or
(iii) Is PCSK9 p-type expressed, where the human is of ASW, GBR, TSI, CLM, JPT, PUR, IBS, FIN or CEU lineage; or
(iv) Is the m-type of PCSK9 expressed, where this human is of LWK, ASW, YRI or CLM lineage; or
(v) Is the e-type of PCSK9 expressed and where this human is of LWK, ASW or YRI lineage; or
(vi) Is PCSK9 expressing h-type, where the human is of PUR, TSI, FIN or CEU lineage; or
(vii) Is PCSK9 expressing aj form, where this human is of LWK, ASW or YRI lineage; or
(viii) to treat and / or prevent PCSK9-mediated diseases or conditions in humans that express the q-type of PCSK9, where the human is of CHS, ASW, JPT, PUR or CHB lineage. Ligand.

In one example, the form is mature.

In one example, the form is prodromal.

In a twentieth aspect: a pharmaceutical composition or kit for treating and / or preventing a PCSK9-mediated condition or disease (eg, as listed in aspects 14 or 15), any of the aforementioned embodiments. Statins and optionally statins (eg, cerovastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fulvastatin, lovastatin, or pravastatin); In combination with a label or instructions for (eg, covering human procedures as listed in aspects 18 or 19); optionally, this label or instructions is a manufacturing authorization number (eg, FDA or EMA). Includes (approval number); optionally, this label or instructions include instructions for administering alirocumab or evolocumab to said human; optionally, this kit includes ligands (and eg, also statins). A composition or kit comprising an IV or injection device that also contains).

In the 21st aspect: a method of generating an anti-human PCSK9 antibody binding site, wherein a plurality of anti-PCSK9 antibody binding sites are obtained, and the antibody binding sites are (i) PCSK9 of the alternative method, or (ii). ) Human PCSK9 selected from the group consisting of f, c, r, p, m, e, h, aj and q types, or its catalytic or C-terminal domain or peptide (corresponding to SEQ ID NOs: 1, 2 or 3). A step of screening for binding to (including sequence-derived amino acid variations), a step of isolating the antibody binding site to bind in this screening step, and optionally f, c, r, p, m, e, h, A method comprising the step of producing a PCSK9 binding fragment of type aj or q or a derivative of an isolated antibody thereof.

In one example, the form is mature.

In one example, the form is prodromal.

In this aspect and the example of the next aspect, the plurality of binding sites comprises or consist of a plurality of 4-chain antibodies or fragments thereof, such as dAb, Fab or scFv. Suitable methods for generating multiple binding sites for screening include phage display (creating a phage display library for antibody binding sites), ribosome display (producing a ribosome display library for antibody binding sites), yeast. Display (producing a yeast display library of antibody binding sites), or immunity of non-human vertebrates (eg, rodents, eg mice or rats), eg Velocimouse ™, Kymouse ™, Xenomouse ( Using an epitope of PCSK9 with Aliva Mouse ™, HuMab Mouse ™, Omnimouse ™, Omnirat ™ or MeMo Mouse ™, and a repertoire of antibody-producing cells (eg, B cells, The repertoire of plasma cells or plasmablasts) and / or the repertoire of isolated antibodies can be mentioned.

In one example, the method involves selecting one or more antibody binding sites, each of which specifically binds to a human PCSK9 epitope containing an amino acid variation from the corresponding sequence of SEQ ID NO: 1, 2 or 3. Including.

In the 22nd aspect: a method of producing an anti-human PCSK9 antibody, wherein a non-human vertebrate (eg, mouse or rat) is (i) PCSK9 of the alternative, or (ii) f, c, r,. An amino acid sequence selected from the group consisting of p, m, e, h, aj and q type amino acid sequences, or its catalytic or C-terminal domain or peptide (amino acid derived from the corresponding sequence of SEQ ID NO: 1, 2 or 3). Immunization with human PCSK9, including variations) and amino acid sequences selected from the group consisting of f, c, r, p, m, e, h, aj and q types, or their catalytic or C Isolation of an antibody that binds to human PCSK9 containing a terminal domain or peptide (including amino acid variations from the corresponding sequence of SEQ ID NO: 1, 2 or 3) and optionally f, c, r, p, m , E, h, aj or q type PCSK9-binding fragment, or a method comprising the step of producing an isolated antibody derivative.

In one example, the form is mature.

In one example, the form is prodromal.

A 23rd aspect: The method of aspect 21 or 22, comprising obtaining a nucleic acid encoding an antibody, fragment, derivative or binding site, and optionally inserting the nucleic acid into an expression vector.

For example, the method comprises isolating cells containing nucleic acids (eg, B cells, plasmablasts, plasma cells or memory cells), which cells are obtained from non-human vertebrates immunized with the PCSK9 epitope.

In a twenty-fourth aspect: a kit of PCSK9 for genotyping humans, selected from (i) the nucleotide sequence encoding PCSK9 of the alternative method, or (ii) the group consisting of SEQ ID NOs: 29-37. (I) 10 or more (i) specifically hybridize to a nucleotide sequence or at least its catalytic domain or C-terminal domain coding sequence, or specifically to an antisense sequence or RNA transcript of the sequence (i) For example, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) Consistent nucleotide sequences, said sequences of contiguous nucleotides not present in SEQ ID NO: 28. Whether it hybridizes to at least one nucleotide present in the selected sequence, or to its antisense sequence or RNA transcript, and / or (ii) SEQ ID NOs: 29-37 (or (i)). Containing at least 10 (eg, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) nucleotide sequences selected from the group consisting of nucleotide sequences). Alternatively, the kit comprises an antisense sequence or RNA version of the contiguous nucleotide (the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28). ..

In some examples, this nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or 29-32, 34. , 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S and is associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, which encodes 670G, a marker of severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, the nucleotide sequence is SEQ ID NO: 29.

In one example, the nucleotide sequence is SEQ ID NO: 30.

In one example, the nucleotide sequence is SEQ ID NO: 31.

In one example, the nucleotide sequence is SEQ ID NO: 32.

In one example, the nucleotide sequence is SEQ ID NO: 33.

In one example, the nucleotide sequence is SEQ ID NO: 34.

In one example, the nucleotide sequence is SEQ ID NO: 35.

In one example, the nucleotide sequence is SEQ ID NO: 36.

In one example, the nucleotide sequence is SEQ ID NO: 37.

In a 25th aspect: a kit for PCSK9 genotyping or phenotyping of humans by the ligand according to any one of aspects 1-19, or by any one of aspects 21-23. A kit containing the antibodies, fragments or derivatives produced.

In the 26th aspect: (i) anti-binding to PCSK9 of the alternative method or (ii) human PCSK9 selected from the group consisting of f, c, r, p, m, e, h, aj and q types. Use of a PCSK9 ligand, the genome of which comprises the nucleotide sequence encoding PCSK9 of (iii) (i); or (iv) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, optionally. To treat and / or prevent PCSK9-mediated diseases or conditions in humans, comprising nucleotide sequences for treating and / or preventing PCSK9-mediated diseases or conditions in humans as listed in aspects 18 or 19. Use in the manufacture of pharmaceuticals.

In one example, the form is mature.

In one example, the form is prodromal.

In a 27th aspect: (i) anti-binding to PCSK9 of the alternative method or (ii) human PCSK9 selected from the group consisting of f, c, r, p, m, e, h, aj and q types. The use of a PCSK9 ligand, said in humans, to treat and / or prevent a disease or condition mediated by PCSK9, in order to optionally target PCSK9 in humans as listed in aspects 18 or 19. Use in the manufacture of pharmaceuticals to target PCSK9.

In one example, the form is mature.

In one example, the form is prodromal.

In some examples, this nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or 29-32, 34. , 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S and is associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, which encodes 670G, a marker of severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, the nucleotide sequence is SEQ ID NO: 29.

In one example, the nucleotide sequence is SEQ ID NO: 30.

In one example, the nucleotide sequence is SEQ ID NO: 31.

In one example, the nucleotide sequence is SEQ ID NO: 32.

In one example, the nucleotide sequence is SEQ ID NO: 33.

In one example, the nucleotide sequence is SEQ ID NO: 34.

In one example, the nucleotide sequence is SEQ ID NO: 35.

In one example, the nucleotide sequence is SEQ ID NO: 36.

In one example, the nucleotide sequence is SEQ ID NO: 37.

This ligand may be any anti-PCSK9 ligand disclosed herein.

In the 28th aspect: the use of aspect 26 or 27, wherein the ligand, human, disease or condition is any one of aspects 1-19.

In a 29th aspect: a method of targeting PCSK9 to treat and / or prevent a PCSK9-mediated disease or condition in humans, wherein (i) the alternative PCSK9 is encoded in humans. A step of administering an anti-PCSK9 ligand comprising a nucleotide sequence or (ii) a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, thereby targeting PCSK9 encoded by said nucleotide sequence. Method.

This ligand may be any anti-PCSK9 ligand disclosed herein.

In a thirtieth aspect: human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q is targeted with the ligand to determine the disease or condition in said human. 29. The method of aspect 29, comprising the steps of treating and / or preventing.

In one example, the form is mature.

In one example, the form is prodromal.

In a thirty-first aspect: a method of treating and / or preventing a disease or condition mediated by PCSK9 in humans, (i) PCSK9 in the alternative or (ii) f, c, r, p, m, Human PCSK9 selected from the group consisting of types e, h, aj and q is targeted by the step of administering to a human a ligand that binds to the PCSK9, thereby treating and / or preventing the disease or condition in this human. A method that includes the steps to be performed.

In one example, the form is mature.

In one example, the form is prodromal.

The ligand may be any anti-PCSK9 ligand disclosed herein.

In the 32nd aspect: The method of aspect 31, wherein the human genome comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37.

In some examples, this nucleotide sequence is selected from the group consisting of SEQ ID NOs: 29-35 and 37; or from the group consisting of SEQ ID NOs: 29-32 and 34-37; or 29-32, 34. , 35 and 37. These are naturally occurring allelic (haplotype) sequences that do not encode 46L and meet the criteria set above. These groups contain variants associated with elevated LDL-C.

In one example, the nucleotide sequence is SEQ ID NO: 34, which encodes 425S and is associated with elevated LDL-C (Pisciotta et al 2006).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31 and 37, which encodes 670G, a marker of severity of coronary atherosclerosis (Chen et al. 2005).

In one example, a nucleotide sequence selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35, 36 and 37; or selected from the group consisting of SEQ ID NOs: 31, 32, 34, 35 and 37. These are allelic (haplotype) sequences, have a naturally occurring combination of differences from SEQ ID NO: 28 (type a), and meet the criteria set above.

In one example, the nucleotide sequence is SEQ ID NO: 29.

In one example, the nucleotide sequence is SEQ ID NO: 30.

In one example, the nucleotide sequence is SEQ ID NO: 31.

In one example, the nucleotide sequence is SEQ ID NO: 32.

In one example, the nucleotide sequence is SEQ ID NO: 33.

In one example, the nucleotide sequence is SEQ ID NO: 34.

In one example, the nucleotide sequence is SEQ ID NO: 35.

In one example, the nucleotide sequence is SEQ ID NO: 36.

In one example, the nucleotide sequence is SEQ ID NO: 37.

In a 33rd aspect: a human is genotyped or genotyped positive for a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or its catalytic or C-terminal domain coding sequence. , Any one of aspects 29-32.

In a thirty-fourth aspect: humans are positive for (i) PCSK9 of the alternative, or (ii) human PCSK9 selected from the group consisting of f, c, r, p, m, e, h, aj and q types. The method of any one of aspects 29-33, which is genotyped or genotyped as positive.

In one example, the form is mature.

In one example, the form is prodromal.

In the 35th aspect: (i) the nucleotide sequence encoding PCSK9 of the alternative method, or (ii) the nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or its catalytic or C-terminal domain coding sequence positive. The method of any one of aspects 29-34, comprising the step of genotyping the human as being.

In a thirty-sixth aspect: (i) PCSK9 of the alternative method or (ii) a human PCSK9 sequence selected from the group consisting of f, c, r, p, m, e, h, aj and q types. The method of any one of aspects 29-35, comprising the step of phenotyping as positive.

In one example, the form is mature.

In one example, the form is prodromal.

In the 37th aspect: humans are heterozygous for the nucleotide sequence of (i) or the nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37 or its catalytic or C-terminal domain coding sequence. , Or genotyped; optionally, the human being has the nucleotide sequence of SEQ ID NO: 28, or its catalytic or C-terminal domain coding sequence, and the nucleotide sequence or SEQ ID NO: (i). The method of any one of aspects 29-36, genotyping or genotyping a nucleotide sequence selected from the group consisting of 29-37 or a catalytic or C-terminal domain coding sequence thereof.

In a 38th aspect: the human genome is homozygous for the nucleotide sequence of (i), or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, or its catalytic or C-terminal domain coding sequence. The method of any one of aspects 29-37, genotyping or genotyping as homozygous.

In a 39th aspect: a ligand is administered to the human for the nucleotide sequence of (i), or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, or its catalytic or C-terminal domain coding sequence. The method of any one of aspects 29-38, comprising the step of genotyping the human prior to which the ligand is determined to be capable of binding PCSK9 encoded by the selected sequence.

In the 40th aspect: The method of any one of aspects 29-39, wherein the ligand, human, disease or condition is described in any one of aspects 1-19.

In the 41st aspect: The method according to any one of aspects 29-40 for treating and / or preventing the condition or disease listed in aspect 14 or 15, said ligand and statin (eg, cesuvastatin). A method comprising the step of administering lovastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fulvastatin, lovastatin, or pravastatin) to humans.

In aspect 42: The method of aspect 41, wherein the ligand and statin are administered separately.

In the 43rd aspect: The method of aspect 41, wherein the ligand and statin are administered simultaneously.

In the 44th aspect: The method of any one of aspects 29-43, wherein the ligand is administered by subcutaneous injection.

In the 45th aspect: a method for PCSK9 genotyping a human nucleic acid sample, selected from the group consisting of (i) a nucleotide sequence encoding PCSK9 of the alternative method or (ii) SEQ ID NOs: 29-37. A method comprising identifying the presence of a nucleotide sequence or its catalytic or C-terminal domain coding sequence in a sample.

In the 46th aspect: a method of determining PCSK9 type of human protein sample, (i) PCSK9 of the alternative method or (ii) f, c, r, p, m, e, h, aj and q type. A method comprising identifying in a sample the presence of human PCSK9 selected from the group consisting of.

In one example, the form is mature.

In one example, the form is prodromal.

In a 47th aspect: The method of aspect 45 or 46, comprising the step of obtaining a sample of serum, blood, feces, hair, tissue, cells, urine or saliva from a human, thereby obtaining a sample of nucleic acid or protein. The method used in the step of identifying the sequence.

In the 48th aspect: A method comprising any one of aspects 45-47, wherein the specific step is performed using the ligand according to any one of aspects 1-19.

In the 49th aspect: a method of treating and / or preventing a cardiovascular disease or condition, or a disease or condition associated with elevated LDL cholesterol (eg, hypercholesterolemia) in a human patient. The step of typifying a patient using the method of any one of embodiments 45-48 and the steps of embodiments 1-19, wherein the patient is or has previously received statin treatment for the disease or condition. A method comprising the step of administering the ligand according to one, wherein the person is treated or the disease or condition is prevented; optionally the statin treatment is also reduced or stopped.

In some examples, the reduction or discontinuation involves reducing the dose and / or frequency of administration of the statin.

In the 50th aspect: a ligand determined or determined to be capable of binding to an amino acid sequence selected from SEQ ID NOs: 4-27, and instructions for performing any one of aspects 46-49. And / or a diagnostic, therapeutic or prophylactic kit comprising a label or instructions indicating or covering administration of a ligand to a human as defined in any one of aspects 1-19.

In the 51st aspect: a nucleotide sequence, or (i) or a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-37, or a nucleotide sequence that specifically hybridizes to its antisense sequence or RNA transcript. A diagnostic, therapeutic or prophylactic kit comprising a nucleic acid probe comprising, and instructions for performing the method of aspects 45, 47 or 48.

In the examples of the invention, the ligand is human PCSK9, eg, one or more (i) PCSK9 of said alternative or (ii) rare PCSK9 variants disclosed herein (eg, one or two). , 3, or more or all mature types f, c, r, p, m, e, h, aj and q) and optionally also specifically bind to type a and / or type a' To do. For example, the ligand specifically binds to mature f and / or c to type a as well. For example, this ligand specifically binds to human PCSK9 containing the mutant E670G and also to human PCSK9 containing the mutants I474V, Q619P or N425S in SEQ ID NO: 1. For example, this ligand specifically binds to human PCSK9 containing the mutant E670G and also to human PCSK9 containing the mutant I474V in SEQ ID NO: 1. For example, this ligand specifically binds to human PCSK9 containing the mutant E670G and also to human PCSK9 containing the mutant Q619P in SEQ ID NO: 1. For example, this ligand specifically binds to human PCSK9 containing the mutant E670G and also to human PCSK9 containing the mutant N425S in SEQ ID NO: 1. Such binding determinations can be made by any antibody binding test known in the art, for example by surface plasmon resonance. The binding to each of these types is, for example, at least 1 mM, 100 nM, 1 nM, 100 pM, 10 pM or 1 pM Kd, respectively.

In one example, the ligand is selected from type a and (i) PCSK9 of the alternative method or (ii) PCSK9 selected from the group consisting of f, c, r, p, m, e, h, aj and q types. The ligand binding to the selected type is at least 60, 70, 80, 90 or 95% Kd (determined by SPR) for binding to type a. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and type f, and the ligand binding to type f is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to types a and c, and the ligand binding to type c is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and r, and the ligand binding to type a is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and p, and the ligand binding to type p is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and m, and the ligand binding to type m is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to types a and e, and the ligand binding to type a is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and h, and the ligand binding to type a is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and aj, and the ligand binding to type a is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In one example, the ligand binds to type a and q, and the ligand binding to type a is Kd (determined by SPR) of at least 60, 70, 80, 90 or 95% for binding to type a. .. In certain embodiments, both types are mature. In certain embodiments, both types are prodromal.

In the examples of the invention, this ligand is a mature form of human PCSK9, eg, one or more of the rare PCSK9 variants disclosed herein (eg, one, two, three, more or all). F, c, r, p, m, e, h, aj and q) and optionally a and / or a'types are also neutralized. For example, this ligand neutralizes mature f and / or c, as well as type a. Neutralization decisions can be made, for example, by any neutralization assay disclosed in US Patent Application Publication No. 20120093818A1 (Amgen, Inc.) or US Patent Application Publication No. 20110065902A1 (Regeneron Pharmaceuticals, Inc.). The ligands of the invention that bind or target PCSK9 are useful, for example, in the therapeutic or prophylactic applications disclosed in US Patent Application Publication No. 20120093818A1 and US Patent Application Publication No. 20110065902A1 and are detailed therein. The disclosure is incorporated herein by reference for its use in the present invention and for possible inclusion within the claims of this specification.

In embodiments where the ligand is used for therapeutic application, the antigen-binding protein is PCSK9 (eg, one or more of the rare variants disclosed herein and optionally also type a and / or type a'). Can inhibit, interfere with or modulate the biological activity of one or more of the. In one embodiment, the ligand specifically binds to human PCSK9 (eg, rare variants disclosed herein and optionally also one or more of types a and / or a'). And / or binding of human PCSK9 (eg, the rare variants disclosed herein and optionally the one or more of type a and / or type a') to LDLR, at least 20%. For example, 20% -40%, 40-60%, 60-80%, 80-85%, or more (eg, by measuring binding in an in vitro competitive binding assay) substantially inhibits. In one example, this ligand is an antibody.

In certain embodiments, the ligand is a rare variant disclosed herein, and optionally for binding to one, two or more of type a and / or type ^a'10-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 , 10 ^-11 , 10 ^-12 , 10 ^{-13 Have} a Kd of less than M (the bond is even tighter). In one example, Kd is determined using SPR.

In certain embodiments, the ligand blocks the binding of LDLR to one or more of the rare PCSK9 variants (and optionally a and / or also a'types) disclosed herein. It has an IC50 of less than 1 microM, 1000nM to 100nM, 100nM to 10nM, 10nM to 1nM, 1000pM to 500pM, 500pM to 200pM, less than 200pM, 200pM to 150pM, 200pM to 100pM, 100pM to 10pM, 10pM to 1pM.

In certain embodiments, the ligand is against one or more of the rare PCSK9 variants disclosed herein (eg, one or more PCSK9 proteins containing a sequence selected from SEQ ID NOs: 4-27). PCSK9 a that is less than 1000, 100, 90, 80, 70, 60, 50, 40, 30, 20 or tenths (ie, more inhibitory) than an IC50 to block LDLR binding. Has an IC50 for blocking the binding of LDLR to type and / or type a'. Furthermore, or instead, for example, this ligand is (i) less than 1 microM, 1000nM-100nM, 100nM-10nM, 10nM-1nM, 1000pM-100pM, 500pM-200pM, less than 200pM, 200pM-100pM, 200pM-100pM. , 100pM to 10pM, 10pM to 1pM, eg, 1 mM to 1 pM (eg, 1 mM to 100 pM; 10 nM to 100 pM; 1 nM to 10 pM; or 100 pM to 1 pM), a and / or a'type, and (ii) ) Less than 1 microM, 1000nM to 100nM, 100nM to 10nM, 10nM to 1nM, 1000pM to 500pM, 500pM to 200pM, less than 200pM, 200pM to 150pM, 200pM to 100pM, 100pM to 10pM, 10pM to 1pM, for example, 1mM to 1pM Blocks the binding of LDLR to one or more PCSK9 proteins, including sequences selected from SEQ ID NOs: 4-27 in the range (eg, 1 mM-100pM; 10nM-100pM; 1nM-10pM; or 100pM-1pM). Have an IC50 for.

In certain embodiments, the ligand is greater than 10%, greater than 20%, greater than 40%, greater than 50%, for binding to PCSK9 comprising sequences selected from SEQ ID NOs: 4-27. Up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95 It binds to type a and / or type a'PCSK9 with a binding affinity (Kd) greater than% or up to greater than 100% (ie, double). Such binding measurements can be made using various binding assays known in the art, eg, using surface plasmon resonance (SPR) [eg, by Biacore ™], or ProteOn XPR36 ™ [Bio. -Rad®] or KinExA® (Sapidyne Instruments, Inc.) may be used.

In one embodiment, surface plasmon resonance (SPR) is performed at 25 ° C. In another embodiment, the SPR is performed at 37 ° C.

In one embodiment, SPR is performed at physiological pH, eg, about pH 7, or pH 7.6 [eg, using Hepes-buffered saline at pH 7.6 (also referred to as HBS-EP)].

In one embodiment, SPR is performed at a physiological salt concentration, eg, 150 mM NaCl.

In one embodiment, SPR is performed at a detergent level of 0.05% by volume or less, eg, in the presence of 0.05% P20 [polysorbate 20; eg, Tween-20 ™] and 3 mM EDTA.

In one example, SPR is performed at 25 ° C. or 37 ° C. in buffer at pH 7.6 with 150 mM NaCl, 0.05% detergent (eg P20) and 3 mM EDTA. The buffer may contain 10 mM Hepes. In one example, SPR is performed in HBS-EP at 25 ° C or 37 ° C. HBS-EP is available from Teknova Inc (California; Catalog No. H8022).

In one example
1. Coupling anti-mouse (or other related vertebrate) IgG (eg, Biacore BR-1008-38) to a biosensor chip (eg, GLM chip), such as by primary amine coupling;
2. Exposure of anti-mouse IgG (vertebrate antibody) to the test IgG antibody to capture the test antibody on the chip;
3. Passing the test antigen over the capture surface of the chip with 1024nM, 256nM, 64nM, 16nM, 4nM and 0nM (ie, buffer alone);
4. For example, under the SPR conditions discussed above (eg, in a physiological buffer at 25 ° C.), surface plasmon resonance is used to determine the affinity of test antibody binding to the test antigen. Ligand affinity for which antibodies are determined using SPR. SPR may be performed using any standard SPR device, eg, by Biacore ™ or by ProteOn XPR36 ™ [Bio-Rad®].

Regeneration of the capture surface may be performed at pH 1.7 with 10 mM glycine. This removes the captured antibody and allows its surface to be used for another interaction. This combined data may be applied to a unique 1: 1 model using standard techniques, eg, using a model specific to the ProteOn XPR36 ™ analysis software.

In certain embodiments, assays or tests of the ligands of the invention are performed substantially at pH 7 (eg, for in vitro tests and assays) and at room temperature or substantially at room temperature.

An example of an IgG2 heavy chain constant domain of an anti-PCSK9 antibody of the invention is an amino acid sequence as set forth in SEQ ID NO: 154 of Figure 3KK of US Patent Application Publication No. 20120093818A1, whose sequence is incorporated herein by reference. Has.

An example of the IgG4 heavy chain constant domain of the anti-PCSK9 antibody of the invention is an amino acid sequence as set forth in SEQ ID NO: 155 of FIG. 3KK of US Patent Application Publication No. 20120093818A1, whose sequence is incorporated herein by reference. Has.

One example of the kappa light chain constant domain of an anti-PCSK9 antibody has an amino acid sequence as set forth in SEQ ID NO: 157 of FIG. 3KK, the sequence of which is incorporated herein by reference.

One example of a lambda light chain constant domain of an anti-PCSK9 antibody has an amino acid sequence as set forth in SEQ ID NO: 156 of Figure 3KK of US Patent Application Publication No. 20120093818A1, whose sequence is incorporated herein by reference.

In the examples of the invention, the ligand also binds to mature PCSK9, eg, one or more rare variants of the mature form disclosed herein, and optionally a and / or a'types.

In the examples of the invention, the ligand is a catalytic domain of PCSK9, eg, one or more rare variants of the mature form disclosed herein and optionally a and / or a'catalytic. Also bind to the domain.

In the examples of the invention, the ligand is in the prodomain of PCSK9, eg, one or more rare variants of the mature form disclosed herein and optionally a and / or a'type prodomain. Also combine.

In some embodiments, the ligand is the V domain of PCSK9, eg, one or more rare variants of the mature form disclosed herein and optionally a and / or a'type V domains. Also binds to. In some embodiments, the ligand is in the V domain of PCSK9 (eg, one or more rare variants of the mature form disclosed herein and optionally a and / or a'type). Also binds and prevents the binding of PCSK9 to LDLR (or, for example, reduced to at least 10%). In some embodiments, the ligand is in the V domain of PCSK9 (eg, one or more rare variants of the mature form disclosed herein and optionally a and / or a'type). Also binds, on the one hand, it does not prevent (or reduce) the binding of PCSK9 to LDLR, but the ligand prevents or reduces harmful activity mediated through PCSK9 on LDLR (eg,). , At least up to 10%).

In the examples of the invention, the ligand is or comprises a fully human antibody. In some examples, the ligand comprises a human variable region or a humanized variable region.

In one example, the ligands of the invention are specifically selected from the group consisting of (i) PCSK9 of the alternative method or (ii) f, c, r, p, m, e, h, aj and q types. It binds to an epitope of human PCSK9, where this epitope contains at least one amino acid not found in type a. For example, this amino acid is selected from the group consisting of 46L, 53V, 425S, 443T, 474V, 619P and 670G (numbered as used in SEQ ID NO: 1). For example, this amino acid is selected from the group consisting of 425S, 443T, 474V, 619P and 670G (numbered as used in SEQ ID NO: 1). For example, this amino acid is selected from the group consisting of 425S and 443T (numbered as used in SEQ ID NO: 1). For example, this amino acid is selected from the group consisting of 474V, 619P and 670G (numbered as used in SEQ ID NO: 1). In one example, the PCSK9 type is mature. In one example, the type of PCSK9 is a precursor. In some examples, this ligand also specifically binds to type a and / or type a'. In certain embodiments, the ligand specifically binds to an epitope of type f PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of type c PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of type r PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of type p PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of m-type PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of e-type PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of h-type PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of type aj PCSK9 that contains at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to an epitope of type q PCSK9 that contains at least one amino acid not found in type a.

In certain embodiments, the ligand specifically binds to the prodomain of human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q. In some examples, the ligand also specifically binds to a-type and / or a'type prodomains. In certain embodiments, the ligand specifically binds to the prodomain of type f PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of type c PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of r-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of p-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of m-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of e-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of h-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of aj-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the prodomain of type q PCSK9, where the epitope comprises at least one amino acid not found in type a.

In certain embodiments, the ligand specifically binds to the catalytic domain of human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q. In one example, the ligand also specifically binds to a-type and / or a'type catalytic domains. In certain embodiments, the ligand specifically binds to the catalytic domain of type f PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of type c PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of r-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of p-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of m-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of e-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of h-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of aj-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the catalytic domain of type q PCSK9, where the epitope comprises at least one amino acid not found in type a.

In certain embodiments, the ligand specifically binds to the C-terminal domain of human PCSK9 selected from the group consisting of f, c, r, p, m, e, h, aj and q types. In some examples, the ligand also specifically binds to the a-type and / or a'type C-terminal domain. In certain embodiments, the ligand specifically binds to the C-terminal domain of type f PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of type c PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of r-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of p-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of m-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of e-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of h-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of type a PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the C-terminal domain of type q PCSK9, where the epitope comprises at least one amino acid not found in type a.

In certain embodiments, the ligand specifically binds to a substrate-binding groove of human PCSK9 selected from the group consisting of types f, c, r, p, m, e, h, aj and q [all by reference. , Incorporated herein by Cunningham et al., Nat Struct Mol Biol. May 2007; 14 (5): pp. 413-9. Epub April 15, 2007, "Structural and biophysical studies of PCSK9 and its ligands." See linked to familial hypercholesterolemia]. In some examples, the ligand also specifically binds to a-type and / or a'type substrate-binding grooves. In certain embodiments, the ligand specifically binds to the substrate-binding groove of type f PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of type c PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of r-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of p-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of m-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of e-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of h-type PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of type a PCSK9, where the epitope comprises at least one amino acid not found in type a. In certain embodiments, the ligand specifically binds to the substrate-binding groove of type q PCSK9, where the epitope comprises at least one amino acid not found in type a.

The entire disclosure is incorporated herein by reference to U.S. Patent Application Publication No. 20120093818A1 (Amgen, Inc.). The patent application discloses the relevant ligands for use in the present invention, as well as examples and methods of producing and testing ligands that can be used with reference to the present invention.

In one example, the ligand is an antibody disclosed in Table 2 of US Patent Application Publication No. 20120093818A1 (Amgen, Inc.), or comprises the antibody, or is a PCSK9 binding derivative thereof.

In certain embodiments, the PCSK9 binding ligands of the present invention are used in US Patent Application Publication No. 20120093818A1 (Amgen, Inc.), eg, in US Patent Application Publication No. 20120093818A1, [0009]-[0014] and [0058]-. Selected from the antigen-binding proteins disclosed in paragraph [0063]. All of these disclosures (including sequences of such proteins), as well as potential inclusion in one or more claims, or use in the present invention, as expressly stated herein, by reference. , Incorporated herein.

In this paragraph, the SEQ ID NOs are as those found in U.S. Patent Application Publication No. 20120093818A1 (Amgen, Inc.), and these sequences are, by reference, as expressly mentioned herein. Also incorporated herein by reference for potential inclusion in one or more claims, or for use in the present invention. In some embodiments, the ligands of the invention comprise isolated antigen-binding proteins that bind to PCSK9, including: A) one or more heavy chain complementarity determining selected from the group consisting of: Regions (CDRH): (i) SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, CDRH1s from CDRH1 in a sequence selected from the group consisting of 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii) SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, CDRH2s derived from CDRH2 in a sequence selected from the group consisting of 81 and 60; (iii) SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, CDRH3s from CDRH3 in sequences selected from the group consisting of 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. ; And (iv) selected from the group consisting of CDRH; B) or less of (i), (ii), and (iii) containing one or more amino acid substitutions, deletions or insertions of 4 or less amino acids. One or more light chain complementarity determining regions (CDRLs): (i) SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46 CDRL1 derived from CDRL1 in a sequence selected from the group; (ii) ) SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36 CDRL2 derived from CDRL2 in a sequence selected from the group consisting of, 37, 38, 39, 40, 42, 44, and 46; (iii) SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46 CDRL3s derived from CDRL3s in the sequences selected from; and (iv) one or less of 4 or less amino acids One or more of (i), (ii), and (iii) CDRL; or C) A) or multiple heavy chain CDRHs and B) containing multiple amino acid substitutions, deletions or insertions. Light chain CDRL. In some embodiments, the isolated antigen-binding protein comprises at least one CDRH in A) and at least one CDRL in B). In some embodiments, the isolated antigen-binding protein comprises at least two CDRHs in A) and at least two CDRLs in B). In some embodiments, the isolated antigen-binding protein comprises said CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3. In some embodiments, the CDRH of A) is selected from at least one of the groups consisting of: (i) in a sequence selected from the group consisting of SEQ ID NOs: 67, 79, 89, and 49: Amino acid sequence of CDRH1 selected from CDRH1; (ii) Amino acid sequence of CDRH2 selected from CDRH2 in the sequence selected from the group consisting of SEQ ID NOs: 67, 79, 89, and 49; (iii) SEQ ID NO: 67, Amino acid sequence of CDRH3 selected from CDRH3 in a sequence selected from the group consisting of 79, 89, and 49; and (iv) contain one or more amino acid substitutions, deletions or insertions of no more than two amino acids. CDRH of (i), (ii) and (iii). In addition, the CDRL of B) is selected from at least one of the following groups: (i) selected from CDRL1 in the sequence selected from the groups consisting of SEQ ID NOs: 12, 35, 32, and 23: CDRL1 amino acid sequence; (ii) CDRL2 amino acid sequence selected from CDRL2 in a sequence selected from the group consisting of SEQ ID NOs: 12, 35, 32, and 23; (iii) SEQ ID NOs: 12, 35, 32, and 23. CDRL3 amino acid sequence selected from CDRL3 in a sequence selected from the group consisting of; and (iv) containing one or more amino acid substitutions, deletions or insertions of two or less amino acids (i), (ii) ) And (iii) CDRL; or C) one or more heavy chain CDRHs in A), and one or more light chain CDRLs in B. In some embodiments, the CDRH of A) is selected from at least one of the following groups: (i) the CDRH1 amino acid sequence of the amino acid sequence of CDRH1 in SEQ ID NO: 67; (ii) sequence. CDRH2 amino acid sequence of the amino acid sequence of CDRH2 in number 67; (iii) CDRH3 amino acid sequence of the amino acid sequence of CDRH3 in SEQ ID NO: 67; and (iv) one or more amino acid substitutions of two or less amino acids, The CDRL of (i), (ii) and (iii) CDRH; B) containing a deletion or insertion is selected from at least one of the following groups: (i) in SEQ ID NO: 12. CDRL1 amino acid sequence of CDRL1 amino acid sequence; (ii) CDRL2 amino acid sequence of CDRL2 amino acid sequence of SEQ ID NO: 12; (iii) CDRL3 amino acid sequence of amino acid sequence of CDRL3 of SEQ ID NO: 12; One or more heavy chain CDRHs of (i), (ii) and (iii) CDRL; or C) A) containing one or more amino acid substitutions, deletions or insertions, and one or more of B Multiple light chain CDRLs. In some embodiments, the antigen-binding protein is A) CDRH1 of the CDRH1 sequence in SEQ ID NO: 67, CDRH2 of the CDRH2 sequence in SEQ ID NO: 67, and CDRH3 of the CDRH3 sequence in SEQ ID NO: 67, And B) CDRL1 of the CDRL1 sequence in SEQ ID NO: 12, CDRL2 of the CDRL2 sequence in SEQ ID NO: 12, and CDRL3 of the CDRL3 sequence in SEQ ID NO: 12. In some embodiments, the antigen-binding protein comprises SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, Heavy chain variable with at least 80% sequence identity with an amino acid sequence selected from the group consisting of 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. Region (VH) and / or SEQ ID NOs: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, Contains a light chain variable region (VL) having at least 80% sequence identity with an amino acid sequence selected from the group consisting of 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. .. In some embodiments, this VH has SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64. , 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60 having at least 90% sequence identity with and / or this amino acid sequence selected from the group. VL is SEQ ID NO: 5,7,9,10,12,13,15,16,17,18,19,20,21,22,23,24,26,28,30,31,32,33,35 , 36, 37, 38, 39, 40, 42, 44, and 46 have at least 90% sequence identity with the amino acid sequence selected from the group. In some embodiments, this VH has SEQ ID NOs: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64. , 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and / or this VL is SEQ ID NO: 5, 7, 9, 10. , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42 , 44, and 46 are selected.

In an example of any aspect of the invention, the PCSK9-targeting or binding ligand is capable of its disclosure (including sequence) as explicitly stated herein, and in one or more claims. AMG145 or 31H4, 16F12, 11F1, 8A3 or 21B12, disclosed in U.S. Patent Application Publication No. 20120093818A1 (Amgen, Inc.), incorporated herein by reference for certain inclusions or for use in the present invention, or Contains or consists of antibodies containing the variable domains of AMG145, 31H4, 16F12, 11F1, 8A3 or 21B12. Preferably, the PCSK9-targeting or binding ligand comprises or consists of AMG145.

In one example, AMG145 or another ligand of the invention is glycosylated and has, for example, human glycosylation (eg, produced by CHO, Cos or Hek293 cells). In one example, the ligands of the invention are produced in CHO.

US Patent Application Publication No. 20110065902A1 (Regeneron Pharmaceuticals, Inc.), the entire disclosure of which is incorporated herein by reference. The patent application discloses examples and methods of generating and testing relevant ligands for use in the present invention, as well as ligands that can be used with reference to the present invention, to determine medical efficacy.

See the following PCT application, the entire disclosure of which is incorporated herein by reference. These disclose examples and methods of generating and testing relevant ligands for use in the present invention, as well as ligands that can be used with reference to the present invention, to determine medical efficacy.
WO2008057457
WO2008057458
WO2008057459
WO2008063382
WO2008133647
WO2009100297
WO2009100318
WO2011037791
WO2011053759
WO2011053783
WO2008125623
WO2011072263
WO2009055783
WO2010029513
WO2011111007
WO2010077854

Each of the antibody ligands for PCSK9 is incorporated herein by reference in their entirety, eg, WO2008 / 057457, WO2008 / 057458, WO2008 / 057459, WO2008 / 063382, WO2008 / 125623, and US Patent Application Publication No. 2008. / 068697.

In some examples, the ligand is an antibody disclosed in Examples of US Patent Application Publication No. 20110065902A1 or contains it (eg, 316P or 300N), or a PCSK9-binding derivative thereof. All of these disclosures, including sequences of such proteins and corresponding nucleotide sequences, are as expressly stated herein, and with respect to possible inclusions in one or more claims, or the present. Incorporated herein by reference for use in the invention. In certain embodiments, the ligand is a variable domain of antibody 316P or 300N disclosed in US Patent Application Publication No. 20110065902A1, or comprises it, or is such an antibody or a PCSK9 binding derivative thereof ( Or include it).

In certain embodiments, the ligand is the variable domain of the antibody alirocumab or SAR236553 / REGN727 (Sanofi Aventis / Regeneron), or contains it, or is such an antibody or a PCSK9 binding derivative thereof ( Or include it). In one example, alirocumab is glycosylated, eg, has human glycosylation (eg, produced by CHO, Cos or Hek293 cells). Preferably, the ligand is alirocumab or SAR236553 / REGN727.

In certain embodiments, the ligand is, or comprises, the variable domain of the antibody evolocumab, or is (or includes) such an antibody or a PCSK9-binding derivative thereof. In some examples, the antibody is glycosylated and has, for example, human glycosylation (eg, produced by CHO, Cos or Hek293 cells). Preferably, the ligand is evolocumab.

In certain embodiments, the ligand is selected from evolocumab, 1D05-IgG2 (Merck & Co.), ALN-PCS02 (Alnylam), RN316 (Pfizer-Rinat) and alirocumab.

In certain embodiments, the ligand is selected from the following (as per US Patent Application Publication No. 2011/0065902, the sequences and definitions incorporated herein by reference):-
1. An antibody or antigen-binding fragment thereof that specifically binds to hPCSK9, wherein the antibody or antigen-binding fragment is the heavy chain and light chain of the amino acid sequence pair of HCVR / LCVR having SEQ ID NO: 218/226. Includes CDR.
2. An antibody or antigen-binding fragment of Concept 1 containing the CDR amino acid sequences of heavy and light chains having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.
3. An antibody or antigen-binding fragment of Concept 2, comprising an HCVR having the amino acid sequence of SEQ ID NO: 218 and an LCVR having the amino acid sequence of SEQ ID NO: 226.
4. An antibody or antigen-binding fragment thereof that binds to the same epitope on hPCSK9 with an antibody comprising the heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.
5. An antibody or antigen-binding fragment thereof that competes for binding to hPCSK9 with an antibody comprising the heavy and light chain CDR amino acid sequences having SEQ ID NOs: 220, 222, 224, 228, 230 and 232.

In certain embodiments, the ligand is selected from the following (as per US Patent Application Publication No. 2012/0093818, the sequences and definitions incorporated herein by reference):-
1. An isolated neutralizing antigen-binding protein that binds to the PCSK9 protein containing the amino acid sequence of SEQ ID NO: 1, where this neutralizing antigen-binding protein exerts the LDLR-lowering effect of PCSK9 on LDLR. Lower, where the antigen-binding protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46, and where the antigen-binding protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 60.
2. An isolated neutralizing antigen-binding protein of Concept 2, wherein the antigen-binding protein is an LDLR non-competitive neutralizing antigen-binding protein.
3. An isolated neutralizing antigen-binding protein of Concept 2, wherein the antigen-binding protein is an LDLR competitive neutralizing antigen-binding protein.
An antigen-binding protein that selectively binds to PCSK9, which binds to PCSK9 at a Kd of less than 100 pM.
5. An antigen-binding protein that binds to the PCSK9 protein of SEQ ID NO: 303 in the first method, where this antigen-binding protein binds to the PCSK9 variant in the second method, where The PCSK9 variant is 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337 of SEQ ID NO: 303. Has at least one point mutation at a position selected from the group consisting of, 519, 521, and 554, where this first method is the first EC50, the first Bmax, or the first. Includes EC50 and 1st Bmax, where this second method comprises a second EC50, 2nd Bmax, or 2nd EC50 and 2nd Bmax, and here of this 1st method. The values differ from those for the second method, where the antigen-binding protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46, and where the antigen-binding protein is SEQ ID NO: An antigen-binding protein containing a heavy chain containing an amino acid sequence of 60.
6. The first method contains a first Bmax, where this second method contains a second Bmax different from the first Bmax, where the PCSK9 variants are: D162R, R164E, An antigen-binding protein of concept 6 having at least one point mutation selected from the group consisting of E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R.
7. An antigen-binding protein of concept 6 that binds to PCSK9 at a position that overlaps with the position where LDLR binds to PCSK9.
8. A method for producing an antigen-binding protein that binds to a PCSK9 protein containing the amino acid sequence of SEQ ID NO: 1, where this antigen-binding protein reduces the LDLR-lowering effect of PCSK9 on LDLR and is antigen-binding. A step of providing a host cell containing a nucleic acid sequence encoding a protein; a step of maintaining the host cell under conditions in which the antigen-binding protein is expressed, wherein the antigen-binding protein is referred to as SEQ ID NO: 46. A method comprising a light chain comprising the amino acid sequence of, wherein the antigen-binding protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 60.
9. A method for treating or preventing a condition associated with elevated serum cholesterol levels in a subject, with an effective amount of isolated neutralizing antigen-binding protein for the subject in need of it. , Which comprises the step of administering simultaneously or sequentially with a drug that increases the availability of the LDLR protein, wherein the isolated antigen-binding protein binds to the PCSK9 protein, which comprises the amino acid sequence of SEQ ID NO: 1. This neutralizing antigen-binding protein reduces the LDLR-lowering effect of PCSK9 on LDLR, where the antigen-binding protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46, and where this antigen-binding property is present. A method in which the protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 60.
10. A method of concept 10 in which a drug that increases the availability of the LDLR protein contains a statin.
11. An antigen-binding protein that binds to PCSK9, where if this antigen-binding protein binds to PCSK9, the antibody is the following residues PCSK9: S153, S188, I189, Q190, S191, D192. , R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154, T187, H193, E195, I196, M201, V202, C223, T228, S235, G236 , A239, G244, M247, I369, S372, C375, or C378, located less than or equal to 8 angstroms, wherein the antigen-binding protein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 46 and. Here, this antigen-binding protein is an antigen-binding protein containing a heavy chain containing the amino acid sequence of SEQ ID NO: 60.

The ligand may be used in the treatment, treatment, prevention and / or diagnosis of one or more diseases or conditions, or susceptibility to them, wherein such diseases or conditions are described in U.S. Patent Application Publication No. 20120093818A1. What is disclosed in No. (Amgen, Inc.) and U.S. Patent Application Publication No. 20110065902A1 (Regeneron Pharmaceuticals, Inc.), eg, the disclosure thereof for inclusion in one or more claims herein. Includes the diseases or conditions disclosed in paragraphs [0375]-[0383] of U.S. Patent Application Publication No. 20120093818A1, which are incorporated herein by reference in their entirety.

The ligand can be administered to a human characterized as described in US Patent Application Publication No. 20120093818A1 (Amgen, Inc.) or US Patent Application Publication No. 20110065902A1.

The ligand may be administered in the form or combination disclosed in US Patent Application Publication No. 20120093818A1 (Amgen, Inc.) or US Patent Application Publication No. 20110065902A1, the disclosure of which is incorporated herein by reference. For example, as described in US Patent Application Publication No. 20120093818A1 (Amgen, Inc.) or US Patent Application Publication No. 20110065902A1 of which the disclosure is incorporated herein by reference {eg, US Patent Application Publication No. As disclosed in paragraphs [0384]-[0412] of 20120093818A1} a ligand with a drug, excipient, diluent or carrier, and the present invention is also a combination of the ligand of the present invention and such additional agents. Also related to the corresponding pharmaceutical composition comprising.

The ligand is found in U.S. Patent Application Publication No. 20120093818A1 (Amgen, Inc.) or U.S. Patent Application Publication No. 20110065902A1, for example, paragraphs of U.S. Patent Application Publication No. 20120093818A1 whose disclosure is incorporated herein by reference. It can be used in the diagnostic method set in [0413]-[0415].

Diagnostic Application In some embodiments, the ligands of the invention are diagnostic tools. This ligand may be used to assay the amount of PCSK9 present in the sample and / or subject. As will be appreciated by those skilled in the art, such ligands need not be neutralizing ligands. In some embodiments, this diagnostic ligand is not a neutralizing ligand. In some embodiments, the diagnostic ligand binds to a different epitope than the neutralizing ligand binds. In some embodiments, the two ligands do not compete with each other.

In some embodiments, the ligands of the invention are assay kits and / for the detection of PCSK9 in mammalian tissues or cells to screen / diagnose diseases or disorders associated with altered levels of PCSK9. Or used or provided in a method. The kit includes a ligand that binds to PCSK9, and a means for binding the ligand to PCSK9 (if present) and optionally indicating PCSK9 protein levels. Various means may be used to indicate the presence of the ligand. For example, a fluorophore, other molecular probe, or enzyme may be linked to the ligand, and the presence of the ligand may be observed in various ways. Methods for screening for such disorders may be involved in the use of the kit, or simply the use of one of the disclosed ligands, and in determining whether the ligand binds to PCSK9 in the sample. As will be appreciated by those skilled in the art, high or elevated levels of PCSK9 result in large amounts of ligand binding to PCSK9 in the sample. Therefore, the degree of ligand binding can be used to determine how much PCSK9 is in the sample. A subject or sample with an amount of PCSK9 in excess of a predetermined amount (eg, an amount or range possessed by a person without a PCSK9-related disorder) can be characterized as having a PCSK9-mediated disorder. In some embodiments, the invention provides a method in which a ligand is administered to a subject taking a statin to determine if the statin has increased the amount of PCSK9 in the subject.

In some embodiments, the ligand is a non-neutralizing ligand and is used to determine the amount of PCSK9 in a subject undergoing ABP and / or statin treatment.

In some embodiments, the ligands of the invention can specifically bind to human PCSK9 (eg, one, two or more rare variants disclosed herein), including: Characterized by at least one of: (i) serum total cholesterol can be reduced by at least about 25-35% compared to before administration, and the reduction can be sustained for at least 24 days; (ii) serum LDL cholesterol It can be reduced by at least about 65-80% relative to pre-dose levels and can be sustained for at least 24 days; (iii) serum LDL cholesterol is reduced by at least about 40-70% relative to pre-dose levels and at least The reduction can be sustained for 60 or 90 days; (iv) serum triglyceride can be reduced by at least about 25-40% relative to pre-dose levels; (v) serum HDL cholesterol is not lowered relative to pre-dose levels or Serum HDL cholesterol reduction is less than 5%. In some embodiments, an isolated nucleic acid molecule is provided, which encodes a ligand. In some embodiments, an expression vector is provided, which comprises a nucleic acid molecule. In some embodiments, a pharmaceutical composition is provided, which may include a ligand and a pharmaceutically acceptable carrier. In some embodiments, the PCSK9 antagonist ligands of the present invention are used to provide methods for treating a disease or condition that is alleviated, ameliorated, inhibited or prevented. The method may include administering a therapeutic amount of the pharmaceutical composition or ligand to a subject in need thereof. In some embodiments, the subject is hypercholesterolemia, hyperlipidemia, LDL aferesis indication, identification of being heterozygous for familial hypercholesterolemia, statin intolerance, statin unregulation, It is a human subject suffering from the risk of developing hypercholesterolemia, dyslipidemia, cholestatic liver disease, nephrotic syndrome, hypothyroidism, obesity, atherosclerosis and cardiovascular disease. In some embodiments, a method of providing treatment or treatment is provided to the subject. In some embodiments, the method comprises reducing serum cholesterol by at least 40-70% over a period of at least 60-90 days. In some embodiments, a method of receiving treatment or treatment is provided, which method may include the step of administering the ligand at a frequency of once every 60-90 days.

In one aspect, the invention is pre-dose, as determined by ALT and AST measurements, with little or no reduction in serum HDL cholesterol and / or with little or unmeasurable effect on liver function. Human precursor protein convertase subtilisin / kexin type 9 (hPCSK9, eg, herein) characterized by the ability to lower human serum LDL cholesterol by 40-80% over 24, 60 or 90 days relative to levels. Whether it is a human antibody or an antigen-binding fragment of a human antibody that specifically binds to and inhibits one, two or more of the disclosed rare variant types and optionally a and / or a'type). , Or the ligand of the present invention containing the same.

In one embodiment, the ligand of the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds to hPCSK9 and is characterized by at least one of the following:
(i) Serum total cholesterol is reduced by at least about 25-35% relative to pre-dose levels and can be sustained for at least 24 days, preferably serum total cholesterol reduction is at least about 30-40%. ;
(ii) Serum LDL cholesterol is reduced by at least about 65-80% relative to pre-dose levels and can be sustained for at least 24 days;
(iii) Serum triglycerides can be reduced by at least about 25-40% relative to pre-dose levels;
(iv) Does not lower serum HDL cholesterol or is less than 5% lower in serum HDL cholesterol relative to pre-dose levels.

For definitions of these terms and any features, see US Patent Application Publication No. 2011/0065902, the disclosure of which is incorporated herein by reference in its entirety.

In one embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds to hPCSK9 and is characterized by at least one of the following:
(i) Serum LDL cholesterol is reduced by at least about 40-70% relative to pre-dose levels and can be sustained for at least 60 or 90 days;
(ii) Serum triglycerides can be reduced by at least about 25-40% relative to pre-dose levels;
(iii) Does not lower serum HDL cholesterol or lowers serum HDL cholesterol by 5% or less relative to pre-dose levels.

In one embodiment, the antibody or antigen binding fragment is characterized as exhibiting an increase in binding affinity (KD) for hPCSK9 at pH 5.5 compared to KD at pH 7.4, as measured by plasmon surface resonance. .. In certain embodiments, the antibody or fragment has at least a 20-fold, at least 40-fold, or at least 50-fold affinity for PCSK9 at an acidic pH relative to a neutral pH, as measured by plasmon surface resonance. Show improvement.

In one embodiment, the antibody or antigen-binding fragment is characterized as showing no improvement in binding affinity for PCSK9 at acidic pH relative to neutral pH as measured by surface plasmon resonance. In certain embodiments, the antibody or fragment exhibits reduced binding affinity at acidic pH.

In another embodiment, the antibody or antigen binding fragment binds to human, human GOF mutant D374Y, cynomolgus monkey, rhesus monkey, mouse, rat and hamster PCSK9.

In one embodiment, the antibody or antigen binding fragment binds to human and monkey PCSK9, but not to mouse, rat or hamster PCSK9.

In one embodiment, the invention is a heavy chain variable region disclosed in any of paragraphs [023]-[037] of U.S. Patent Application Publication No. 2011/0065902, the disclosure of which is incorporated herein by reference. Includes an antibody or antigen-binding fragment of an antibody comprising (HCVR), light chain variable region (LCVR), HCDR1, HCDR2, HCDR3.

In a related embodiment, the invention comprises an antibody or antigen-binding fragment of an antibody that specifically binds to hPCSK9, wherein the antibody or fragment is sequence number (US Patent Application Publication No. 2011/0065902). (Use numbering): 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190 / 192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386 / 394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594 / 596, 598/600, 602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, Heavy and light chain contained within a heavy and light chain sequence pair selected from the group consisting of 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744. Contains the light chain CDR domain. In one embodiment, the CDR sequences are SEQ ID NOs: 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 122/130, 138/140, 142/144, 218/ 226, 234/236, 238/240, 242/250, 258/260, 262/264, 314/3
It is contained in HCVR and LCVR selected from the amino acid sequence pairs of 22, 330/332 and 334/336. In a more specific embodiment, this CDR sequence is contained within the HCVR / LCVR sequence selected from SEQ ID NO: 90/92 or 218/226.

In one example, the invention comprises a ligand of the invention, wherein the ligand comprises or consists of a recombinant human antibody or fragment thereof that specifically binds to hPCSK9 and a pharmaceutically acceptable carrier. It is characterized by a pharmaceutical composition containing. In one embodiment, the invention features a composition that is a combination of a ligand of the invention (eg, an antibody or an antigen-binding fragment of an antibody) and a second therapeutic agent. The second therapeutic agent is cholesterol synthesis by inhibiting any agent that is advantageously combined with the ligands of the invention, such as 3-hydroxy-3-methylglutaryl (HMG) -coenzyme A (CoA) reductase. Drugs that can induce cellular depletion of, such as cellobastatin, atorvastatin, simvastatin, pitabastatin, rosuvastatin, fulvastatin, lovastatin, pravastatin; drugs that can inhibit cholesterol uptake or bile acid reabsorption; lipoproteins Agents capable of increasing catabolism (eg, niacin); and / or activators of LXR transcription factors responsible for cholesterol excretion, such as 22-hydroxycholesterol.

In one example, the invention provides a method for inhibiting hPCSK9 activity using an anti-PCSK9 ligand of the invention (eg, an antibody of the invention or an antigen-binding portion of an antibody), the therapeutic method of which is the invention. Includes the step of administering a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antigen-binding fragment of the antibody. The disorder being treated is any disease or condition that is ameliorated, alleviated, inhibited or prevented by the removal, inhibition or reduction of PCSK9 activity. Specific populations that can be treated by the therapeutic methods of the present invention include subjects to which LDL aferesis is indicated, PCSK9-activated mutations (functional mutations, acquisition of "GOF"), heterozygous subjects. Subjects with familial hypercholesterolemia (heFH); Subjects with statin intolerant or statin-uncontrolled primary hypercholesterolemia; and subjects at risk of developing prophylactically treatable hypercholesterolemia Can be mentioned. Other indications include type 2 diabetes, cholestatic liver disease (primary biliary cirrhosis), nephrotic syndrome, hypothyroidism, dyslipidemia associated with secondary causes such as obesity; and atherosclerosis. Prevention and treatment of atherosclerosis and cardiovascular disease.

In certain embodiments of the methods of the invention, the ligands of the invention (eg, anti-hPCSK9 antibodies or antibody fragments of the invention) are elevated total cholesterol, non-HDL cholesterol, LDL cholesterol, and / or apolipoprotein B (eg, apolipoprotein B). It is useful for lowering apolipoprotein B100).

The ligands of the invention (eg, antibodies or antigen-binding fragments) may be used alone or in combination with a second agent, eg, an HMG-CoA reductase inhibitor and / or another lipid lowering agent. May be used.

Treatment Population The present invention provides a therapeutic method for treating a human patient in need of the composition or ligand of the present invention. Modifications in lifestyle and conventional drug treatment often succeed in lowering cholesterol levels, but not all patients can achieve the recommended target cholesterol levels using such an approach. .. Various conditions, such as familial hypercholesterolemia (FH), appear to be resistant to reduced LDL-C levels, despite the active use of conventional treatments. Homozygous and heterozygous familial hypercholesterolemia (hoFH, heFH) is a condition associated with immature atherosclerotic vascular disease. However, patients diagnosed with hoFH are extremely unresponsive to conventional drug therapy, limiting treatment options. Specifically, treatment with statins that lower LDL-C by inhibiting cholesterol synthesis and upregulating the LDL receptor in the liver is effective in patients with the absence or deficiency of the LDL receptor. May be scarce. Patients with genotyped hoFH treated with the highest dose of statins have recently been reported to have a mean LDL-C reduction of less than about 20%. The addition of ezetimibe 10 mg / day to this regimen resulted in a total reduction in LDL-C levels of 27%, which is also far from optimal. Similarly, many patients may be statin-unresponsive, poorly controlled by statin treatment, or intolerant to statin treatment; in general, these patients use alternative treatments to cholesterol. Control cannot be achieved. There is still considerable unmet medical need for new treatments that can address the shortcomings of current treatment options.

Specific populations treatable by the therapeutic methods of the invention include patients to whom LDL aferesis is indicated, subjects with PCSK9 activation (GOF) mutations, heterozygous familial hypercholesterolemia (heFH); statins. Subjects with intolerant or uncontrolled primary hypercholesterolemia; and subjects at risk of developing hypercholesterolemia that can be treated prophylactically.

Therapeutic Administration and Formulations The present invention provides therapeutic compositions comprising the anti-PCSK9 ligands, antibodies or antigen-binding fragments thereof of the present invention. Administration of the therapeutic composition according to the invention is expected to be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. To. A number of suitable formulations can be found in Formulations known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) -containing vesicles [eg LIPOFECTINT ™], DNA conjugates, anhydrous absorption pastes, etc. Examples include oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of preferablys for parenteral excipients" PDA (1998) J Pharm Sci Technol 52: 238-311.

The dose may be changed according to the age and size of the subject to be administered, the target disease, condition, route of administration and the like. Using this ligand, eg, the antibodies of the invention, in adult patients, various PCSK9-related conditions and diseases, including hypercholesterolemia, LDL and apolipoprotein B-related disorders, and dyslipidemia, etc. When treated, a single dose of the ligand or antibody of the invention is usually about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. It is advantageous to administer it intravenously. The frequency and duration of treatment may be adjusted depending on the severity of the condition.

Various delivery systems are known and may be used to administer the pharmaceutical compositions of the invention, thus the compositions of the invention are, for example, encapsulated in liposomes, microparticles, microcapsules, suddenly. Recombinant cells capable of expressing mutant viruses, providing ligands by receptor-mediated endocytosis [see, eg, Wu et al. (1987) J. Biol. Chem. 262: 4429-4432]. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, eg, by instillation or bolus injection, by absorption through the lining of the epithelial or mucosal skin (eg, oral mucosa, rectum and intestinal mucosa, etc.) and other biological activities. It may be administered with the substance. Administration may be systemic or topical.

The pharmaceutical composition can also be delivered in vesicles, especially liposomes [Langer (1990) Science 249: 1527-1533; Treat et al. (1989), Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler. (Eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., Pp. 317-327; see ibid.]

In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used [Langer, supra; see Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201]. In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition and therefore requires only a systemic dose (eg, Goodson, in Medical Applications of Controlled Release, supra). See Volume 2, pp. 115-138, 1984).

Injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injection, infusion, and the like. These injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared, for example, by dissolving, suspending or emulsifying the above-mentioned antibodies or salts thereof in sterile aqueous or oily media customarily used for injection. Aqueous vehicles for injection include, for example, saline, isotonic solutions containing glucose and other auxiliaries, which are suitable solubilizers such as alcohols (eg ethanol), polyvalent. It may be used in combination with alcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, polysolvate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hardened castor oil)] and the like. As the oil-based medium, for example, sesame oil, soybean oil and the like are adopted. These may be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol and the like. The injection thus prepared is preferably filled in a suitable ampoule. The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen-type delivery devices can be readily used in applications in the delivery of the pharmaceutical compositions of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices typically utilize replaceable cartridges containing pharmaceutical compositions. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. In this way the pen-type delivery device can be reused. For disposable pen-type delivery devices, there are no replaceable cartridges. Instead, the disposable pen-type delivery device is pre-filled with the pharmaceutical composition and held in a storage container within the device. When the pharmaceutical composition in the storage container is empty, discard the entire device.

A large number of reusable pen-type and self-injector delivery devices are applied in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples are not limited to them at all, but to name a few, AUTOPEN ™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC ™ Pen (Disetronic Medical Systems, Burghdorf). , Switzerland), HUMALOG MIX 75/25 ™ Pen, HUMALOG ™ Pen, HUMALIN 70/30 ™ Pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN ™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ™ (Novo Nordisk, Copenhagen, Denmark), BD ™ Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT ™, OPTIPEN PRO (Trademark) Trademarks), OPTIPEN STARLET ™, and OPTICLIKT ™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices applied to the subcutaneous delivery of the pharmaceutical compositions of the present invention are, but are not limited to, SOLOSTAR ™ Pens (sanofi-aventis), FLEXPEN ™. ) (Novo Nordisk) and KWIKPEN ™ (Eli Lilly).

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage form in unit doses suitable to match the dose of active ingredient. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories and the like. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per unit dose dosage form; especially in the form of injectables, the aforementioned antibody is contained in about 5 to about 100 mg. It is preferably contained in about 10 to about 250 mg in other dosage forms.

The present invention provides a method in which a ligand, eg, an antibody or antibody fragment of the invention, is useful in treating hypercholesterolemia associated with various conditions associated with hPCSK9. Anti-PCSK9 ligands, such as the antibodies or antibody fragments of the invention, are particularly useful in the treatment of hypercholesterolemia and the like. Combination therapies include, for example, (1) drugs that induce cellular depletion of cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG) -colesevelam A (CoA) reductase, such as cesevelam. Lovastatin, atorvastatin, simvastatin, pitabastatin, rosuvastatin, fulvastatin, lovastatin, pravastatin; (2) drugs that inhibit cholesterol uptake or bile acid reabsorption; In addition to the anti-PCSK9 ligands of the invention in combination with one or more of the agents; as well as activators of LXR transcription factors responsible for cholesterol excretion, such as 22-hydroxycholesterol, or fixed combinations such as ezetimib. Simvasstatin; statins and bile resins (eg, cholestyramine, cholestyl, colesevelam), fixed combinations of statins in addition to niacin (eg, niacin and lovastatin); or other lipid-lowering agents, such as omega-3- fatty acids. Combinations with ethyl ester (eg, omacol) may be mentioned.

Tailor Made Antibodies to Rare PCSK9 Variant Profiles As outlined above, the invention is based on the genetic segments commonly found in humans of ethnic populations in which variant PCSK9 is found to meet the selection criteria of the invention. Further, by selecting an antibody-based ligand having a variable domain, the possibility of tailor-making human treatment is included. An example is shown below for a ligand containing an antibody VH domain derived from recombinant human VH3-23.

The inventor analyzed the frequency and distribution of various human VH3-23 alleles and realized the desirable situation of using ligands based on human VH3-23 alleles, including SNPrs56069819. This SNP corresponds to the change from leucine at position 24 in the encoded protein sequence to valine at that position (change in L24V), which is at coordinate 106268889 on human chromosome 14.

Figure 2 shows the cumulative allele frequency distribution across a database of 1000 genome projects of human VH3-23 alleles, including SNP rs56069819 [such alleles mean "C" and are the most frequent alleles. (This does not include this SNP) meant "A"]. According to this figure, VH3-23 alleles, including SNP rs56069819, are present at a cumulative frequency of 11% across all human ethnic populations taken as a whole, but specific specific human ethnic subpopulations (ASW, LWK, YRI). , CEU and GBR) indicate that such alleles are present above the average cumulative frequency. Shown in this figure are human PCSK9 variant types [denoted as "Variants"] found in various subpopulations above the average appearance of human VH3-23 alleles, including SNP rs56069819. Table 7 shows the VH3-23 variants and the SNPs they contain, as well as their cumulative alleles found in the 1000 Genomes Project database.

In particular, human VH3-23 alleles, including SNP rs56069819, were found in the CEU population almost twice as often as 11% in all populations. For the ASW and YRI populations, the frequency exceeded a quarter of the population. Thus, advantageously, the invention allows the selection of an antibody or a ligand containing an antibody fragment, which antibody or fragment is derived from recombination of a human VH gene segment, a human D gene segment and a human JH gene segment. Containing the VH domain, this VH gene segment contains a nucleotide sequence containing SNP rs56069819 (dbSNP numbered, numbered as listed above).

In one example, further tailoring the procedure by selecting such a ligand that specifically binds to human PCSK9 selected from the following types: f, c, m, e, h, p, q and aj Is possible, and such types are found in the human populations ASW, LWK, YRI, CEU and GBR.

In one example, the ligand specifically binds to human PCSK9 containing the mutant I474V in SEQ ID NO: 1 (this human is of ASW, LWK, YRI, CEU or GBR lineage). Optionally, this human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In some examples, the ligand is further an antibody or fragment comprising a VH domain from the recombination of the human gene segment VH3-23 * 04.

In one example, the ligand specifically binds to human PCSK9 containing the mutant I474V in SEQ ID NO: 1 (eg, f-type, r-type, p-type, e-type or aj-type), and humans have ASW, LWK, YRI, CEU or GBR pedigree. Optionally, the human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in the human, eg, to lower cholesterol or maintain previously lowered cholesterol levels in the human.

In one example, the ligand specifically binds to human PCSK9 containing the mutant E670G in SEQ ID NO: 1 (eg, c-type, r-type or q-type), and humans have ASW, LWK, YRI, CEU or GBR pedigree. Is. Optionally, the human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in the human, eg, to lower cholesterol or maintain previously lowered cholesterol levels in the human.

In one example, the ligand specifically binds to human PCSK9 containing the mutation Q619P in SEQ ID NO: 1 (eg, type h) and the human is of ASW, LWK, YRI, CEU or GBR lineage. Optionally, the human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in the human, eg, to lower cholesterol or maintain previously lowered cholesterol levels in the human.

In one example, the ligand specifically binds to human PCSK9 containing the mutant N425S in SEQ ID NO: 1 (eg, type e) and the human is of ASW, LWK, YRI, CEU or GBR lineage. Optionally, the human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in the human, eg, to lower cholesterol or maintain previously lowered cholesterol levels in the human.

In one example, the ligand specifically binds to human PCSK9 containing the mutant R46L in SEQ ID NO: 1 (eg, type aj), and this human is of ASW, LWK, YRI, CEU or GBR lineage. Optionally, this human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in said humans, eg, to increase cholesterol levels in said humans.

In one example, the ligand specifically binds to human PCSK9 containing the mutant A53V in SEQ ID NO: 1 (eg, p-type or q-type), and the human is of ASW, LWK, YRI, CEU or GBR lineage. .. Optionally, this human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in said humans, eg, to increase cholesterol levels in said humans.

In one example, the ligand specifically binds to human PCSK9 containing the mutant A443T in SEQ ID NO: 1 (eg, m-type or h-type), and the human is of ASW, LWK, YRI, CEU or GBR lineage. .. Optionally, this human comprises a nucleotide sequence encoding PCSK9 comprising mutation I474V in gene segment VH3-23 * 04 and / or SEQ ID NO: 1. In one example, the ligand is further an antibody or fragment containing a VH domain from the recombination of the human gene segment VH3-23 * 04. Optionally, this ligand is to treat or prevent dyslipidemia in said humans, eg, to increase cholesterol levels in said humans.

In one example, the VH gene segment is VH3-23 * 04, which is a commonly found variant containing SNP rs56069819 in the human populations ASW, LWK, YRI, CEU and GBR.

In one example, the ligand treats and / or prevents PCSK9-mediated diseases or conditions in humans expressing human PCSK9 selected from the following types: f, c, m, e, h, p, q and aj: To do.

In one example, the ligand is to treat and / or prevent a PCSK9-mediated disease or condition in humans of ASW, LWK, YRI, CEU or GBR pedigree.

In certain embodiments, the ligand expresses a human of ASW lineage [this human expresses PCSK9 selected from f, c, m, e, h, p and q, or this human expresses Table 6 (Table 7). ) Contains the corresponding nucleotide or amino acid sequence] to treat and / or prevent PCSK9-mediated diseases or conditions. Optionally, this ligand comprises a recombinantly derived VH domain of human VH3-23 * 04.

In certain embodiments, the ligand is a human of LWK lineage [this human expresses PCSK9 selected from f, c, m, e and h, or this human is set in Table 6 (Table 7). Including the corresponding nucleotide or amino acid sequence] to treat and / or prevent PCSK9-mediated diseases or conditions. Optionally, this ligand comprises a recombinantly derived VH domain of human VH3-23 * 04.

In certain embodiments, the ligand is a human of YRI lineage [this human expresses PCSK9 selected from f, c, m, e and h, or this human is set in Table 6 (Table 7). Including the corresponding nucleotide or amino acid sequence] to treat and / or prevent PCSK9-mediated diseases or conditions. Optionally, this ligand comprises a recombinantly derived VH domain of human VH3-23 * 04.

In certain embodiments, the ligand is a human of CEU pedigree [this human expresses PCSK9 selected from f, c, p and aj, or this human expresses the correspondence set in Table 6 (Table 7). Containing nucleotide or amino acid sequences] to treat and / or prevent PCSK9-mediated diseases or conditions. Optionally, this ligand comprises a recombinantly derived VH domain of human VH3-23 * 04.

In certain embodiments, the ligand is a human of GBR pedigree [this human expresses PCSK9 selected from f, c and p, or this human expresses the corresponding nucleotides set in Table 6 (Table 7). Or to contain an amino acid sequence] to treat and / or prevent a PCSK9-mediated disease or condition. Optionally, this ligand comprises a recombinantly derived VH domain of human VH3-23 * 04.

In one example, the ligand is alirocumab.

In any configuration or other embodiment of the TOI herein, as described in more detail above, the invention is based on an alternative human VH gene segment or a V _K , V _λ or constant region gene. Based on the segment [see further Table 9 for representative variants], tailor-made ligands for the genotype and / or phenotype of human recipients are provided.

For example, the ligand of the present invention comprises or consists of an antibody comprising a VH domain from the recombination of the human VH gene segment, the human D gene segment and the human JH gene segment, wherein the VH gene segment is: (I) IGHV1-18 * 01 and this human genome contain the human IGHV1-18 * 01 nucleotide sequence, or this human is a variable derived from the recombination of human IGHV1-18 * 01. Does it express a domain-containing antibody; or (ii) the IGHV1-46 * 01 and this human genome contain the human IGHV1-46 * 01 nucleotide sequence, or this human is a set of human IGHV1-46 * 01 It expresses an antibody containing a variable domain derived from the alternation.

For example, the ligand of the present invention comprises or comprises an antibody comprising a human VL gene segment and a recombinantly derived VL domain of the human JL gene segment, wherein the VL gene segment is selected from the group consisting of: (i) IGKV4-1 * 01 and this human genome contain the human IGKV4-1 * 01 nucleotide sequence, or this human expresses an antibody comprising a recombinantly derived variable domain of human IGKV4-1 * 01. (Ii) IGLV2-14 * 01 and this human genome contain the human IGLV2-14 * 01 nucleotide sequence, or this human contains a variable domain derived from the recombination of human IGLV2-14 * 01. Does it express an antibody; or (iii) IGKV1-13 * 02 and this human genome contain the human IGKV1-13 * 02 nucleotide sequence, or this human is from a recombinant of human IGKV1-13 * 02 Express antibodies containing the variable domain.

For example, the inventor has identified the possibility of tackling the rare IGH-gamma-1 SNP 204D (with a cumulative frequency of 0.296) and 206L (with a cumulative frequency of 0.283) individually or in combination. .. These residues are part of the CH3 domain and therefore they form part of the antibody Fc region. Therefore, these CH3 variations and patient compatibility are particularly beneficial for the reasons discussed above. Thus, in this example, the ligand of the invention comprises an antibody comprising the Asp corresponding to position 204 of SEQ ID NO: 42 or the human gamma-1 heavy chain constant region containing Leu corresponding to position 206 of SEQ ID NO: 42. Or consisting of, this human genome contains a gamma-1 heavy chain constant region nucleotide sequence encoding such Asp or Leu, or this human contains a human gamma-1 constant region containing such Asp or Leu. Expresses an antibody containing. An example of such a ligand is alirocumab.

In another example, the inventor identified the potential for IGH-gamma-2 SNPs. This included examining Fc region variations-in this regard, the inventor focused on positions 161 and 257 in the Fc region. Therefore, in this example, the ligands of the present invention are Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, and 161 of SEQ ID NO: 44. A human gamma containing an amino acid selected from the group consisting of Val corresponding to the position Val and Ala corresponding to the 257th position of SEQ ID NO: 44-Contains or consists of an antibody containing a heavy chain constant region, wherein the human genome Contains a gamma-2 heavy chain constant region nucleotide sequence encoding such a selected amino acid, or the human expresses an antibody comprising a human gamma-2 constant region containing such a selected amino acid. .. Examples of such ligands are evolocumab or bokoshizumab.

In another example, the inventor worked on variations of the human kappa constant region. Thus, in this example, the ligand of the invention comprises or comprises an antibody comprising a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50. And here the human genome contains a kappa light chain constant region nucleotide sequence encoding such Val or Cys, or this human contains a human kappa light chain constant region containing such Val or Cys. Express the antibody containing. Examples of such ligands are alirocumab or bocosizumab.

In another example, the inventor worked on a variation of the human lambda constant region. Thus, in this example, the ligand of the invention comprises or consists of an antibody comprising a human IGLC2 * 01 light chain constant region; and where the human genome comprises or comprises a human IGLC2 * 01 nucleotide sequence. This human expresses an antibody containing the human light chain IGLC2 * 01 constant region. An example of such a ligand is evolocumab.

Further exemplary ligands are in paragraphs 1-17 as follows.
1. (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) antibodies or antibodies for use in methods of targeting PCSK9 in humans. It is an antibody fragment
Here, the antibody or fragment comprises a human gamma heavy chain constant region containing the first amino acid encoded by the human gamma heavy chain constant region gene segment SNP, and the antibody or fragment is I474V, Q619P, N425S and in SEQ ID NO: 1. It specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing a C-terminal domain containing a mutation selected from the group consisting of E670G (eg, selected from I474V and E670G). Here, the human comprises a nucleotide sequence encoding the PCSK9 amino acid sequence and comprises a human gamma heavy chain constant region gene segment comprising said SNP, or a human comprises a human gamma constant region comprising said first amino acid. An antibody or antibody fragment that expresses an antibody.

In one embodiment of (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one alternative, paragraph 1 provides:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, the antibody or fragment comprises a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 or Leu corresponding to position 206 of SEQ ID NO: 42, and the antibody or fragment comprises SEQ ID NO: In 1, it specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G), where this human is the amino acid. An antibody or antibody that comprises a nucleotide sequence encoding a sequence and contains an IGHG1 * 01 human heavy chain constant region gene segment, or that this human expresses an antibody comprising a human gamma-1 constant region containing Asp, Leu, etc. fragment.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G. In another embodiment, the mutation is N425S. In another embodiment, the mutation is Q619P.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example the following is provided:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, the antibody or fragment comprises a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, and this antibody or fragment contains SEQ ID NO: In 1, it specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutant I474V, where this human contains the nucleotide sequence encoding the amino acid sequence , And an antibody or antibody fragment containing the IGHG1 * 01 human heavy chain constant region gene segment. Optionally, this antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

Another example provides:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, the antibody or fragment comprises a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, and this antibody or fragment contains SEQ ID NO: In 1, it specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutant E670G, where this human contains the nucleotide sequence encoding the amino acid sequence. , And IGHG1 * 01 human heavy chain constant region gene segment, an amino acid or an amino acid fragment. Optionally, this antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment is a precursor comprising a C-terminal domain containing a mutation selected from the group consisting of I474V, Q619P, N425S and E670G in SEQ ID NO: 1 (eg, selected from I474V and E670G). It has been determined to specifically bind to the protein convertase subtilisin / kexin type 9 (PCSK9), where the antibody or fragment corresponds to Asp corresponding to position 204 of SEQ ID NO: 42 and position 206 of SEQ ID NO: 42. The human gamma-1 heavy chain constant region containing Leu, and said human, is (i) IGHG1 * 01 human heavy chain constant region gene segment and (ii) the mutation in SEQ ID NO: 1 (eg, I474V or E670G). Includes a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising a C-terminal domain comprising.

2. The antibody or antibody fragment of paragraph 1 wherein the antibody comprises a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42.

3. The antibody or antibody fragment of paragraph 1 or 2 where the antibody comprises the IGHG1 * 01 human heavy chain constant region.

4. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The antibody or antibody fragment of any one of paragraphs 1-3, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

5. The method is PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). Including the step of determining that a human comprises a nucleotide sequence encoding a variant protein, optionally, this determining step is performed prior to administration of the antibody to human, any one antibody of paragraphs 1-4 or Antibody fragment.

6. The step of determining includes assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. Antibody or antibody fragment of.

7. The step of assaying is a biological sample and
Can it be specifically hybridized in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 to identify it? At least one oligonucleotide probe containing a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence, wherein the nucleic acid is present in the selected sequence not present in SEQ ID NO: 28. At least one nucleotide encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide or antisense sequence. Oligonucleotide probes that form complexes, if sequences are present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The antibody or antibody of paragraph 6 comprising the step of detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. fragment.

8. The assaying step comprises or optionally comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. The antibody or antibody fragment of paragraph 6 or 7, wherein the step of assaying is performed in a multiplex format.

9. Any one antibody of paragraphs 1-8, wherein the antibody or antibody fragment is further determined to be substantially resistant to statin treatment, or for administration to a further determined human. Or an antibody fragment.

10. An antibody or antibody of any one of paragraphs 1-9, for administration of an antibody or antibody fragment to a human who has received, has received, or has become less responsive to statin treatment. fragment.

11. The antibody or antibody fragment of paragraph 9 or 10, wherein the antibody or antibody fragment is for administration to a human separately or simultaneously with the statin treatment.

12. The antibody or antibody fragment of any one of paragraphs 6-10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human has been shown to be heterozygous for a nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutation (eg, I474V or E670G), and optionally, where the human further comprises a nucleotide of SEQ ID NO: 28. Paragraph 1 which is shown to contain a sequence or the human is shown to be homozygous for a nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. Any one of ~ 12 antibodies or antibody fragments.

14. The human being has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( Any one antibody or antibody fragment of paragraphs 1-13 diagnosed with at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

15. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The antibody or antibody fragment of any one of paragraphs 1-14 that treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

16. An antibody or antibody fragment of any one of paragraphs 1-15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

17. The antibody or antibody fragment of any one of paragraphs 1-16, wherein the antibody or antibody fragment is for administration by intravenous or subcutaneous administration and / or is included in the preparation for injection.

Further exemplary methods are in paragraphs 1-18 as follows:
1. (a) a method of lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
An antibody or antibody fragment that specifically binds to the human precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. The antibody or fragment comprises a human gamma-1 heavy chain constant region comprising Asp corresponding to position 204 of SEQ ID NO: 42 or Leu corresponding to position 206 of SEQ ID NO: 42 and said by the human. , (I) IGHG1 * 01 human heavy chain constant region gene segment (or this human expresses an antibody containing a human gamma-1 heavy chain constant region containing such Asp and Leu) and (ii) SEQ ID NO: 1 A method comprising a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain comprising the mutation (eg, I474V or E670G) in. In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example the following is provided:-
A method of lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
It comprises the step of administering to said human an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutation I474 in SEQ ID NO: 1. This antibody or fragment comprises a human gamma-1 heavy chain constant region comprising Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, wherein the human is (i) IGHG1. * 01 A method comprising a human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutation I474 in SEQ ID NO: 1. .. Optionally, the antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

In the embodiment of (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

Another example provides:-
A method of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
It comprises the step of administering to said human an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutant E670G in SEQ ID NO: 1. This antibody or fragment comprises a human gamma-1 heavy chain constant region comprising Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, wherein the human is (i) IGHG1. * 01 A method comprising a human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutant E670G in SEQ ID NO: 1. .. Optionally, this antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

In the embodiment of (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment comprises a human gamma-1 heavy chain constant region comprising Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, where The human is the precursor protein converting enzyme subtilisin / kexin containing (i) IGHG1 * 01 human heavy chain constant region gene segment and (ii) C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1. Contains a nucleotide sequence encoding type 9 (PCSK9).

2. The method of paragraph 1, comprising selecting a human comprising said nucleotide sequence of (ii), wherein the human is the human of paragraph 1 prior to said administration.

3. The method of paragraph 1 or 2, wherein the antibody comprises a human gamma-1 heavy chain constant region comprising Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42.

4. The method of paragraph 1, 2 or 3 where the antibody comprises the IGHG1 * 01 human heavy chain constant region.

5. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The method of any one of paragraphs 1-4, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

6. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The method of any one of paragraphs 1-5, comprising the step of determining to include the nucleotide sequence encoding the variant protein, optionally, this determining step is performed prior to administration of the antibody to this human. ..

7. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 6 methods.

8. The step of assaying is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G), or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The method of paragraph 7, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 comprising a C-terminal domain comprising a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

9. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of paragraph 7 or 8, wherein the step of assaying is performed in a multiplex format.

10. The method of any one of paragraphs 1-9, wherein the human has been determined to be substantially resistant to statin treatment, or further determined.

11. The method of any of paragraphs 1-10, wherein the human has received or has received statin treatment or is less responsive to statin treatment.

12. The method of paragraph 10 or 11, wherein the antibody or antibody fragment is administered to a human separately or simultaneously with the statin treatment.

13. The method of any one of paragraphs 7-9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

14. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of paragraphs 1-13, wherein the human is shown to be homozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

15. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( The method of any one of paragraphs 1-14, diagnosed as having at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

16. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The method of any one of paragraphs 1-15, which treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

17. The method of any one of paragraphs 1-16, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

18. The method of any one of paragraphs 1-17, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in the injectable preparation.

Further exemplary ligands are in paragraphs 1-17 as follows.
1. (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) antibodies or antibodies for use in methods of targeting PCSK9 in humans. It is an antibody fragment
Here, the antibodies are Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, Val corresponding to position 161 of SEQ ID NO: 44, and Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala corresponding to position 257 of SEQ ID NO: 44, and this antibody or fragment contains the PCSK9 mutation in SEQ ID NO: 1 (eg, I474V or It specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing E670G), where this human contains a nucleotide sequence encoding said amino acid sequence and is IGHG2. * 01 An antibody or antibody fragment that comprises a human heavy chain constant region gene segment, or that this human expresses an antibody comprising a human gamma-2 constant region, including such Pro, Asn, Phe, Val and Ala.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments of (b), the antibody or fragment treats or lowers cholesterol levels in the human domain or maintains previously lowered cholesterol levels.

In one example, the following is provided:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, this antibody is Pro corresponding to the 72nd position of SEQ ID NO: 44, Asn corresponding to the 75th position of SEQ ID NO: 44, Phe corresponding to the 76th position of SEQ ID NO: 44, Val corresponding to the 161st position of SEQ ID NO: 44, and Subtilisin / Kexin, a precursor protein convertase containing the human gamma-2 heavy chain constant region containing Ala corresponding to position 257 of SEQ ID NO: 44, and the antibody or fragment containing the C-terminal domain containing the mutant I474V in SEQ ID NO: 1. An antibody or antibody that specifically binds to a type 9 (PCSK9) amino acid sequence, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG2 * 01 human heavy chain constant region gene segment. fragment. Optionally, this antibody or fragment comprises the IGHG2 * 01 human heavy chain constant region gene segment.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In another example, the following is provided:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, this antibody is Pro corresponding to the 72nd position of SEQ ID NO: 44, Asn corresponding to the 75th position of SEQ ID NO: 44, Phe corresponding to the 76th position of SEQ ID NO: 44, Val corresponding to the 161st position of SEQ ID NO: 44, and A precursor protein convertase subtilisin / kexin containing the human gamma-2 heavy chain constant region containing Ala corresponding to position 257 of SEQ ID NO: 44, and the antibody or fragment containing the C-terminal domain containing the mutant E670G in SEQ ID NO: 1. An antibody or antibody that specifically binds to a type 9 (PCSK9) amino acid sequence, wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises an IGHG2 * 01 human heavy chain constant region gene segment. fragment. Optionally, this antibody or fragment comprises the IGHG2 * 01 human heavy chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment is here: Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, SEQ ID NO: 44. Contains a human gamma-2 heavy chain constant region containing Val corresponding to position 161 of and Ala corresponding to position 257 of SEQ ID NO: 44, and said human is (i) IGHG2 * 01 human heavy chain constant region gene segment and (ii) Contain a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1.

2. The antibody is Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, Val and sequence corresponding to position 161 of SEQ ID NO: 44. The antibody or antibody fragment of paragraph 1 comprising the human gamma-2 heavy chain constant region containing Ala corresponding to position 257 of number 44.

3. The antibody or antibody fragment of paragraph 1 or 2 where the antibody comprises the IGHG2 * 01 human heavy chain constant region.

4. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The antibody or antibody fragment of any one of paragraphs 1-3, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

5. The method is PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). Including the step of determining that a human comprises a nucleotide sequence encoding a variant protein, optionally, this determining step is performed prior to administration of the antibody to human, any one antibody of paragraphs 1-4 or Antibody fragment.

6. The step of determining includes assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. Antibody or antibody fragment of.

7. The step of assaying is a biological sample and
Can it be specifically hybridized in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 to identify it? At least one oligonucleotide probe containing a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence, wherein the nucleic acid is present in the selected sequence not present in SEQ ID NO: 28. At least one nucleotide encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide or antisense sequence. Oligonucleotide probes that form complexes, if sequences are present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The antibody or antibody of paragraph 6 comprising the step of detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. fragment.

8. The assaying step comprises or optionally comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. The antibody or antibody fragment of paragraph 6 or 7, wherein the step of assaying is performed in a multiplex format.

9. Any one antibody of paragraphs 1-8, wherein the antibody or antibody fragment is further determined to be substantially resistant to statin treatment, or for administration to a further determined human. Or an antibody fragment.

10. An antibody or antibody of any one of paragraphs 1-9, for administration of an antibody or antibody fragment to a human who has received, has received, or has become less responsive to statin treatment. fragment.

11. The antibody or antibody fragment of paragraph 9 or 10, wherein the antibody or antibody fragment is for administration to a human separately or simultaneously with the statin treatment.

12. The antibody or antibody fragment of any one of paragraphs 6-8, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutation (eg, I474V or E670G), and optionally the human further has the nucleotide sequence of SEQ ID NO: 28. Any one antibody of paragraphs 1-12, which is shown to contain or the human is shown to be homozygous for a nucleotide sequence encoding a PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. Or an antibody fragment.

14. The human being has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( Any one antibody or antibody fragment of paragraphs 1-13 diagnosed with at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

15. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The antibody or antibody fragment of any one of paragraphs 1-14 that treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

16. An antibody or antibody fragment of any one of paragraphs 1-15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

17. The antibody or antibody fragment of any one of paragraphs 1-16, wherein the antibody or antibody fragment is for administration by intravenous or subcutaneous administration and / or is included in the preparation for injection.

Further exemplary methods are in paragraphs 1-18 as follows:
1. (a) a method of lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
An antibody or antibody fragment that specifically binds to the human precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment comprises the step of administering, where the antibody or fragment corresponds to Pro at position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, SEQ ID NO: It contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Val corresponding to position 161 of 44 and Ala corresponding to position 257 of SEQ ID NO: 44, wherein the human is (i) IGHG2 *. 01 Human heavy chain constant region gene segment or this human expresses an antibody comprising a human gamma-2 heavy chain constant region such as Pro, Asn, Phe, Val and Ala, and (ii) said in SEQ ID NO: 1. A method comprising a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising a C-terminal domain comprising a mutation (eg, I474V or E670G).

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example the following is provided:-
A method of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
A step of administering to the human an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutation I474 in SEQ ID NO: 1 is included herein. This antibody or fragment corresponds to Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, and Val corresponding to position 161 of SEQ ID NO: 44. And a human gamma-2 heavy chain constant region containing Ala corresponding to position 257 of SEQ ID NO: 44, wherein the human is (i) IGHG2 * 01 human heavy chain constant region gene segment, and (ii) SEQ ID NO: 1. A method comprising a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain comprising said mutant I474 in. Optionally, the antibody or fragment comprises the IGHG2 * 01 human heavy chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

Another example provides:-
A method of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
A step of administering to the human an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutant E670G in SEQ ID NO: 1, wherein This antibody or fragment corresponds to Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, and Val corresponding to position 161 of SEQ ID NO: 44. And a human gamma-2 heavy chain constant region containing Ala corresponding to position 257 of SEQ ID NO: 44, wherein the human is (i) IGHG2 * 01 human heavy chain constant region gene segment, and (ii) SEQ ID NO: A method comprising a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutant E670G in 1.

Optionally, this antibody or fragment comprises the IGHG2 * 01 human heavy chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment is here: Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, SEQ ID NO: 44. Contains a human gamma-2 heavy chain constant region containing Val corresponding to position 161 of and Ala corresponding to position 257 of SEQ ID NO: 44, and said human is (i) IGHG2 * 01 human heavy chain constant region gene segment and (ii) Contain a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1.

2. The method of paragraph 1, comprising the step of selecting the human containing the nucleotide sequence of (ii) prior to the step of administration.

3. This antibody includes Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, Phe corresponding to position 76 of SEQ ID NO: 44, Val corresponding to position 161 of SEQ ID NO: 44, and The method of paragraph 1 or 2, comprising the human gamma-1 heavy chain constant region containing Ala corresponding to position 257 of SEQ ID NO: 44.

4. The method of paragraph 1, 2 or 3 where the antibody comprises the IGHG2 * 01 human heavy chain constant region.

5. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The method of any one of paragraphs 1-4, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

6. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The method of any one of paragraphs 1-5, comprising the step of determining to include the nucleotide sequence encoding the variant protein, optionally, this determining step is performed prior to administration of the antibody to this human. ..

7. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 6 methods.

8. The step of assaying is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G), or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The method of paragraph 7, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 comprising a C-terminal domain comprising a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

9. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of paragraph 7 or 8, wherein the step of assaying is performed in a multiplex format.

10. The method of any one of paragraphs 1-9, wherein the human has been determined to be substantially resistant to statin treatment, or further determined.

11. The method of any of paragraphs 1-10, wherein the human has received or has received statin treatment or is less responsive to statin treatment.

12. The method of paragraph 10 or 11, wherein the antibody or antibody fragment is administered to a human separately or simultaneously with the statin treatment.

13. The method of any one of paragraphs 7-9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

14. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of paragraphs 1-13, wherein the human is shown to be homozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

15. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( The method of any one of paragraphs 1-14, diagnosed as having at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

16. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The method of any one of paragraphs 1-15, which treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

17. The method of any one of paragraphs 1-16, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

18. The method of any one of paragraphs 1-17, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in the injectable preparation.

Further exemplary ligands are in paragraphs 1-17 as follows.
1. (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) antibodies or antibodies for use in methods of targeting PCSK9 in humans. It is an antibody fragment
Here, the antibody or fragment comprises a human kappa light chain constant region containing Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50, and this antibody or fragment is in SEQ ID NO: 1. It specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G), where this human is the amino acid sequence. An antibody or antibody that comprises a nucleotide sequence encoding the above and contains an IGKC * 01 human light chain constant region gene segment, or in which a human expresses an antibody comprising a human kappa light chain constant region such as Val and Cys. fragment.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example the following is provided:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, the antibody or fragment comprises a human kappa light chain constant region containing Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50, and this antibody or fragment is in SEQ ID NO: 1. It specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing mutant I474V, where this human comprises a nucleotide sequence encoding said amino acid sequence. And an antibody or antibody fragment containing the IGKC * 01 human light chain constant region gene segment. Optionally, the antibody or fragment comprises the IGKC * 01 human light chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In another example, the following is provided:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, the antibody or fragment comprises a human kappa light chain constant region containing Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50, and this antibody or fragment is in SEQ ID NO: 1. It specifically binds to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutant E670G, where this human comprises a nucleotide sequence encoding said amino acid sequence And an antibody or antibody fragment containing the IGKC * 01 human light chain constant region gene segment. Optionally, this antibody or fragment comprises the IGKC * 01 human light chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50, wherein said human. Is a precursor protein convertase subbutyricin / kexin type 9 containing (i) IGKC * 01 human heavy chain constant region gene segment and (ii) C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1. Contains a nucleotide sequence encoding (PCSK9).

2. The antibody or antibody fragment of paragraph 1 wherein the antibody comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50.

3. The antibody or antibody fragment of paragraph 1 or 2 where the antibody comprises the IGKC * 01 human kappa chain constant region.

4. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The antibody or antibody fragment of any one of paragraphs 1-3, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

5. The method is PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). Including the step of determining that a human comprises a nucleotide sequence encoding a variant protein, optionally, this determining step is performed prior to administration of the antibody to human, any one antibody of paragraphs 1-4 or Antibody fragment.

6. The step of determining includes assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. Antibody or antibody fragment of.

7. The step of assaying is a biological sample and
Can it be specifically hybridized in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 to identify it? At least one oligonucleotide probe containing a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence, wherein the nucleic acid is present in the selected sequence not present in SEQ ID NO: 28. At least one nucleotide encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide or antisense sequence. Oligonucleotide probes that form complexes, if sequences are present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The antibody or antibody of paragraph 6 comprising the step of detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. fragment.

8. The assaying step comprises or optionally comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. The antibody or antibody fragment of paragraph 6 or 7, wherein the step of assaying is performed in a multiplex format.

9. Any one antibody of paragraphs 1-8, wherein the antibody or antibody fragment is further determined to be substantially resistant to statin treatment, or for administration to a further determined human. Or an antibody fragment.

10. An antibody or antibody of any one of paragraphs 1-9, for administration of an antibody or antibody fragment to a human who has received, has received, or has become less responsive to statin treatment. fragment.

11. The antibody or antibody fragment of paragraph 9 or 10, wherein the antibody or antibody fragment is for administration to a human separately or simultaneously with the statin treatment.

12. The antibody or antibody fragment of any one of paragraphs 6-10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutation (eg, I474V or E670G), and optionally the human further has the nucleotide sequence of SEQ ID NO: 28. Any one antibody of paragraphs 1-12, which is shown to contain or the human is shown to be homozygous for a nucleotide sequence encoding a PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. Or an antibody fragment.

14. The human being has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( Any one antibody or antibody fragment of paragraphs 1-13 diagnosed with at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

15. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The antibody or antibody fragment of any one of paragraphs 1-14 that treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

16. An antibody or antibody fragment of any one of paragraphs 1-15, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

17. The antibody or antibody fragment of any one of paragraphs 1-16, wherein the antibody or antibody fragment is for administration by intravenous or subcutaneous administration and / or is included in the preparation for injection.

Further exemplary methods are in paragraphs 1-18 as follows:
1. (a) a method of lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
An antibody or antibody fragment that specifically binds to the human precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. The antibody or fragment comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50, wherein the antibody or fragment comprises the above. Humans (i) IGKC * 01 human light chain constant region gene segment (or this human expresses an antibody comprising a human kappa light chain constant region containing such Val and Cys), and (ii) SEQ ID NO: 1 A method comprising a nucleotide sequence encoding a precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising a C-terminal domain comprising the mutation (eg, I474V or E670G) in.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example the following is provided:-
A method of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
It comprises the step of administering to said human an antibody or antibody fragment that specifically binds to the precursor protein converting enzyme subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing mutation I474 in SEQ ID NO: 1. This antibody or fragment comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50, wherein the human is (i) IGKC *. 01 A method comprising a human light chain constant region gene segment and (ii) a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutant I474 in SEQ ID NO: 1. .. Optionally, this antibody or fragment comprises the IGKC * 01 human light chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

Another example provides:-
A method of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans.
A step of administering to the human an antibody or antibody fragment that specifically binds to the precursor protein converting enzyme subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutant E670G in SEQ ID NO: 1 is included herein. This antibody or fragment comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50, wherein the human is (i) IGKC *. 01 A method comprising a human light chain constant region gene segment and (ii) a nucleotide sequence encoding the precursor protein convertase subbutyricin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutant E670G in SEQ ID NO: 1. Optionally, the antibody or fragment comprises the IGKC * 01 human light chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment is specific for the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 and Cys corresponding to position 87 of SEQ ID NO: 50, where it is determined to bind to. The human is a precursor protein convertase subtilisin / kexin containing (i) IGKC * 01 human heavy chain constant region gene segment and (ii) C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1. Contains a nucleotide sequence encoding type 9 (PCSK9).

2. The method of paragraph 1, comprising the step of selecting the human containing the nucleotide sequence of (ii) prior to the step of administration.

3. The method of paragraph 1 or 2, wherein the antibody comprises a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 50 or Cys corresponding to position 87 of SEQ ID NO: 50.

4. The method of paragraph 1, 2 or 3 where the antibody comprises the IGKC * 01 human kappa chain constant region.

5. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The method of any one of paragraphs 1-4, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

6. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The method of any one of paragraphs 1-5, comprising the step of determining to include the nucleotide sequence encoding the variant protein, optionally, this determining step is performed prior to administration of the antibody to this human. ..

7. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 6 methods.

8. The step of assaying is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G), or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The method of paragraph 7, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 comprising a C-terminal domain comprising a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

9. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of paragraph 7 or 8, wherein the step of assaying is performed in a multiplex format.

10. The method of any one of paragraphs 1-9, wherein the human has been determined to be substantially resistant to statin treatment, or further determined.

11. The method of any of paragraphs 1-10, wherein the human has received or has received statin treatment or is less responsive to statin treatment.

12. The method of paragraph 10 or 11, wherein the antibody or antibody fragment is administered to a human separately or simultaneously with the statin treatment.

13. The method of any one of paragraphs 7-9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

14. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of paragraphs 1-13, wherein the human is shown to be homozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

15. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( The method of any one of paragraphs 1-14, diagnosed as having at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

16. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The method of any one of paragraphs 1-15, which treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

17. The method of any one of paragraphs 1-16, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

18. The method of any one of paragraphs 1-17, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in the injectable preparation.

Further exemplary ligands are in paragraphs 1-15 as follows.
1. (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) antibodies or antibodies for use in methods of targeting PCSK9 in humans. It is an antibody fragment
Here, the antibody or fragment comprises a human IGLC2 * 01 lambda light chain constant region, and the antibody or fragment is a precursor protein comprising the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. It specifically binds to the amino acid sequence of the convertase subtilisin / kexin type 9 (PCSK9), where the human comprises a nucleotide sequence encoding said amino acid sequence and a human IGLC2 * 01 lambda light chain constant region gene segment. An antibody or antibody fragment comprising, or in which this human expresses an antibody comprising the human IGLC2 * 01 lambda light chain constant region.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the following is provided:-
Antibodies or antibody fragments for use in methods that either (a) lower or maintain previously lowered cholesterol levels in humans who require it, or (b) target PCSK9 in humans. Wherever this antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region and the antibody or fragment comprises the C-terminal domain comprising mutant I474V in SEQ ID NO: 1 Subtilisin / An antibody that specifically binds to the amino acid sequence of kexin type 9 (PCSK9), wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises a human IGLC2 * 01 lambda light chain constant region gene segment. Or an antibody fragment. Optionally, the antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In another example, the following is provided:-
Antibodies or antibody fragments for use in methods of (a) lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) targeting PCSK9 in humans. And
Here, the antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region, and the antibody or fragment comprises the C-terminal domain containing the mutant E670G in SEQ ID NO: 1 for the precursor protein convertase subtilisin / kexin type 9. An antibody or antibody fragment that specifically binds to the amino acid sequence of (PCSK9), wherein the human comprises a nucleotide sequence encoding said amino acid sequence and comprises a human IGLC2 * 01 lambda light chain constant region gene segment. .. Optionally, this antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

In one example, the antibody or antibody fragment specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. Where the antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region, wherein said human is (i) IGLC2 * 01 human heavy chain constant region gene segment, and (ii). ) Includes a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1.

2. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The antibody or antibody fragment of paragraph 1 determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

3. The method is PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The antibody or antibody fragment of paragraph 1 or 2, comprising determining that a human comprises a nucleotide sequence encoding a variant protein, optionally, this determining step is performed prior to administration of the antibody to human.

4. The step of determining includes assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. Antibody or antibody fragment of.

5. The step of assaying is a biological sample and
Can it be specifically hybridized in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 to identify it? At least one oligonucleotide probe containing a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence, wherein the nucleic acid is present in the selected sequence not present in SEQ ID NO: 28. At least one nucleotide encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide or antisense sequence. Oligonucleotide probes that form complexes, if sequences are present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The antibody or antibody of paragraph 4 comprising the step of detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. fragment.

6. The assaying step comprises or optionally includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. The antibody or antibody fragment of paragraph 4 or 5, wherein the step of assaying is performed in a multiplex format.

7. The antibody of any one of paragraphs 1-6, wherein the antibody or antibody fragment is further determined to be substantially resistant to statin treatment, or for administration to a further determined human. Or an antibody fragment.

8. The antibody or antibody in any one of paragraphs 1-7, for administration to a human whose antibody or antibody fragment has received or has received statin treatment or is less responsive to statin treatment. fragment.

9. The antibody or antibody fragment of paragraph 7 or 8, wherein the antibody or antibody fragment is for administration to a human separately or simultaneously with the statin treatment.

10. An antibody or antibody fragment of any one of paragraphs 4-8, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutation (eg, I474V or E670G), and optionally the human further has the nucleotide sequence of SEQ ID NO: 28. Any 1 of paragraphs 1-10, which is shown to contain or the human is shown to be homozygous for a nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutant I474V or E670G in SEQ ID NO: 1. Two antibodies or antibody fragments.

12. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( Any one antibody or antibody fragment of paragraphs 1-11 diagnosed with at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

13. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The antibody or antibody fragment of any one of paragraphs 1-12 that treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

14. An antibody or antibody fragment of any one of paragraphs 1-13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

15. The antibody or antibody fragment of any one of paragraphs 1-14, wherein the antibody or antibody fragment is for administration by intravenous or subcutaneous administration and / or is included in the preparation for injection.

Further exemplary methods are in paragraphs 1-16 as follows:
1. A method of lowering or maintaining previously lowered cholesterol levels in humans who require it, such as the PCSK9 mutation (eg, I474V or) in SEQ ID NO: 1 for said human. It comprises the step of administering an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing E670G), wherein the antibody or fragment is human IGLC2 * 01. Lambda light chain constant region, wherein said human expresses an antibody comprising (i) IGLC2 * 01 human light chain constant region gene segment, (or this human contains human IGLC2 * 01 lambda light chain constant region. ), And (ii) a method comprising a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain comprising said mutation (eg, I474V or E670G) in SEQ ID NO: 1.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In one example the following is provided:-
A method of lowering or maintaining previously lowered cholesterol levels in humans in need thereof, comprising the C-terminal domain comprising mutation I474 in SEQ ID NO: 1 for said human. It comprises administering an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9), wherein the antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region and Here, the human is the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing (i) IGLC2 * 01 human light chain constant region gene segment and (ii) C-terminal domain containing the mutant I474 in SEQ ID NO: 1. A method comprising a nucleotide sequence encoding). Optionally, this antibody or fragment comprises the IGLC2 * 01 human light chain constant region.

Another example provides:-
A method of lowering or maintaining previously lowered cholesterol levels in humans in need thereof, comprising the C-terminal domain comprising the mutant E670G in SEQ ID NO: 1 for said human. It comprises administering an antibody or antibody fragment that specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9), wherein the antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region and Here, the human is the precursor protein convertase subbutyricin / kexin type 9 containing (i) IGLC2 * 01 human light chain constant region gene segment and (ii) C-terminal domain containing the mutant E670G in SEQ ID NO: 1. A method comprising a nucleotide sequence encoding PCSK9). Optionally, the antibody or fragment comprises the IGLC2 * 01 human light chain constant region.

In one example, the antibody or antibody fragment specifically binds to the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. Where the antibody or fragment comprises the human IGLC2 * 01 lambda light chain constant region, wherein said human is (i) IGLC2 * 01 human heavy chain constant region gene segment and (ii). Includes a nucleotide sequence encoding the precursor protein convertase subtilisin / kexin type 9 (PCSK9) comprising the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1.

2. The method of paragraph 1, comprising the step of selecting the human containing the nucleotide sequence of (ii) prior to the step of administration.

3. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The method of paragraph 1 or 2, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

4. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The method of any one of paragraphs 1-3, comprising the step of determining to include the nucleotide sequence encoding the variant protein, optionally, this determining step is performed prior to administration of the antibody to this human. ..

5. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 4 methods.

6. The assay process is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G) or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The method of paragraph 5, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 comprising a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

7. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of paragraph 5 or 6, wherein the step of assaying is performed in a multiplex format.

8. The method of any one of paragraphs 1-7, wherein the human has been determined to be substantially resistant to statin treatment, or further determined.

9. The method of any of paragraphs 1-8, wherein the human has received or has received statin treatment or has reduced responsiveness to statin treatment.

10. The method of paragraph 8 or 9, wherein the antibody or antibody fragment is administered to a human separately or simultaneously with the statin treatment.

11. The method of any one of paragraphs 5-7, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

12. Heterozygousness was shown for the nucleotide sequence in which the human encodes the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of paragraphs 1-11, wherein the human is shown to be homozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

13. The human being has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( The method of any one of paragraphs 1-12, diagnosed as having at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

14. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The method of any one of paragraphs 1-13, which treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

15. The method of any one of paragraphs 1-14, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

16. The method of any one of paragraphs 1-15, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in the injectable preparation.

Further exemplary ligands are in paragraphs 1-15 as follows.
1. (a) lowering or maintaining previously lowered cholesterol levels in humans in need of it, or (b) antibodies or antibodies for use in methods of targeting PCSK9 in humans An antibody fragment comprising a human variable domain derived from a recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment is defined in any one of clauses 80-90. And this antibody or fragment is specific to the amino acid sequence of the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. Here, the human comprises a nucleotide sequence encoding said amino acid sequence and comprises said VH gene segment, or a set of said human VH gene segment, human D gene segment and human JH gene segment. An antibody or antibody fragment that expresses an antibody containing a VH domain derived from a mutant.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

2. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The antibody or antibody fragment of paragraph 1 determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

3. The method is PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The antibody or antibody fragment of paragraph 1 or 2, comprising determining that a human comprises a nucleotide sequence encoding a variant protein, optionally, this determining step is performed prior to administration of the antibody to human.

4. The step of determining includes assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. Antibody or antibody fragment of.

5. The step of assaying is a biological sample and
Can it be specifically hybridized in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 to identify it? At least one oligonucleotide probe containing a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence, wherein the nucleic acid is present in the selected sequence not present in SEQ ID NO: 28. At least one nucleotide encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide or antisense sequence. Oligonucleotide probes that form complexes, if sequences are present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The antibody or antibody of paragraph 4 comprising the step of detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. fragment.

6. The assaying step comprises or optionally includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. The antibody or antibody fragment of paragraph 4 or 5, wherein the step of assaying is performed in a multiplex format.

7. The antibody of any one of paragraphs 1-6, wherein the antibody or antibody fragment is further determined to be substantially resistant to statin treatment, or for administration to a further determined human. Or an antibody fragment.

8. The antibody or antibody in any one of paragraphs 1-7, for administration to a human whose antibody or antibody fragment has received or has received statin treatment or is less responsive to statin treatment. fragment.

9. The antibody or antibody fragment of paragraph 7 or 8, wherein the antibody or antibody fragment is for administration to a human separately or simultaneously with the statin treatment.

10. An antibody or antibody fragment of any one of paragraphs 4-8, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the PCSK9 mutation (eg, I474V or E670G), and optionally the human further has the nucleotide sequence of SEQ ID NO: 28. Any one antibody of paragraphs 1-10, which is shown to contain or the human is shown to be homozygous for a nucleotide sequence encoding a PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. Or an antibody fragment.

12. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( Any one antibody or antibody fragment of paragraphs 1-11 diagnosed with at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

13. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The antibody or antibody fragment of any one of paragraphs 1-12 that treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

14. An antibody or antibody fragment of any one of paragraphs 1-13, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

15. The antibody or antibody fragment of any one of paragraphs 1-14, wherein the antibody or antibody fragment is for administration by intravenous or subcutaneous administration and / or is included in the preparation for injection.

Further exemplary methods are in paragraphs 1-16 as follows:
1. (a) a method of lowering or maintaining previously lowered cholesterol levels in humans who need it, or (b) a method of targeting PCSK9 in humans,
An antibody or antibody fragment that specifically binds to the human precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or E670G) in SEQ ID NO: 1. This antibody or fragment comprises a human variable domain derived from a recombination of a human VH gene segment, a human D gene segment and a human JH gene segment, wherein the VH gene segment comprises Clause 80. Defined in any one of ~ 90, wherein the human (i) comprises the VH gene segment or is a recombination of the human VH gene segment, the human D gene segment and the human JH gene segment. It expresses an antibody containing the VH domain of origin and (ii) encodes the precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1. A method comprising a nucleotide sequence to be made.

In certain embodiments, the mutation is I474V. In another embodiment, the mutation is E670G.

In certain embodiments (b), the antibody or fragment treats or lowers cholesterol levels in humans or maintains previously lowered cholesterol levels.

2. The method of paragraph 1, comprising the step of selecting the human containing the nucleotide sequence of (ii) prior to the step of administration.

3. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The method of paragraph 1 or 2, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

4. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). The method of any one of paragraphs 1-3, comprising the step of determining to include the nucleotide sequence encoding the variant protein, optionally, this determining step is performed prior to administration of the antibody to this human. ..

5. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 4 methods.

6. The assay process is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G) or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. The method of paragraph 5, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 comprising a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

7. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of paragraph 5 or 6, wherein the step of assaying is performed in a multiplex format.

8. The method of any one of paragraphs 1-7, wherein the human has been determined to be substantially resistant to statin treatment, or further determined.

9. The method of any of paragraphs 1-8, wherein the human has received or has received statin treatment or has reduced responsiveness to statin treatment.

10. The method of paragraph 8 or 9, wherein the antibody or antibody fragment is administered to a human separately or simultaneously with the statin treatment.

11. The method of any one of paragraphs 5-7, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

12. Heterozygousness was shown for the nucleotide sequence in which the human encodes the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of paragraphs 1-11, wherein the human is shown to be homozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

13. The human being has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( The method of any one of paragraphs 1-12, diagnosed as having at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

14. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease. The method of any one of paragraphs 1-13, which treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

15. The method of any one of paragraphs 1-14, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

16. The method of any one of paragraphs 1-15, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in the injectable preparation.

In one alternative, instead of the method of "lowering cholesterol levels or maintaining previously lowered cholesterol levels" as mentioned herein, this method is a "human PCSK9-mediated condition or Treat and / or prevent disease (eg, associated with hypercholesterolemia, or hypocholesterolemia, or any other PCSK9-mediated disease or condition disclosed herein). " , This mutation is instead selected from the group R46L, A53V, N425S, A443T, I474V, Q619P and E670G. Thus, in this alternative method, any such embodiment is disclosed as documented herein, but with the mutations listed to be selected from this group. In one example, this mutation is R46L. In one example, this mutation is A53V. In one example, this mutation is N425S. In one example, this mutation is A443T. In one example, this mutation is I474V. In one example, this mutation is Q619P. In one example, this mutation is E670G.

Regimen
A: The present invention further provides the following regimens, ligands and kits.
1. Rare variant A method for treating a human PCSK9-mediated disease or condition in humans by targeting human PCSK9, which specifically binds to the PCSK9 variant in humans. Includes the step of administering a ligand (eg, an antibody or fragment) determined to be; where this human expresses the PCSK9 variant, or the human genome is a nucleotide sequence encoding the PCSK9 variant. Includes; where the human is treated for the disease or condition, the method.

In one alternative, Clause 1 provides:-
A method for targeting a rare variant human PCSK9, the step of administering to this human a ligand (eg, an antibody or fragment) determined to specifically bind to the PCSK9 variant. Includes; where the human expresses the PCSK9 variant, or the human genome comprises a nucleotide sequence encoding the PCSK9 variant. In certain embodiments, the human is treated for the disease or condition.

Variant PCSK9 may be, for example, any rare variant as described herein.

For example, the following is provided:
1a. A method for treating PCSK9-mediated diseases or conditions in humans by targeting PCSK9 containing the C-terminal domain amino acid gene polymorphism (compared to SEQ ID NO: 1), the human being. To a ligand (eg, antibody) determined to specifically bind to PCSK9 containing the C-terminal domain containing the PCSK9 mutation (eg, I474V or 670G) (numbered according to SEQ ID NO: 1). Or fragment) is administered; where the human expresses the PCSK9, or the human genome comprises a nucleotide sequence encoding the PCSK9; where the human is the disease or condition. How to be treated about.

For example, the following is provided:
1b. A method of targeting PCSK9 containing a C-terminal domain amino acid gene polymorphism (compared to SEQ ID NO: 1), wherein the PCSK9 mutation (eg, I474V or 670G) (SEQ ID NO: 1) is used in this human. Including the step of administering a ligand (eg, an antibody or fragment) determined to specifically bind to PCSK9 containing a C-terminal domain containing (numbered according to 1); where this human expresses the PCSK9. Or, the human genome comprises a nucleotide sequence encoding said PCSK9; optionally, the method by which the human is treated for said disease or condition.

In certain embodiments, the determination of the specific binding is by reference to the binding assay data, eg, using SPR or ELISA. The determination may refer to the information in the printed publication, eg, using knowledge of the data presented in the present invention or another patent application, or in a bibliographic article. Once such information is available (eg, without further testing of binding), one of ordinary skill in the art will identify relevant humans whose genotype or phenotype is compatible with the binding specificity of the ligand (instructions of the invention). Can be treated (by).
This antibody or fragment may be in any configuration, example, embodiment, embodiment, clause or paragraph herein.

In certain embodiments, the method selects a human comprising the nucleotide sequence encoding PCSK9 [where this human is said human in clause 1 (eg, 1a)] prior to the administration step. Includes steps.

2. The method of Clause 1, comprising determining, for example, using SPR or ELISA, that the ligand specifically binds to the PCSK9 prior to the administration step.

3. The method of Clause 1 or 2, wherein the specific binding to PCSK9 is a binding with a dissociation constant (Kd) of 1 nM or less, eg, 100, 10 or 1 pM or less.

4. The method of any of clauses 1-3 (eg, clause 1a), wherein the condition is an increase in LDL-cholesterol or is caused by an increase in LDL-cholesterol.

5. The method of Clause 4 (eg, independent of Clause 1a), where the condition is lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, stroke. , Stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

6. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. Any one method of Clauses 1-5 (eg, independent of Clause 1a) determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

7. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). Including a step of determining to include a nucleotide sequence encoding a variant protein, optionally, this determining step is performed prior to administration of this antibody to this human, clauses 1-6 (eg, clauses 1a). Is independent) any one way.

8. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 7 ways.

9. The assay process is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G), or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. Clause 8 method, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

10. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. Section 8 or 9 where the step of assaying is performed in a multiplex format.

11. The method of any one of Clauses 1-10, wherein the human is determined to be substantially resistant to statin treatment, or further determined.

12. The method of any of Clauses 1-11, wherein the human has received, has received, or has become less responsive to statin treatment.

13. The method of Clause 11 or 12, wherein the antibody or antibody fragment is administered to a human separately or simultaneously with the statin treatment.

14. The method of any one of clauses 8-10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

15. Heterozygous was shown for the nucleotide sequence in which the human encodes the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of Clauses 1-14, wherein the human is shown to be homozygous for a nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

16. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( Any one method of clauses 1-15, diagnosed as having at least one condition selected from, for example, lameness associated with elevated cholesterol) and hypertension.

17. The antibody or antibody fragment is a lipid disorder, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral blood vessels. Any one method of Clauses 1-16 that treats or reduces the risk in said humans of at least one condition selected from disease, lameness (eg, lameness associated with elevated cholesterol) and hypertension.

18. The method of any one of clauses 1-17, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

19. The method of any one of Clauses 1-18, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in an injectable preparation.

20. A ligand (eg, antibody or fragment) for use in any one of Clauses 1-19, wherein the ligand specifically binds to PCSK9.

21. A kit containing the ligand of Clause 20 and instructions for performing any one of Clauses 1-19.

B: The present invention further provides the following regimens, ligands and kits.
1. A way to lower or maintain previously lowered cholesterol levels in humans who need it:
A step of performing the initial treatment of the human during the initial treatment period by administering an anti-human PCSK9 ligand (eg, an antibody or fragment) to the human, wherein (i) the ligand is the PCSK9. It was determined to specifically bind to PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or 670G) (numbered according to SEQ ID NO: 1); (ii) humans express said PCSK9. Or this human genome contains the nucleotide sequence encoding PCSK9, and (iii) the human has undergone or has undergone statin treatment to lower or maintain cholesterol levels; Initial treatment with steps involving single or multiple doses of the ligand to humans;
b. (i) End statin treatment or (ii) refrain from statin treatment in humans; or (iii) with the step of determining to reduce statin treatment after the initial treatment period described above;
c. The step of continuing to administer the ligand to the patient after the expiration of the period, thereby lowering or maintaining previously lowered cholesterol levels in the human.
Including methods.

In certain embodiments, the determination of the specific binding is by reference to binding assay data, eg, using SPR or ELISA. The determination may refer to the information in the printed publication, eg, using knowledge of the data presented in the present invention or another patent application, or in a bibliographic article. Once such information is available (eg, without further testing of binding), one of ordinary skill in the art will identify relevant humans whose genotype or phenotype is compatible with the binding specificity of the ligand (instructions of the invention). Can be treated (by).

The antibody or fragment may be in accordance with any of the configurations, examples, embodiments, embodiments, clauses or paragraphs herein.

A pharmaceutically effective amount of the ligand is administered.

In certain embodiments, the method comprises selecting a human comprising the nucleotide sequence encoding PCSK9, where the human is mentioned in Clause 1, prior to the administering step. ..

In some cases, the initial treatment period is 7 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or 1 year. Is.

2. The method of Clause 1 where the person has, or is at risk of, or has an increased risk of statin-related memory impairment or statin-related neurodegenerative condition (eg, statin-related diabetes). ..

3. Prior to the initial treatment, the human has or has a statin-related memory disorder or statin-related neurodegenerative condition, or has diabetes (eg, statin-related diabetes), or The method of Clause 1 or 2, which includes the step of determining that the risk is increasing.

4. The method according to clause 2 or 3, wherein after step (b) [eg, during step (c)] includes the step of determining that the memory disorder or said neurodegenerative condition has been ameliorated.

5. The method of any one of clauses 1-4, wherein the person is over 40 years of age (eg, 50 or older, 55 or older, 60 or older, or 65 or older, or 70 or older).

6. Step 1 to include step (c) determining to increase the dose of the ligand to be administered after the initial treatment period and administering the increased dose to the human. Any one of the 5 methods.

7. The method of any one of Clauses 1-6, wherein step (b) comprises determining that a human is intolerant or refractory to treatment with a statin.

8. The method of any one of clauses 1-7, wherein the initial treatment comprises administration of statin, fenofibrate [eg, Tricor ™ or Lofibra ™] or ezetimibe to humans in addition to the ligand.

9. Clause 1-, wherein step (b) comprises a step of ending or reducing statin, fenofibrate [eg, Tricor ™ or Lofibra ™] or ezetimibe treatment during step (c). Any one of the eight methods.

10. The method of any one of Clauses 1-9, comprising step increasing the ligand dose during step (c) (ie, increasing the dose relative to the initial treatment dose).

11. The human has received a high dose of statin treatment prior to initial treatment, where step (c) is the step of applying a lower (eg, medium or low) dose of statin treatment in addition to the ligand. Any one method of clauses 1-10, including.
Those skilled in the art are familiar with the meaning of high-dose, medium-dose and low-dose treatments (and methods that are determined according to each patient's body mass, eg). For example, the statin is selected from rosuvastatin, atorvastatin and simvastatin.
For example, the daily dose of statin is:-Low is 10-20 mg (eg 10 mg); Medium is> 20 and <60 mg (eg 40 mg); High is 60-100 mg (eg 80 mg) ..

12. The human receives a medium dose of statin treatment prior to initial treatment, where step (c) comprises applying a lower (eg, lower) dose of statin treatment in addition to the ligand. Or one method of clauses 1-10 without administration of statins.

13. The method of any one of Clauses 1-10, wherein the human receives a low dose of statin treatment prior to initial treatment, where step (c) does not administer statin in addition to said ligand.

14. The method of any one of Clauses 1-13, comprising determining, for example, using SPR or ELISA, that the ligand specifically binds to PCSK9 prior to initial treatment.

15. The method of any one of clauses 1-14, wherein the specific binding to PCSK9 is a binding with a dissociation constant (Kd) of 1 nM or less, eg, 100, 10 or 1 pM or less.

16. Humans have lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( For example, the method of any of Clauses 1-15, at risk of a condition selected from (e.g., lameness associated with elevated cholesterol) and hypertension, or suffering from them.

17. The method of Clause 16 in which step (c) treats or reduces the risk of the condition in humans.

18. Humans have PCSK9 containing the C-terminal domain containing the mutation (eg, I474V or E670G) in SEQ ID NO: 1 and / or precursor protein convertase subtilisin / kexin 9 encoded by the nucleotide sequence of SEQ ID NO: 29 or 30. The method of any one of clauses 1-17, determined to contain a nucleotide sequence encoding a type (PCSK9) variant protein.

19. Humans have PCSK9 containing a C-terminal domain containing the mutation (eg, I474V or E670G) and / or precursor protein convertase subtilisin / kexin type 9 (PCSK9) containing the mutation (eg, I474V or E670G). Any one method of Clauses 1-18, comprising the step of determining to include the nucleotide sequence encoding the variant protein, optionally, this determining step is performed prior to administration of this antibody to this human. ..

20. The step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1. 19 methods.

21. The step of assaying is a biological sample and
Can it be specifically hybridized and identified in a biological sample to a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation in SEQ ID NO: 1 (eg, I474V or E670G)? The selected sequence in which the nucleic acid is absent in SEQ ID NO: 28, the at least one oligonucleotide probe comprising a sequence of at least 10 contiguous nucleotides that specifically hybridizes to the antisense of the sequence. PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) in SEQ ID NO: 1 by hybridizing to at least one nucleotide present therein or to an antisense sequence. Oligonucleotide probes forming a complex when at least one nucleotide sequence encoding is present; and / or
b. Containing at least 10 consecutive nucleotide sequences of the nucleotide sequence encoding PCSK9 containing the C-terminal domain containing the mutation in SEQ ID NO: 1 (eg, I474V or E670G), or antisense of said contiguous nucleotides. At least one oligonucleotide probe comprising a sequence, wherein the sequence of contiguous nucleotides comprises at least one nucleotide present in the selected sequence that is not present in SEQ ID NO: 28, thereby in SEQ ID NO: 1. If a nucleotide sequence encoding PCSK9 containing a C-terminal domain containing a mutation (eg, I474V or E670G) is present, the step of contacting with an oligonucleotide probe to form a complex; and detecting the presence of the complex. Clause 20 method, comprising detecting the presence or absence of a complex, thereby determining that a human comprises PCSK9 comprising a C-terminal domain comprising a mutation (eg, I474V or E670G) in SEQ ID NO: 1.

22. The assaying step involves nucleic acid amplification and, optionally, one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification and / or this. The method of clause 20 or 21 where the step of assaying is performed in a multiplex format.

23. The method of any one of Clauses 1-22, wherein the human is determined to be substantially resistant to statin treatment, or further determined.

24. The method of any one of clauses 20-22, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

25. Heterozygousness was shown for the nucleotide sequence in which the human encodes the PCSK9C terminal domain containing the mutation (eg, I474V or E670G), and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 28. The method of any one of Clauses 1-24, wherein the human is shown to be homozygous for the nucleotide sequence encoding the PCSK9C terminal domain containing the mutant I474V or E670G in SEQ ID NO: 1. ..

26. The human has lipid disorders, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, hypercholesterolemia, heart attack, stroke, coronary heart disease, atherosclerosis, peripheral vascular disease, lameness ( For example, any one method of clauses 1-25, diagnosed as having at least one condition selected from lameness associated with elevated cholesterol) and hypertension.

27. The method of any one of clauses 1-26, wherein the nucleotide sequence is SEQ ID NO: 29 or 30.

28. The method of any one of clauses 1-27, wherein said ligand (eg, antibody or antibody fragment) is administered by intravenous or subcutaneous administration and / or is included in an injectable preparation.

29. A ligand (eg, antibody or fragment) for use in any one of Clauses 1-28, which specifically binds to PCSK9.

30. A kit containing the ligand of Clause 29 and instructions for performing any one of Clauses 1-28.

In an example of any aspect of the invention, the ligand (eg, antibody or fragment, eg, alirocumab, bocobizumab or evolocumab, or antibody or fragment comprising the variable domain of 316P or 31H4) is 75-150 mg to humans 2 Administered in weekly doses (eg, 75-150 mg once or twice over 2 weeks). In one example, this ligand is for such administration to humans.

Determining specific binding of the ligand of the present invention to the PCSK9 variant Method for determining binding SPR
Binding of antibodies to the PCSK9 variant was performed by SPR using the ProteOn XPR36 ™ Array system (BioRad). Anti-human IgG surfaces (Jackson Labs 109-005-008) were made with GLC Biosensor chips by primary amine coupling. The test antibody was captured as a ligand on this surface. The PCSK9 variant was used as an analyte and passed over the captured antibody at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. The binding curve was doubly referenced with buffer injectate (ie 0 nM) to remove baseline drift and injectable artifacts. Regeneration of the capture surface uses 100 mM phosphate, which removes the captured antibody, allowing another cycle of capture and binding. The generated coupling sensorgrams were analyzed using a 1: 1 model specific to the ProteOn XPR36 Array system analysis software. This assay is performed at 25 ° C. using 1 × HBS-EP (Teknova) as an electrophoresis buffer.

data
The three antibodies were tested and the binding data obtained are shown below [Table 3]. Antibodies 316P and 31H4 are antibodies disclosed in US Patent Application Publication No. 20110065902A1 (these antibodies and their variable domain sequences are possible uses in the present invention and in the claims herein. Incorporated herein by reference for possible inclusion). Antibody 316P is a heavy chain variable domain derived from recombination of human VH3-23 * 04 and JH2 * 01 (with D), and light derived from recombination of human V _K 4-1 * 01 and J _K 2 * 01. Includes chain variable domain.

Eborokumab is a recombinantly derived VH of human IGHG2 * 01 heavy chain and human IGLC2 * 01 lambda light chain, human IGHV1-18 * 01 and IGHJ6 * 01 (with D segment), and human IGLV2-14 * 01 and IGU2. * 01 Contains Vλ derived from recombination.

Results This result shows that all antibodies tested bind equally to the PCSK9 variant, and any binding variation seen here is within experimental error for such strong affinity interactions. Was shown.

Therefore, the present invention determines that an antibody having the following profile can specifically bind to one or more variants of the present invention:-
1. Antibodies (eg, 316P or alirocumab) that are recombinantly derived heavy chain variable domains of human IGHV3-23 * 04 and IGHJH2 * 01 (with D), as well as human IGKV4-1 * 01 and IGKJ2 * 01. An antibody containing a light chain variable domain derived from the recombination of
2. Antibodies (eg, evolocumab), human heavy chain variable domains derived from recombination of human IGHV1-18 * 01 and IGHJ6 * 01 (with D segment), and human IGLV2-14 * 01 and IGLJ2 * 01. An antibody comprising a light chain variable domain derived from the recombination of.

Therefore, according to the present invention, one of ordinary skill in the art will have the necessary determination of specific binding of the ligand to the PCSK9 variant. The application of this determination is described above in the context of the methods and other aspects of the invention.

References:
Horton et al, Trends Biochem Sci. 2007 Feb; 32 (2): 71-7. Epub 2007 Jan 9, Molecular biology of PCSK9: its role in LDL metabolism.
Seidah and Prat, J Mol Med (Berl). 2007 Jul; 85 (7): 685-96. Epub 2007 Mar 10, The proprotein convertases are potential targets in the treatment of dyslipidemia.
Benjannet et al, J Biol Chem. 2004 Nov 19; 279 (47): 48865-75. Epub 2004 Sep 9, NARC-1 / PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.
Lagace et al, J Clin Invest. 2006 Nov; 116 (11): 2995-3005, Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.
Maxwell et al, Proc Natl Acad Sci US A. 2005 Feb 8; 102 (6): 2069-74. Epub 2005 Jan 27, Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment.
Park et al, J Biol Chem. 2004 Nov 26; 279 (48): 50630-8. Epub 2004 Sep 22, Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin / kexin type 9a in mouse liver.
Rashid et al, Proc Natl Acad Sci US A. 2005 Apr 12; 102 (15): 5374-9. Epub 2005 Apr 1, Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9.
Kotowski et al, Am J Hum Genet. 2006 Mar; 78 (3): 410-22. Epub 2006 Jan 20, A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol.
Chen et al, J Am Coll Cardiol. 2005 May 17; 45 (10): 1611-9. Epub 2005 Apr 21, A common PCSK9 haplotype, comprising the E670G coding single nucleotide polymorphism, is a novel genetic marker for plasma low-density lipoprotein cholesterol levels and severity of coronary atherosclerosis.
Pisciotta et al, Atherosclerosis. 2006 Jun; 186 (2): 433-40. Epub 2005 Sep 23, Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia.
Zhao et al, Am J Hum Genet. 2006 Sep; 79 (3): 514-23. Epub 2006 Jul 18, Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.
Seidah et al, Proc Natl Acad Sci US A. 2003 Feb 4; 100 (3): 928-33. Epub 2003 Jan 27, The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation ..

(Example 2)
IL4 receptor alpha (IL4Ra; CD124)
Interleukin-4 (also known as IL-4, B cell stimulator or BSF-1) was originally characterized by its ability to stimulate 9 on B cells in response to low concentrations of antibodies to surface immunoglobulins. It was. IL-4 has been shown to possess a broad spectrum of biological activity, including proliferative stimulation of T cells, mast cells, granulocytes, megakaryocytes and erythrocytes. IL-4 induces the expression of class II major histocompatibility complex molecules in resting B cells and enhances the secretion of IgE and IgGI isotypes by stimulated B cells.

In one example, the invention is a method of treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof and specifically binds to the human IL4RA protein to said human. Provided is a method comprising the step of administering a ligand (eg, an antibody or antibody fragment). The present invention also provides a corresponding ligand.

The present invention provides anti-IL4Ra ligands; as well as IL4Ra binding or targeting ligands as described herein. This ligand has various usefulness. Some of these ligands are other antagonists of IL4Ra activity in affinity purification of IL4Ra (particularly human IL4Ra or its ligands) to genotype or phenotype humans, for example in specific binding assays. It is useful in screening assays to identify. Some ligands of the invention are useful for inhibiting the binding of IL4Ra to IL4 or for inhibiting IL4Ra-mediated activity.

Anti-IL4Ra ligands (eg, antibodies and antisense RNA) have been developed based on the targeting and neutralization of so-called "wild" human IL4Ra, which is a commonly present type (eg, SEQ ID NO: 67). See). Although such treatments are useful in human patients carrying this type of human IL4RA, we have targeted a rare but also naturally occurring type of IL4Ra in the human population. Was considered useful for investigating the possibility of. In this way, the inventor is a useful target for human treatment, prevention and diagnosis (protein or nucleic acid level) that is mediated by IL4RA activity or appropriate for diseases and conditions associated thereto. We have reached an insight into the natural appearance and distribution of rare human IL4Ra types that may function. This provides tailor-made treatment, prevention and diagnosis, especially in humans lacking the common IL4Ra gene or protein.

Those skilled in the art are aware that SNPs or other changes that translate into amino acid variations can result in variability in the activity and / or higher-order structure of the human target to be targeted. This has led to great interest in personalized medicine that uses knowledge of genotyping and protein and nucleotide variability to more effectively tailor-make patient care and diagnosis. Accordingly, the present invention provides tailor-made medicines and studies specifically directed to the rare variant of the IL4Ra polymorph. Such forms or "alleles" (at the nucleotide level) contain one or more changes at the nucleotide and amino acid levels from the corresponding common types of nucleotide and amino acid sequences, i.e., protein targets in humans. There are one or more non-synonymous changes at the nucleotide level that translate into one or more corresponding changes in.

Moreover, the inventor surprisingly found that rare natural forms are present in humans much less frequently than common forms, but are nevertheless shown in large and ethnically diverse human populations. We have noticed that there are usually many human examples per ethnic group shown. Thus, by targeting such rare types, the inventor provides effective treatment, prevention or diagnosis across many human ethnic groups, thereby expanding the usefulness of the invention. I noticed.

With this recognition, the inventor is concerned with guiding the selection of anti-IL4Ra ligands for administration to human patients for the treatment and / or prevention of IL4Ra-mediated or related diseases or conditions. I noticed that there are various industrial and medical applications. In this method, the patient receives tailor-made drugs and ligands for their needs as determined by the composition of the patient's genes or phenotypes. Along with that, the present invention provides patient genotyping and / or phenotyping associated with such treatments, thereby allowing the drug to be appropriately adapted to the patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment (eg, poor efficacy and / or side effects) with drugs or ligands that are not suitable for the patient, and avoids drug prescribing mistakes and waste. ..

To develop this idea, in this non-limiting example, the inventor decided to determine a set of human IL4Ra variants based on the following criteria, but these criteria were noticed by the inventor. Provides medical drugs and diagnostics useful for tailor-made needs in the human population. We have selected variants that have at least three of the following four criteria:
Naturally occurring human IL4Ra variation with a cumulative human allele frequency of 35% or less;
Naturally occurring human IL4Ra variation with a total human genotype frequency of about 50% or less;
Naturally occurring human IL4Ra variations found in many different human populations [using standard categorization of the 1000 Genomes Project; see Table 12 below]; and many such different races A naturally occurring human IL4Ra variation found in many individuals distributed across populations.

Based on these criteria, the inventor identified the variants listed in Table 11 below. In our choice, as an additional or alternative study, the nucleotide variation that produced the amino acid variation in the corresponding IL4Ra type, as opposed to the silent variation that did not alter the amino acid residue in the target protein (ie, non-). Included selections for (synonymous variations).

In one example, the invention is a method of treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, with respect to said human, I75V, E400A, C431R in SEQ ID NO: 67. , S503P, Q576R and S752A. A method comprising the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to a human IL4RA protein comprising a mutation selected from the group. As further explained below, these amino acid variations are found in the naturally occurring IL-4Ra variants in humans found in many populations. The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

One example provides a ligand (eg, an antibody or antibody fragment) for treating or reducing the risk of an IL4Ra-mediated disease or condition in a human in need thereof, the method of which is described in the human. It comprises the step of administering the ligand, wherein the ligand specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. .. The human comprises a nucleotide sequence encoding the IL4RA protein, comprising said mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In one example, the invention is a method of targeting IL4Ra in humans, the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 to said human. Provided is a method comprising the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to a human IL4RA protein comprising. The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In some cases, this human has or is at risk for an IL4Ra-mediated disease or condition. In some examples, this method treats or reduces the risk of IL4Ra-mediated disease or condition in humans.

In one example, a ligand (eg, an antibody or antibody fragment) for targeting IL4Ra in humans is also provided, the method comprising administering the ligand to the human, wherein the ligand. Specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The human comprises a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. In some cases, this human has or is at risk for an IL4Ra-mediated disease or condition. In some examples, this method treats or reduces the risk of IL4Ra-mediated disease or condition in humans.

In certain embodiments, (i) the antibody or fragment comprises a VH domain from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment comprising Framework 1 of SEQ ID NO: 40. Encoding and where the human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40. And here (ii) the human comprises a nucleotide sequence encoding the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. ..

Further, or instead, in one example, (i) this antibody or fragment comprises the Leu at position 189 found in SEQ ID NO: 73 or the Arg at position 289 found in SEQ ID NO: 73, a human gamma-4 heavy chain constant region. And here the human comprises the IGHG4 * 01 human heavy chain constant region gene segment, or this human is at position 189 Leu found in SEQ ID NO: 73 or position 289 found in SEQ ID NO: 73. Human gamma containing Arg-4 antibodies containing the heavy chain constant region; and where (ii) said human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Contains a nucleotide sequence encoding the IL4RA protein containing the mutation.

The biological activity of IL-4 is mediated by specific cell surface receptors of IL-4. Human IL-4 receptor alpha (hlL-4Ra) is described, for example, in US Pat. Nos. 5,599,905, 5,767,065, and 5,840,869. Antibodies to hlL-4R are described in US Pat. No. 5,717,072.

Methods using antibodies against hIL-4R are described in US Pat. Nos. 5,714,146; 5,985,280; and 6,716,587.

The human IL-4Ra (also known as IL-4Ra) subunit (Swiss Prot accession number P24394) is a 140 kDa type 1 membrane protein that binds to human IL-4 with high affinity [Andrews et al. J. Biol. Chem ( 2002) 277: 46073-46078]. The IL-4 / IL-4Ra complex is a dimer with either a common gamma chain (γc, CD132) or IL-13R alpha 1 (IL-13Ra1) subunit via a domain on IL-4. To create two different signaling complexes, each commonly referred to as type I and type II receptors. Alternatively, IL-13 can bind to IL-13Ra1 to form the IL-13 / IL-13Ra1 complex, which supplements the IL-4Ra subunit to form a type II receptor complex. Thus, IL-4Ra mediates the biological activity of both IL-4 and IL-13 (reviewed by Gessner et al., Immunobiology, 201: 285, 2000). In vitro studies have shown that IL-4 and IL-13 have a number of cell types, such as T cells, B cells, eosinophils, mast cells, basal cells, airway smooth muscle cells, respiratory epithelial cells, and lung fibers. It has been shown to activate effector function in blast and endothelial cells (reviewed by Steinke et al., Resp Res, 2:66, 2001, and by Willis-Karp, Immunol Rev, 202: 175, 2004). ..

IL-4 is essential for the development of inflammation of the airways present in asthma through its binding to its receptor (IL-4R), which induces IgE synthesis in B cells and differentiation of T cells into the Th2 phenotype. .. Dupilumab is a monoclonal antibody designed for the treatment of atopic diseases. Dupilumab binds to the alpha subunit of the interleukin-4 receptor. Through blocking IL-4R alpha, dupilumab modulates the signaling of both the IL-4 and IL-13 pathways involved in the pathophysiology of allergic diseases. The anti-IL4Ra ligands, antibodies and fragments according to the invention may also be useful, for example, to treat or reduce the risk of allergic diseases.

In some examples, this ligand, antibody or fragment is to treat or reduce the risk of allergic asthma, eosinophilic asthma or atopic dermatitis (or treat or reduce that risk), optionally. This antibody is dupilumab.

In addition to its role in asthma, IL-4Ra has been associated with a number of other pathologies, eg, the anti-IL4Ra ligands, antibodies, and fragments according to the invention, eg, these diseases or Can be used to treat or reduce the risk of any one of the conditions):

Chronic obstructive pulmonary disease (COPD) includes a population of patients with varying degrees of chronic bronchitis, peripheral airway disease, and emphysema, and is progressive and irreversible with poor response to current asthma-based treatments. Characterized by decreased lung function. The causes behind COPD are not yet fully understood. The Dutch Hypothesis suggests that there is a common susceptibility to COPD and asthma, and therefore similar mechanisms can contribute to the pathogenicity of both disorders (Sluiter et al., Eur Respir J). Zheng et al. [J Clin Invest, 106 (9): 1081-93, 2000] found that overexpression of IL-13 in the lungs of mice resulted in emphysema, increased mucus production and inflammation, and aspects of human COPD. Demonstrated that it affected. Furthermore, AHR (IL-13-dependent response in a mouse model of allergic inflammation) has been shown to be a precursor to pulmonary dysfunction in smokers [Tashkin et al., Am J Respir Crit Care Med, 153 ( 6 Pt 1): 1802-11, 1996]. It was also determined that there is an association between genetic polymorphisms in the IL-13 promoter and susceptibility to developing COPD [Van Der Pouw Kraan et al., Genes Immun, 3 (7): 436-9, 2002]. Therefore, the symptom is that the IL-4 / IL-13 pathway (and especially IL-13) plays an important role in the pathogenicity of COPD.

In addition to asthma, the IL-4 / II-13 pathway is associated with other fibrotic conditions, such as systemic sclerosis [Hasegawa et al., J Rheumatol, 24 (2): 328-32, 1997], pulmonary fibrosis. Disease [Hancock et al., Am J Respir Cell Mol Biol, 18 (1): 60-5, 1998], Parasite-induced liver fibrosis [Fallon et al., J Immunol, 164 (5): 2585-91, 2000 Chiaramonte et al., J Clin Invest, 104 (6): 777-85, 1999; Chiaramonte Hepatology 34 (2): 273-82, 2001], and cystic fibrosis [Hauber et al., J. Cyst Fibr 2: It was related to p. 189, 2003].

IL-4, and to some extent IL-13, are important for B cell-mediated activities such as B cell proliferation, immunoglobulin secretion, and Fcepsilon R expression. Clinical applications of IL-4Ra inhibitors include, for example, use in allergy therapy to suppress IgE synthesis (including, for example, atopic dermatitis and food allergies), and graft therapy to prevent graft rejection. Use in, as well as suppression of delayed or contact hypersensitivity reactions.

Il-4R antagonists can also be found to be used as adjuvants for allergic immunotherapy and as vaccine adjuvants.

IL-4Ra gene polymorphisms in the human population have been described (reviewed by Gessner et al., Immunobiology, 201: 285, 2000), and some populations are associated with IgE levels or clinical atopy. It is reported in. For example, V75R576 IL-4Ra was associated with allergic asthma and improved the function of IL-4Ra [Risma et al. J. Immunol. 169 (3): 1604-1610, 2002].

The anti-IL4Ra ligands, antibodies and fragments according to the invention optionally neutralize IL-4Ra with high titers, eg, as detailed in Examples of European Patent No. 2604628. Neutralization means inhibition of biological activity mediated by IL-4Ra. The ligands, antibodies and fragments according to the invention can neutralize one or more activities mediated by IL-4Ra. Inhibition of this biological activity prevents the gamma chain (or IL-13Ra) and related soluble ligands, such as either IL-4 or IL-13, from forming a signaling complex with IL-4Ra. It can be mediated by things.

Neutralization of IL-4 or IL-13 signaling through its IL-4Ra-containing receptor complex can be measured by inhibition of IL-4 or IL-13-stimulated TF-1 cell proliferation.

The ligands, antibodies or fragments according to the invention are, for example, dupilumab, or disclosures thereof (including sequences such as VH, VL and heavy and light chain sequences) are possible in one or more claims herein. Any of those disclosed in WO 08/054606 (Regeneron), European Patent No. 2604628 (Medimmune), WO 01/92340 (immnunex) or WO 05/047331 (Immunex), which is incorporated herein by reference for sexual inclusion. Or one. According to a further aspect of the ligand, antibody or fragment according to the invention, it can bind to human interleukin-4 receptor alpha (hlL-4Ra) and cynomolgus monkey interleukin-4 receptor alpha (cyIL-4Ra). The cDNA sequence of cynomolgus monkey IL-4Ra is shown in European Patent No. 2604628 as SEQ ID NO: 455. In certain embodiments, the antibody or fragment is a human antibody or fragment. A ligand, antibody or fragment according to the invention is, for example, an antibody or fragment comprising VH and / or VL of dupilumab, or a heavy and / or light chain of dupilumab. In one embodiment, the ligand, antibody or fragment comprises a VH domain comprising SEQ ID NO: 69. In one embodiment, the ligand, antibody or fragment comprises a VL domain comprising SEQ ID NO: 70. In one embodiment, the ligand, antibody or fragment comprises a heavy chain comprising SEQ ID NO: 71. In one embodiment, the ligand, antibody or fragment comprises a light chain comprising SEQ ID NO: 72.

In certain embodiments, the ligand, antibody or fragment of the invention comprises an Fc region, wherein the Fc region is numbered with an EU index as shown in Kabat, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L , 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V , 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S , 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L , 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H , 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C , 331Y, 331H, 331R, 331N, 3 Group consisting of 31D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W Contains at least one unnatural amino acid residue selected from. Optionally, this Fc region may contain additional and / or alternative non-natural amino acid residues known to those of skill in the art (eg, US Pat. No. 5,624,821; 6,277,375; 6,737,056; See PCT International Publication Nos. WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217).

Ligands, antibodies or fragments according to the invention are incorporated herein by reference for possible inclusion of the disclosed diseases and conditions in one or more claims herein. Disclosed in any of 054606 (Regeneron), European Patent No. 2604628 (Medimmune), WO 01/92340 (immnunex) and WO 05/047331 (Immunex), eg, treating or preventing or reducing the risk of any disease or condition. To (or to treat, prevent, or reduce that risk).

Further included by the present invention is the use of its ligand, antibody or fragment in the manufacture of a medicament for use in attenuating or inhibiting an IL-4Ra-mediated disease or disorder in humans. IL-4Ra-mediated or related disorders treated with the ligands, antibodies or fragments of the invention include, for example, arthritis (including septic arthritis), herpes-like diseases, chronic idiopathic urticaria, strong skin. Disease, hypertrophic scar formation, whipple disease, benign prostatic hyperplasia, lung disorders, such as mild, moderate or severe asthma, inflammatory disorders, such as inflammatory bowel disease, allergic reactions, Kawasaki disease, sickle red disease, Examples include Churgstrauss syndrome, Graves' disease, pregnancy hypertensive nephropathy, Sjogren's syndrome, autoimmune lymphocyte proliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune vasculitis, tuberculosis, and nephrosis.

Specific conditions in which the ligands, antibodies or fragments of the invention can be used for treatment or diagnosis include: asthma, COPD (eg, chronic bronchitis, peripheral airway disease or pulmonary emphysema), inflammatory bowel disease, fibrotic condition (eg, eg). To prevent systemic sclerosis, pulmonary fibrosis, parasite-induced liver fibrosis, or cystic fibrosis), allergies (eg, atopic dermatitis, house mite allergies, pet allergies or food allergies), transplant rejection Transplantation therapy, suppression of delayed hypersensitivity or contact hypersensitivity reactions (as an adjuvant for allergic immunotherapy or as a vaccine adjuvant). In some cases, this method and ligand is to treat or prevent (or reduce the risk of) nasal polyps and / or sinusitis.

Therefore, the ligands, antibodies or fragments of the present invention are useful as therapeutic agents in the treatment of conditions involved in the expression and / or activity of IL-4, IL-13 or IL-4Ra. In particular, one embodiment is a method of treatment comprising administering to a patient in need of an effective amount of a ligand, antibody or fragment of the invention, wherein the functional consequences of IL-4Ra activation are. It will be reduced. Another embodiment is, among other things, (i) a step of identifying a patient exhibiting expression or activity of IL-4, IL-13 or IL-4Ra, and (ii) an effective amount of a ligand, antibody or or of the invention. A method of treatment involving the administration of a fragment to the patient, where the functional consequences of IL-4Ra activation are diminished. An effective amount according to the invention is the severity of at least one symptom of the particular disease or disorder being treated by modulating (eg, reducing) the functional consequences of IL-4Ra activation. Is an amount that modulates (eg, reduces or weakens), but does not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, in an amount effective for a patient in need thereof. One or more ligands, antibodies or fragments of the invention, alone or in a therapeutic regimen known in the art or in combination with another suitable medicament described herein. A method that comprises the step of administration, such that the severity of at least one symptom of the disorder is reduced as a result. Another embodiment of the invention is, among other things, a method of antagonizing at least one effect of IL-4Ra, contacting or contacting an effective amount of one or more ligands, antibodies or fragments of the invention. The at least one effect of IL-4Ra is antagonized, eg, the ability of IL-4Ra to form a complex (a precursor to active signaling) with IL-4. The method.

The ligands, antibodies or fragments of the invention may be used, for example, in combination with other therapeutic agents for the treatment of cancer. Suitable agents that may be used in combination include those disclosed in European Patent No. 2604628 (eg, paragraph [0287]), the disclosure of which is incorporated herein by reference.

Tailor Made Antibodies to Rare IL4Ra Variant Profiles As outlined herein (eg, in the context of PCSK9 in Example 1), the present invention finds that the TOI type of variant meets the selection criteria of the present invention. It further includes the possibility of tailoring human treatment by selecting antibody-based ligands with variable and / or constant domains based on the genetic segments found in many humans of the ethnic population. This also applies mutatis mutandis when the TOI is human IL4Ra, as in this example. Thus, all disclosures herein regarding tailor-making of variable and / or constant domains apply to this embodiment with respect to IL4Ra and can be combined with use in one or more claims herein. is there.

As described in Example 1, in some examples, a ligand comprising an antibody VH domain from a recombination of a human IGHV gene segment comprising a nucleotide selected at a position in HCDR1 or FW3 that is variable in human is provided. (That is, SNPs are present in humans here).

Further information is provided in Table 4, which also shows variations at these locations and variant distributions across the 1000 Genomes Project database associated with many human populations.

In other embodiments, as described in more detail above, the invention is based on an alternative human VH gene segment, or V _K , V _λ or constant region gene segment, and the genotype of the human recipient and /. Alternatively, provide tailor-made ligands for the phenotype [see further Table 9 for representative variants].

In one example, according to this guidance, the selected ligand is dupilumab.

In a further example, therefore:-
(i) Here, the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a human JH segment, which human VH segment is SEQ ID NO: 40. The human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human comprises Framework 1 of SEQ ID NO: 40. Express the VH domain.
In one alternative, this example provides (i):-
Here, this ligand contains a VH domain derived from recombination of a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a human JH segment, and this human VH segment contains SNP rs56069819. And, here, the human comprises a VH gene segment containing SNP rs56069819.
(ii) Here, this ligand comprises a human VH segment IGHV3-7 * 01, a human D gene segment and a recombination-derived VH domain of a human JH segment, wherein the human has an IGHV3-7 * 01 VH gene segment. Included or this human expresses the VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment.
(iii) The ligand comprises the human Vκ segment IGKV1-12 * 01 and the recombinantly derived Vκ domain of the human Jκ segment, and the human comprises the IGKV1-12 * 01 Vκ gene segment, or the human contains the human Vκ segment. It expresses the Vκ domain derived from recombination of IGKV1-12 * 01 and human Jκ segment.
(iv) The ligand comprises the Vκ domain from the recombination of the human Vκ segment and the human Jκ segment, and the human Vκ segment encodes (i) CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, said human. Does it contain the Vκ gene segment encoding CDR3 containing Pro at position 7 shown in SEQ ID NO: 36, or does human express the Vκ domain containing CDR3 containing Pro at position 7 shown in SEQ ID NO: 36? Or (ii) the Vκ gene segment encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38 and the human encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38, or Humans express the Vκ domain containing FW3, which contains Ser at position 15 shown in SEQ ID NO: 38.
(v) The ligand contains a human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human is (i) IGHG1 * 01 human. An antibody containing a heavy chain constant region gene segment or containing a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4 is expressed. ..
(vi) The ligands are Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 and Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala at position 257 shown in SEQ ID NO: 6, and whether the human contains (i) IGHG2 * 01 human heavy chain constant region gene segment. , Or human, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Val or the antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown in SEQ ID NO: 6.
The (vii) ligand comprises a human kappa chain constant region containing Val at position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16, and the human is (i) IGKC1 * 01 human kappa chain constant region. An antibody containing a gene segment or containing a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16 is expressed.
(viii) the ligand comprises a human IGLC1 * 01 lambda chain constant region, said human containing (i) a human IGLC1 * 01 lambda chain constant region gene segment, or a human containing a human IGLC1 * 01 lambda chain constant region. Expresses an antibody containing.
(ix) The ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human. An antibody comprising a heavy chain constant region gene segment or containing a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73 is expressed. ..

For example, as in Example (ix), the inventor identified the possibility of addressing variations of IGH-gamma-4 and identified the usefulness of variations 189L and 289R individually or in combination. This is because these residues are part of the CH3 domain and, as such, they form part of the antibody Fc region. Therefore, these CH3 variations and patient compatibility are particularly beneficial for the reasons discussed above. Thus, in this example, the ligand of the invention comprises or comprises an antibody comprising a human gamma-4 heavy chain constant region comprising Leu corresponding to position 189 of SEQ ID NO: 73 or Arg corresponding to position 289 of SEQ ID NO: 73. Consisting of that antibody, and where this human genome contains a gamma-4 heavy chain constant region nucleotide sequence encoding such Leu and / or Arg, or this human is such Leu and / Alternatively, it expresses an antibody containing the human gamma-4 constant region containing Arg. An example of such a ligand is dupilumab.

Determination of specific binding of the ligand of the invention to the IL4RA variant
Specific binding of the ligand of the present invention to the IL4RA variant may be carried out using the SPR method described in Example 1.

Embodiments are provided as follows:-

How to use VH's TaylorMade
1. A method for treating or reducing the risk of IL4Ra-mediated disease or condition in humans in need thereof, for the human beings I75V, E400A, C431R, S503P, Q576R in SEQ ID NO: 67. And the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to a human IL4Ra protein containing a mutation selected from the group consisting of S752A;
Here (i) this ligand contains a VH domain derived from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40. And here, the human comprises a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here ( ii) The method, wherein the human comprises a nucleotide sequence encoding the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In one alternative, Clause 1 provides:-
A method of targeting IL4Ra in humans for human IL4Ra proteins containing mutations selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 to said humans. Including the step of administering a specifically binding ligand (eg, an antibody or antibody fragment).
Here (i) this ligand contains a VH domain derived from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40 and Here, said human contains a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here ( ii) The method, wherein the human comprises a nucleotide sequence encoding the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In some cases, this human has or is at risk for an IL4Ra-mediated disease or condition.

In some examples, this method treats or reduces the risk of IL4Ra-mediated disease or condition in humans. Optionally, the disease is asthma.

Optionally, the disease is atopic dermatitis. Optionally, the disease is nasal polyps and / or sinusitis.

In some examples of the methods or ligands of the invention, this IL4Ra comprises a mutant I75V.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant E400A.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant C431R.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant S503P.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant Q576R.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant S752A.

2. The method of Clause 1, wherein IL4Ra contains mutations I75V and Q576R in SEQ ID NO: 67 and, optionally, the disease is asthma. Optionally, the disease is atopic dermatitis. Optionally, the disease is nasal polyps and / or sinusitis.

Q576R and I75V are associated with airway inflammatory diseases such as asthma. For example, Clin Exp Allergy. August 2003; 33 (8): 1111-7, "Polymorphisms in the interleukin-4 and interleukin-4 receptor alpha chain genes confer susceptibility to asthema and atopy in a Caucasian population", Beghe B Et al. (Refers to I75V instead of I50V); J asthma. April 2010; 47 (3): pp. 238-44. Doi: 10.3109 / 02770900903509099, "Association and gene-gene interactions of eight common single-nucleotide polymorphisms with pediatric asthma in middle china ”, Wu X et al.; Clin Exp Allergy. October 2004; 34 (10): pp. 1570-5,“ Haplotypes of the interleukin-4 receptor alpha chain gene associate with susceptibility to and severity of atopic asthma ”. , Hytonen AM et al. (All incorporated herein by reference).

3. The method of Clause 1 or 2, wherein the nucleotide sequence of (ii) comprises a nucleotide mutation-3223T (dB SNP numbering).

This variation is associated with airway inflammatory diseases such as asthma.

4. The method of any of the aforementioned provisions, wherein each said human VH gene segment comprises SEQ ID NO: 39.

5. The method of any of the aforementioned provisions, wherein the VH gene segment contained in the human is a germline VH gene segment.

6. The method of any of the aforementioned provisions, comprising the step of selecting a human comprising the nucleotide sequence of (ii) prior to the said step of administration, which is the human of Clause 1.

7. A nucleotide sequence encoding the IL4Ra protein in which a human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 and / or I75V, E400A in SEQ ID NO: 67. , C431R, S503P, Q576R and S752A, the method of any of the aforementioned provisions, determined to include the IL4Ra protein containing the mutation selected from the group.

8. A nucleotide sequence in which humans encode an IL4Ra protein containing said mutation selected from the group consisting of (a) I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, and / or sequence (b). Including the step of determining to include the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A at number 67, and optionally, this determining step is this human. The method of any of the aforementioned provisions performed prior to administration of this antibody against.

9. A human-derived biological sample for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Clause 8 method, including the step of assaying.

10. The step to be assayed is at least 10 of the nucleotide sequence encoding the biological sample and the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A step of contacting with at least one oligonucleotide probe comprising a sequence of contiguous nucleotides or comprising an antisense sequence of said contiguous nucleotide, wherein the sequence of contiguous nucleotides is SEQ ID NO: Containing a nucleotide sequence encoding the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in 67, thereby from I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. If at least one nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group is present, a step of forming a complex and a step of detecting the presence or absence of this complex, wherein the complex is present. The step of detecting the presence of the complex determines that the human comprises an IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Clause 9 method, including steps.

11. The assaying step comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification, and / or here. The method of Clause 9 in which this assaying step is performed in a multiplex format.

12. The method of Clause 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human has been shown to be heterozygous for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. , Optionally, the human is shown to further comprise the nucleotide sequence of SEQ ID NO: 68, or the human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The method of any of the aforementioned provisions, which is shown to be homozygous for a nucleotide sequence encoding an IL4Ra protein containing the mutation.

14. The method of any of the aforementioned provisions, wherein the human is substantially resistant to, or is further determined to be, to the treatment of asthma.

15. The method of any of the aforementioned provisions, wherein the person has been or has been treated for asthma or has had reduced responsiveness to asthma treatment.

16. Is the disease or condition an inflammatory disease or condition; an atopic disease or condition; a respiratory disease or condition; a disease or condition associated with elevated IgE; or an increased IL-4 and / or IL-13 activity? The method of any of the aforementioned provisions that is a disease or condition associated with.

17. The disease or condition is an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome , Endocytoma, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, aspergillus tumor, aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophil pneumonia, Idiopathic pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous acidobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary radiomyopathy, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary arterial embolization , Pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis, any The method of the above clause.

18. The human has an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, cystic Fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome, endothelium Tumor, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, Aspergillus tumor, Aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophilic pneumonia, idiopathic Pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous mycobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary bacteriolysis, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary artery embolization, pneumonia At least one condition selected from the group consisting of pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary vein obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. The method of any of the aforementioned provisions diagnosed as having.

19. The antibody or antibody fragment is an inflammatory disease or condition of the airway, chronic obstructive pulmonary disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation. , Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, medial disorder, diaphragm disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress Syndrome, Endocytoma, Memoroma, Transplant rejection, Transplant vs. host disease, Lung cancer, Allergic rhinitis, Allergy, Aspergillosis, Aspergillus tumor, Aspergillosis, Bronchial dilatation, Chronic bronchitis, Pulmonary emphysema, Eosinophilic pneumonia , Idiopathic pulmonary fibrosis, Invasive pneumococcal disease, Influenza, Non-tuberculous acid bacillus, Pulmonary fluid, Dust pneumonia, Subcutaneous emphysema, Pneumonia, Pulmonary radiculopathy, Alveolar proteinosis, Pulmonary ulcer, Pulmonary edema, Pulmonary artery At least one selected from the group consisting of embolization, pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. The method of any of the aforementioned provisions that treats or reduces the risk of one condition in said human.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs1805010, rs1805011, rs1805012, rs1805015, rsl801275 and rs1805016.

21. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. The method of any of the aforementioned clauses, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 69.

Ligand using VH tailor made
1. A ligand (eg, antibody or antibody fragment) for use in a method of treating or reducing the risk of an IL4Ra-mediated disease or condition (eg, asthma) in humans in need thereof, SEQ ID NO: 67. It specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A; where (i) this ligand is the human VH segment, the human D gene segment. And the VH domain from the recombination of the human JH segment, which encodes Framework 1 of SEQ ID NO: 40, wherein said human encodes Framework 1 of SEQ ID NO: 40. A VH gene segment containing, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and where (ii) said human expresses I75V, E400A, C431R, in SEQ ID NO: 67, A ligand comprising a nucleotide sequence encoding the IL4RA protein containing the mutation selected from the group consisting of S503P, Q576R and S752A.

In one alternative, paragraph 1 provides:-
A ligand (eg, antibody or antibody fragment) for use in methods of targeting IL4Ra in humans, a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Specifically binds to human IL4RA proteins, including;
Here (i) this ligand contains a VH domain from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40. And here, said human contains a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here. (ii) A ligand comprising the nucleotide sequence in which the human encodes the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In some cases, this human has or is at risk for an IL4Ra-mediated disease or condition.

In some examples, this method treats or reduces the risk of IL4Ra-mediated disease or condition in humans.

2. The ligand of paragraph 1, wherein IL4Ra contains the mutations I75V and Q576R in SEQ ID NO: 67, and optionally the disease is asthma.
The ligand of paragraph 1 or 2 in which the nucleotide sequence of (ii) comprises the nucleotide mutation-3223T (dB SNP numbering).
The ligand of any of the aforementioned paragraphs, wherein each said human VH gene segment comprises SEQ ID NO: 39.

3. The ligand of any of the above paragraphs, wherein the VH gene segment contained in the human is a germline VH gene segment.

4. The method comprises selecting a human comprising the nucleotide sequence of (ii) prior to the administering step, wherein the human is the human of paragraph 1, the ligand of any of the aforementioned paragraphs. ..

5. A nucleotide sequence encoding the IL4Ra protein in which a human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 and / or I75V, E400A in SEQ ID NO: 67. , C431R, S503P, Q576R and S752A, the ligands of any of the aforementioned paragraphs, determined to include the IL4Ra protein containing the mutation selected from the group.

6. The method is (a) a nucleotide sequence encoding an IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, and / or ( b) Including the step of determining to include the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, optionally this determining step The ligand of any of the aforementioned paragraphs, which is performed prior to administration of this antibody to this human.

7. A human-derived biological sample for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The ligand of paragraph 8, which comprises the step of assaying.

8. The step to be assayed is at least 10 of the nucleotide sequence encoding the biological sample and the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A step of contacting with at least one oligonucleotide probe comprising a sequence of contiguous nucleotides or comprising an antisense sequence of said contiguous nucleotide, wherein the sequence of contiguous nucleotides is SEQ ID NO: Containing a nucleotide sequence encoding the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in 67, thereby from I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. If at least one nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group is present, a step of forming a complex and a step of detecting the presence or absence of this complex, wherein the complex is present. The step of detecting the presence of the complex determines that the human comprises an IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The ligand of paragraph 9, including the steps.

9. The step of assaying comprises nucleic acid amplification and, optionally, one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification, and / or here. The ligand of paragraph 9 in which this assaying step is performed in a multiplex format.

10. The ligand of paragraph 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. , Optionally, the human is shown to further comprise the nucleotide sequence of SEQ ID NO: 68, or the human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A ligand of any of the aforementioned paragraphs, which is shown to be homozygous for a nucleotide sequence encoding an IL4Ra protein containing the mutation.

12. The ligand of any of the aforementioned paragraphs, wherein the human is substantially resistant to, or is further determined to be, resistant to asthma treatment.

13. The ligand of any of the preceding paragraphs in which the human has been or has been treated for asthma or has had reduced responsiveness to asthma treatment.

14. Is the disease or condition an inflammatory disease or condition; an atopic disease or condition; a respiratory disease or condition; a disease or condition associated with elevated IgE; or an elevated IL-4 and / or IL-13 activity? A ligand in any of the aforementioned paragraphs that is a disease or condition associated with.

15. The disease or condition is an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome , Endometrioma, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, aspergillus tumor, aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophil pneumonia, Idiopathic pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous acidobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary radiomyopathy, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary arterial embolization , Pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis, any Lungs in the above paragraph.

16. The human has an inflammatory disease or condition of the airway, chronic obstructive pulmonary disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, cystic Fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome, endothelium Tumor, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, Aspergillus tumor, Aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophilic pneumonia, idiopathic Pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous acid-acid bacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary bacillosis, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary artery embolization, pneumonia At least one condition selected from the group consisting of disease, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary vein obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. Lungs in any of the aforementioned paragraphs diagnosed as having.

17. The antibody or antibody fragment is an inflammatory disease or condition of the airway, chronic obstructive pulmonary disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation. , Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, medial disorder, diaphragm disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress Syndrome, Endocytoma, Memoroma, Transplant rejection, Transplant vs. host disease, Lung cancer, Allergic rhinitis, Allergy, Aspergillosis, Aspergillus tumor, Aspergillosis, Bronchial dilatation, Chronic bronchitis, Pulmonary emphysema, Eosinophilic pneumonia , Idiopathic pulmonary fibrosis, Invasive pneumococcal disease, Influenza, Non-tuberculous acid bacillus, Pulmonary fluid, Dust pneumonia, Subcutaneous emphysema, Pneumonia, Pulmonary radiculopathy, Alveolar proteinosis, Pulmonary ulcer, Pulmonary edema, Pulmonary artery At least one selected from the group consisting of embolization, pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of one condition in said human.

18. The ligand of any of the preceding paragraphs, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs1805010, rs1805011, rs1805012, rs1805015, rsl801275 and rs1805016.

19. Ligand of any of the aforementioned paragraphs, wherein the antibody or antibody fragment is for inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

20. The ligand of any of the aforementioned paragraphs, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 69.

Method using tailor made in C area
1. A method for treating or reducing the risk of IL4Ra-mediated disease or condition in humans in need thereof, for the human beings I75V, E400A, C431R, S503P, Q576R in SEQ ID NO: 67. And the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to a human IL4Ra protein containing a mutation selected from the group consisting of S752A;
Here, the ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, wherein the human is (i). IGHG4 * 01 Human gamma-4 heavy chain constant region containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. The nucleotide that expresses the antibody comprising; and where (ii) the human encodes the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A method that includes an array.

One alternative provides clause 1 and below:-
A method of targeting IL4Ra in humans, specific for the human IL4Ra protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 to the human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment);
Here, the ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, wherein the human is (i). IGHG4 * 01 Human gamma-4 heavy chain constant region containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. The nucleotide that expresses the antibody comprising; and where (ii) the human encodes the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A method that includes an array.

In some cases, this human has or is at risk for an IL4Ra-mediated disease or condition. In some examples, this method treats or reduces the risk of IL4Ra-mediated disease or condition in humans.

In some examples of the methods or ligands of the invention, this IL4Ra comprises a mutant I75V.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant E400A.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant C431R.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant S503P.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant Q576R.

In some examples of the methods or ligands of the invention, this IL4Ra comprises the mutant S752A.

2. The method of Clause 1, wherein IL4Ra contains the mutations I75V and Q576R in SEQ ID NO: 67 and, optionally, the disease is asthma.

3. The method of Clause 1 or 2, wherein the nucleotide sequence of (ii) comprises a nucleotide mutation-3223T (dB SNP numbering).

4. The method of any of the aforementioned provisions, wherein the human gamma-4 heavy chain constant region comprises SEQ ID NO: 73.

5. The method of any of the aforementioned provisions, wherein the human expresses an antibody comprising a human IGHG4 * 01 heavy chain constant region.

6. The method of any of the aforementioned provisions, comprising the step of selecting a human comprising the nucleotide sequence of (ii) prior to the said step of administration, which is the human of Clause 1.

7. A nucleotide sequence encoding the IL4Ra protein in which a human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 and / or I75V, E400A in SEQ ID NO: 67. , C431R, S503P, Q576R and S752A, the method of any of the aforementioned provisions, determined to include the IL4Ra protein containing the mutation selected from the group.

8. A nucleotide sequence in which humans encode an IL4Ra protein containing said mutation selected from the group consisting of (a) I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, and / or sequence (b). Including the step of determining to include the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A at number 67, and optionally, this determining step is this human. The method of any of the aforementioned provisions performed prior to administration of this antibody against.

9. Human-derived biological for the nucleotide sequence in which the determination step encodes the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Clause 8 method, including step of assaying a sample.

10. The step to be assayed is at least 10 of the nucleotide sequence encoding the biological sample and the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A step of contacting with at least one oligonucleotide probe comprising a sequence of contiguous nucleotides or comprising an antisense sequence of said contiguous nucleotide, wherein the sequence of contiguous nucleotides is SEQ ID NO: Containing a nucleotide sequence encoding the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in 67, thereby from I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. If at least one nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group is present, a step of forming a complex and a step of detecting the presence or absence of this complex, wherein the complex is present. The step of detecting the presence of the complex determines that the human comprises an IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. Clause 9 method, including steps.

11. The assaying step comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification, and / or here. The method of Clause 9 in which this assaying step is performed in a multiplex format.

12. The method of Clause 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human has been shown to be heterozygous for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. , Optionally, the human is shown to further comprise the nucleotide sequence of SEQ ID NO: 68, or the human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The method of any of the aforementioned provisions, which is shown to be homozygous for a nucleotide sequence encoding an IL4Ra protein containing the mutation.

14. The method of any of the aforementioned provisions, wherein the human is substantially resistant to, or is further determined to be, to the treatment of asthma.

15. The method of any of the aforementioned provisions, wherein the person has been or has been treated for asthma or has had reduced responsiveness to asthma treatment.

16. Is the disease or condition an inflammatory disease or condition; an atopic disease or condition; a respiratory disease or condition; a disease or condition associated with elevated IgE; or an increased IL-4 and / or IL-13 activity? The method of any of the aforementioned provisions that is a disease or condition associated with.

17. The disease or condition is an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome , Endometrioma, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, aspergillus tumor, aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophil pneumonia, Idiopathic pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous acidobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary radiomyopathy, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary arterial embolization , Pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis, any The method of the above clause.

18. The human has an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, cystic Fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome, endothelium Tumor, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, Aspergillus tumor, Aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophilic pneumonia, idiopathic Pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous mycobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary bacteriolysis, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary artery embolization, pneumonia At least one condition selected from the group consisting of pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary vein obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. The method of any of the aforementioned provisions diagnosed as having.

19. The antibody or antibody fragment is an inflammatory disease or condition of the airway, chronic obstructive pulmonary disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation. , Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, medial disorder, diaphragm disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress Syndrome, Endocytoma, Memoroma, Transplant rejection, Transplant vs. host disease, Lung cancer, Allergic rhinitis, Allergy, Aspergillosis, Aspergillus tumor, Aspergillosis, Bronchial dilatation, Chronic bronchitis, Pulmonary emphysema, Eosinophilic pneumonia , Idiopathic pulmonary fibrosis, Invasive pneumococcal disease, Influenza, Non-tuberculous acid bacillus, Pulmonary fluid, Dust pneumonia, Subcutaneous emphysema, Pneumonia, Pulmonary radiculopathy, Alveolar proteinosis, Pulmonary ulcer, Pulmonary edema, Pulmonary artery At least one selected from the group consisting of embolization, pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. The method of any of the aforementioned provisions that treats or reduces the risk of one condition in said human.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs1805010, rs1805011, rs1805012, rs1805015, rsl801275 and rs1805016.

21. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

Ligand using tailor-made constant region
1. A ligand (eg, antibody or antibody fragment) for use in a method of treating or reducing the risk of an IL4Ra-mediated disease or condition (eg, asthma) in humans in need thereof, SEQ ID NO: 67. It specifically binds to a human IL4RA protein containing a mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A; where (i) this ligand is found at position 189 in SEQ ID NO: 73. Contains a human gamma-4 heavy chain constant region containing Arg at position 289 found in Leu or SEQ ID NO: 73, and where the human comprises (i) IGHG4 * 01 human heavy chain constant region gene segment. Alternatively, this human expresses an antibody comprising a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73; and where (ii) said human. Ligand comprising the nucleotide sequence encoding the IL4RA protein comprising the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67.

In one alternative, paragraph 1 provides:-
A ligand (eg, antibody or antibody fragment) for use in methods of targeting IL4Ra in humans, suddenly selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. It specifically binds to the human IL4RA protein containing the mutation;
Here (i) the ligand comprises a human gamma-4 heavy chain constant region comprising Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, wherein said human. (i) IGHG4 * 01 Human gamma-4 heavy chain determination containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. The IL4RA protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 is expressed by expressing an antibody containing a normal region; A ligand that contains the encoding nucleotide sequence.

In some cases, this human has or is at risk for an IL4Ra-mediated disease or condition.

In some examples, this method treats or reduces the risk of IL4Ra-mediated disease or condition in humans.

2. The ligand of paragraph 1 in which IL4Ra contains the mutations I75V and Q576R in SEQ ID NO: 67 and optionally the disease is asthma.

3. The ligand of paragraph 1 or 2 where the nucleotide sequence of (ii) comprises the nucleotide mutation-3223T (dB SNP numbering).

4. The ligand of any of the aforementioned paragraphs, wherein the human gamma-4 heavy chain constant region comprises SEQ ID NO: 73.

5. The ligand of any of the preceding paragraphs in which human expresses an antibody containing the human IGHG4 * 01 heavy chain constant region.

6. The method comprises selecting a human comprising the nucleotide sequence of (ii) prior to the administering step, wherein the human is the human of paragraph 1, the ligand of any of the aforementioned paragraphs. ..

7. A nucleotide sequence encoding the IL4Ra protein in which a human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67 and / or I75V, E400A in SEQ ID NO: 67. , C431R, S503P, Q576R and S752A, the ligands of any of the aforementioned paragraphs, determined to include the IL4Ra protein containing the mutation selected from the group.

8. The method is (a) a nucleotide sequence encoding an IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, and / or ( b) Including the step of determining to include the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67, optionally this determining step The ligand of any of the aforementioned paragraphs, which is performed prior to administration of this antibody to this human.

9. A human-derived biological sample for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The ligand of paragraph 8, which comprises the step of assaying.

10. The step of assaying is at least 10 of the nucleotide sequence encoding the biological sample and the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A step of contacting with at least one oligonucleotide probe comprising a sequence of contiguous nucleotides or comprising an antisense sequence of said contiguous nucleotide, wherein the sequence of contiguous nucleotides is SEQ ID NO: Containing a nucleotide sequence encoding the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in 67, thereby from I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. If at least one nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group is present, a step of forming a complex and a step of detecting the presence or absence of this complex, wherein the complex is present. The step of detecting the presence of the complex determines that the human comprises an IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. The ligand of paragraph 9, including the steps.

11. The assaying step comprises nucleic acid amplification and optionally one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification, and / or here. The ligand of paragraph 9 in which this assaying step is performed in a multiplex format.

12. The ligand of paragraph 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human has been shown to be heterozygous for the nucleotide sequence encoding the IL4Ra protein containing the mutation selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. , Optionally, the human is shown to further comprise the nucleotide sequence of SEQ ID NO: 68, or the human is selected from the group consisting of I75V, E400A, C431R, S503P, Q576R and S752A in SEQ ID NO: 67. A ligand of any of the aforementioned paragraphs, which is shown to be homozygous for a nucleotide sequence encoding an IL4Ra protein containing the mutation.

14. The ligand of any of the aforementioned paragraphs, wherein the human is substantially resistant to, or is further determined to be, resistant to asthma treatment.

15. The ligand of any of the preceding paragraphs in which the human has been or has been treated for asthma or has had reduced responsiveness to asthma treatment.

16. Is the disease or condition an inflammatory disease or condition; an atopic disease or condition; a respiratory disease or condition; a disease or condition associated with elevated IgE; or an increased IL-4 and / or IL-13 activity? A ligand in any of the aforementioned paragraphs that is a disease or condition associated with.

17. The disease or condition is an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome , Endocytoma, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, aspergillus tumor, aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophil pneumonia, Idiopathic pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous acidobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary radiomyopathy, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary arterial embolization , Pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis, any Lungs in the above paragraph.

18. The human has an inflammatory disease or condition of the airway, chronic obstructive lung disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation, cystic Fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, mediastinal disorder, diaphragmatic disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress syndrome, endothelium Tumor, sarcoma, transplant rejection, transplant-to-host disease, lung cancer, allergic rhinitis, allergy, asbestos pneumonia, Aspergillus tumor, Aspergillosis, bronchial dilatation, chronic bronchitis, lung emphysema, eosinophilic pneumonia, idiopathic Pulmonary fibrosis, invasive pneumococcal disease, influenza, non-tuberculous mycobacteria, pleural effusion, dust pneumonia, subcutaneous emphysema, pneumonia, pulmonary bacteriolysis, alveolar proteinosis, pulmonary charcoal, pulmonary edema, pulmonary artery embolization, pneumonia At least one condition selected from the group consisting of pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary vein obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. Lungs in any of the aforementioned paragraphs diagnosed as having.

19. The antibody or antibody fragment is an inflammatory disease or condition of the airway, chronic obstructive pulmonary disease, asthma, pneumonia, irritable pneumonia, lung invasion with eosinophilia, environmental lung disease, pneumonia, bronchial dilatation. , Cystic fibrosis, interstitial lung disease, primary pulmonary hypertension, pulmonary thromboembolism, pleural disorder, medial disorder, diaphragm disorder, hypoventilation, hyperventilation, sleep apnea, acute respiratory distress Syndrome, Endocytoma, Memoroma, Transplant rejection, Transplant vs. host disease, Lung cancer, Allergic rhinitis, Allergy, Aspergillosis, Aspergillus tumor, Aspergillosis, Bronchial dilatation, Chronic bronchitis, Pulmonary emphysema, Eosinophilic pneumonia , Idiopathic pulmonary fibrosis, Invasive pneumococcal disease, Influenza, Non-tuberculous acid bacillus, Pulmonary fluid, Dust pneumonia, Subcutaneous emphysema, Pneumonia, Pulmonary radiculopathy, Alveolar proteinosis, Pulmonary ulcer, Pulmonary edema, Pulmonary artery At least one selected from the group consisting of embolization, pulmonary inflammation, pulmonary histocytoproliferative disease X, pulmonary hypertension, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary venous obstructive disease, rheumatic pulmonary disease, sarcoidosis, and Wegner's granulomatosis. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of one condition in said human.

20. The ligand of any of the preceding paragraphs, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rsl805010, rsl805011, rsl805012, rsl805015, rsl801275 and rsl805016.

21. The ligand of any of the aforementioned paragraphs, wherein the antibody or antibody fragment is for inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

In any other embodiment or embodiment, clause or paragraph example associated with IL4Ra, the mutation in IL4Ra is 75V and this human is of AFR or ASN lineage, eg, this human lineage is Selected from ASW, LWK, CHS or JPT. All of these pedigrees have an average frequency of over 47% for 75V.

In any other embodiment or embodiment, clause or paragraph example relating to IL4Ra, the IL4Ra mutation is 400A and the human is of AFR pedigree, eg, this human pedigree is ASW, LWK and YRI. Is selected from. All of these pedigrees exceed the average frequency for 400A.

In any other embodiment or embodiment, clause or paragraph example relating to IL4Ra, the IL4Ra mutation is 431R and this human is of AFR or AMR pedigree, eg, this human pedigree is YRI, Selected from CLM, MXL, PUR or CEU. All of these pedigrees exceed the average frequency for 431R.

In any other embodiment or embodiment, clause or paragraph example relating to IL4Ra, the IL4Ra mutation is 752A and this human is of AFR pedigree, eg, this human pedigree is ASW, LWK and Selected from YRI. All of these pedigrees have a frequency of 37-39%, which is greater than the average frequency for 752A.

The frequency refers here to the 1000 Genomes Database (version 20110521).

In any other embodiment or embodiment, clause or paragraph example relating to IL4Ra, the IL4Ra mutation is 75V and this human is of AFR or ASN lineage, eg, this human lineage is ASW, Selected from LWK, CHS or JPT. All of these pedigrees have an average frequency of over 47% for 75V.

SNP rs1805010 [encoding 75V; see Table 11] is present in humans with an average cumulative frequency of 47% according to the 1000 Genomes Phase I database, but AFR, ASN, ASW, Higher in the LWK, MXL, CHS and JPT populations; therefore, in certain embodiments, this human is of AFR or ASN lineage, eg, ASW, LWK, MXL, CHS or JPT lineage. Thus, in certain embodiments, if the ligand (eg, antibody or fragment) specifically binds to an IL4R protein containing a mutant I75V, the human comprises SNP rs1805010; and optionally this human is AFR. Or ASN pedigree, eg, a human of ASW, LWK, MXL, CHS or JPT pedigree. For example, this human is of AFR pedigree. For example, this human is of ASN pedigree. For example, this human is of ASW pedigree. For example, this human is of LWK pedigree. For example, this human is of ASW pedigree. For example, this human is of MXL pedigree. For example, this human is of CHS pedigree. For example, this human is of JPT pedigree.

SNP rs1805011 [encoding 400A; see Table 11] is present in humans with an average cumulative frequency of 22% according to the 1000 Genomes Phase I database, but AFR, ASW, LWK, And higher in the YRI population; therefore, in some embodiments, this human is of AFR pedigree, eg, ASW, LWK, and YRI pedigree. Thus, in certain embodiments, if the ligand (eg, antibody or fragment) specifically binds to an IL4R protein containing a mutant E400A, the human comprises SNP rs1805011; and optionally this human is AFR. Pedigree, eg, humans of ASW, LWK and YRI pedigree. For example, this human is of AFR pedigree. For example, this human is of ASW pedigree. For example, this human is of LWK pedigree. For example, this human is of YRI pedigree.

SNP rsl805012 [encoding 431R; see Table 11] is present in humans with an average cumulative frequency of 10% according to the 1000 Genomes Phase I database, but AFR, AMR, EUR, Higher in the YRI, CLM, MXL, PUR, CEU and GBR populations; therefore, in certain embodiments, the human is of AFR, AMR or EUR pedigree, eg, YRI, CLM, MXL, PUR, CEU or GBR pedigree. .. Thus, in certain embodiments, if the ligand (eg, antibody or fragment) specifically binds to an IL4R protein containing a mutant C431R, the human comprises SNP rsl805012; and optionally this human is AFR. , AMR or EUR pedigree, eg, humans of YRI, CLM, MXL, PUR, CEU or GBR pedigree. For example, this human is of AFR pedigree. For example, this human is of AMR pedigree. For example, this human is of EUR pedigree. For example, this human is of YRI pedigree. For example, this human is of CLM pedigree. For example, this human is of MXL pedigree. For example, this human is of PUR pedigree. For example, this human is of CEU pedigree. For example, this human is of GBR pedigree.

SNP rsl805015 [encoding 503P; see Table 11] is present in humans with an average cumulative frequency of 21% according to the 1000 Genomes Phase I database, but AFR, ASW, LWK, Higher in the YRI, PUR and GBR populations; therefore, in certain embodiments, the human is of AFR or ASW pedigree, eg, LWK, YRI, PUR or GBR pedigree. Thus, in certain embodiments, if the ligand (eg, antibody or fragment) specifically binds to an IL4R protein containing a mutant S503P, the human comprises SNP rsl805015; and optionally this human is AFR. Or ASW pedigree, eg, a human of LWK, YRI, PUR or GBR pedigree. For example, this human is of AFR pedigree. For example, this human is of ASW pedigree. For example, this human is of LWK pedigree. For example, this human is of YRI pedigree. For example, this human is of PUR pedigree. For example, this human is of GBR pedigree.

SNP rsl801275 [encoding 576R; see Table 11] is present in humans with an average cumulative frequency of 35% according to the 1000 Genomes Phase I database, but AFR, ASW, LWK and Higher in the YRI population; therefore, in certain embodiments, this human is of AFR pedigree, eg, ASW, LWK or YRI pedigree. Thus, in certain embodiments, if the ligand (eg, antibody or fragment) specifically binds to an IL4R protein containing the mutant Q576R, the human comprises SNP rsl801275; and optionally this human is AFR. Pedigree, eg, humans of ASW, LWK or YRI pedigree. For example, this human is of AFR pedigree. For example, this human is of ASW pedigree. For example, this human is of LWK pedigree. For example, this human is of YRI pedigree.

SNP rsl805016 [encoding 752A; see Table 11] is present in humans with an average cumulative frequency of 12% according to the 1000 Genomes Phase I database, but AFR, ASW, LWK and Higher in the YRI population; therefore, in certain embodiments, this human is of AFR pedigree, eg, ASW, LWK or YRI pedigree. Thus, in certain embodiments, if the ligand (eg, antibody or fragment) specifically binds to an IL4R protein containing the mutant S752A, the human comprises SNP rsl805016; and optionally this human is AFR. Pedigree, eg, humans of ASW, LWK or YRI pedigree.

IGHV3-23 SNP rs56069819 is present in humans with an average cumulative frequency of 11% according to the 1000 genome Phase I database, but AFR (23%), EUR (13%), ASW (27%), LWK ( Higher in the 15%), YRI (28%), CEU (19%) and GBR (15%) populations; therefore, in certain embodiments, anti-TOI (eg, PCSK9 or IL4Ra) antibodies or fragments are in SEQ ID NO: 40. Framework 1 or contains a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a recombination-derived VH domain of a human JH segment, and the human VH segment contains SNP rs56069819. If so, this human is of AFR, EUR, ASW, LWK, YRI, CEU or GBR pedigree. For example, this human is of AFR pedigree. For example, this human is of EUR pedigree. For example, this human is of ASW pedigree. For example, this human is of LWK pedigree. For example, this human is of YRI pedigree. For example, this human is of CEU pedigree. For example, this human is of GBR pedigree.

(Example 3)
Nav1.7 (SCN9A; ETHA; FEB3B; GEFSP7; NE-NA; NENA; PN1; SFNP)
J Clin Invest. December 2007; 117 (12): 3603-9, "Mutations in sodium-channel gene SCN9A cause a spectrum of human genetic pain disorders", Drenth JP & Waxman SG (incorporated herein by reference). See). Voltage-gate sodium channels play an important role in the generation and conduction of action potentials and are therefore important for electrical signaling by most excitatory cells. Sodium channels are endogenous membrane proteins that modulate large α subunits (forming voltage-sensitive and ion-selective pores) and smaller auxiliary β subunits (channel kinetics and voltage dependence). Can be rated). To date, we are aware of nine isoforms of sodium channel α subunits (Nav1.1 to Nav1.9), each with its own distribution of central and peripheral nervous systems. The four closely related sodium channels (Nav1.1, -1.2, -1.3, and -1.7) are a series of four genes located within the cluster on chromosome 2q24.3 (SCN1A, SCN2A, SCN3A, respectively). And SCN9A). Nav1.7 has an important role in the sensation of pain. In addition, KO studies and animal pain models suggest that Nav1.7 plays an important role in inflammatory pain.

Nav1.7 is located in peripheral nerves and plays an important role in action potential generation in these cells. Recent genetic studies have identified dysfunction of Nav 1.7 in three different human pain disorders. Function-acquiring missense mutations in Nav1.7 have been shown to result in primary congenital insensitivity (PE) and paroxysmal severe pain (PEPD), whereas nonsense mutations in Nav1.7 are Nav1. Loss of function in 7 and a condition known as channelopathy-associated insensitivity to pain (CIP) (affected individuals do not feel physical pain, a rare disorder). Recent studies have shown that different types of channel disorders (diseases caused by dysfunction of ion channel subunits or proteins that regulate them) are all involved in the same Nav1.7 sodium channel, behind these disorders. It was shown to be.

Nav1.7 is encoded by SCN9A, an 113.5-kb gene (OMIM 603415) containing 26 exons (Fig. 1A, Drenth & Waxman). This encoded sodium channel is composed of 1977 amino acids, organized into 4 domains, each with 6 transmembrane segments, and predominates in dorsal root ganglion (DRG) and exchange ganglion neurons. It is expressed in (Fig. 1B, Drenth & Waxman). Immunohistochemical studies have shown that Nav1.7 is located at the distal end of the wire-like process of neurons known as neurites, near the impulse trigger zone where neuronal firing begins. Interestingly, the majority of Nav1.7-expressing DRG neurons are pain-detectable (noxious), suggesting a role for this sodium channel in the pathogenicity of pain.

Cell. June 5, 2014; 157 (6): pp. 1393-404. doi: 10.1016 / j.cell. 2014.03.064. Epub May 22, 2014; "A monoclonal antibody that targets a NaV1.7 channel" "voltage sensor for pain and itch relief", Lee JH et al. And 2011 (both incorporated herein by reference) describe the production of monoclonal antibodies against human 1.7.

In one example, the invention is a method of treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof and is specific to the human Nav1.7 protein relative to said human. Provided are methods that include the step of administering a ligand that binds specifically (eg, an antibody or antibody fragment). The present invention also provides a corresponding ligand.

The present invention provides anti-Nav1.7 ligands; as well as Nav1.7 binding or targeting ligands as described herein. This ligand has various usefulness. Some of these ligands have been used in affinity purification of Nav1.7 (particularly human Nav1.7 or its ligands) to genotype or phenotype humans, for example in specific binding assays. It is useful in screening assays to identify other antagonists with .7 activity. Some ligands of the invention are useful for inhibiting Nav1.7-mediated activity.

Anti-Nav1.7 ligands (eg, antibodies and antisense RNA) have been developed based on the targeting and neutralization of so-called "wild" human Nav1.7, which is a commonly present type (eg,). See SEQ ID NO: 75). Although such treatments are useful for human patients carrying this type of human Nav1.7, the inventor, although a rare type of Nav1.7 in the human population, is also naturally present. It was considered useful for investigating the possibility of type targeting. In this way, the inventor is a useful target for human treatment, prevention and diagnosis (protein or) that is mediated by Nav1.7 activity or is relevant for diseases and conditions associated thereto. We have reached an insight into the natural appearance and distribution of rare human Nav1.7 types that can function (at the nucleic acid level). This provides tailor-made treatment, prevention and diagnosis, especially in humans lacking the common Nav1.7 gene or protein.

Those skilled in the art are aware that SNPs or other changes that translate into amino acid variations can result in variability in the activity and / or higher-order structure of the human target to be targeted. This has led to great interest in personalized medicine that uses knowledge of genotyping and protein and nucleotide variability to more effectively tailor-make patient care and diagnosis. Accordingly, the present invention provides tailor-made medicines and studies specifically directed to the rare Nav1.7 polymorphic variant. Such forms or "alleles" (at the nucleotide level) contain one or more variations at the nucleotide and amino acid levels from the corresponding common types of nucleotide and amino acid sequences, i.e., one or more non-. There are synonymous (also known as "missense") changes (translating into one or more corresponding changes in a protein target in humans).

Moreover, the inventor surprisingly found that rare natural forms are present in humans much less frequently than common forms, but are nevertheless shown in large and ethnically diverse human populations. We have noticed that there are usually many human examples per ethnic group shown. Thus, by targeting such rare types, the inventor provides effective treatment, prevention or diagnosis across many human ethnic groups, thereby expanding the usefulness of the invention. I noticed.

With this recognition, the inventor relates to guiding the selection of anti-Nav 1.7 ligands for administration to human patients for the treatment and / or prevention of Nav 1.7-mediated or related diseases or conditions. It has been confirmed that the present invention has significant industrial and medical applications. In this method, the patient receives tailor-made drugs and ligands for their needs as determined by the composition of the patient's genes or phenotypes. Along with that, the present invention provides patient genotyping and / or phenotyping associated with such treatments, thereby allowing the drug to be appropriately adapted to the patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment (eg, poor efficacy and / or side effects) with drugs or ligands that are not suitable for the patient, and avoids drug prescribing mistakes and waste. ..

To develop this idea, in this non-limiting example, the inventor decided to determine a series of human Nav1.7 variants based on the following criteria, but these were noticed by the inventor. Criteria provide medical drugs and diagnostics useful for tailor-made needs in the human population. We have selected variants that have at least three of the following four criteria:
Naturally occurring human Nav1.7 variation with a cumulative human allele frequency of 35% or less;
Naturally occurring human Nav1.7 variation with a total human genotype frequency of about 50% or less;
Naturally occurring human Nav 1.7 variations found in many different human populations [using standard categorization of the 1000 Genomes Project; see Table 12 below]; and many such A naturally occurring human Nav 1.7 variation found in many individuals distributed across different ethnic groups.

Based on these criteria, the inventor identified the variants listed in Table 12 below (see Further Variants). In our choice, as an additional or alternative study, the nucleotide variation that produced the amino acid variation in the corresponding Nav1.7 type (ie, as opposed to the silent variation that does not alter the amino acid residue in the target protein). , Non-synonymous variations) included choices.

In one example, the invention is a method of treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof, with respect to said human, 136I, 216S, 241T, 395K. , 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is Y) Amino acids other than), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I) ), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R) Selected from the group consisting of 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Provided is a method comprising the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to a human Nav1.7 protein containing an amino acid. Further, as explained below, these amino acid variations are found in the naturally occurring Nav1.7 variant in humans found in many populations. The humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R). ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (Here X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, Includes a nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group consisting of 1002L, 1161W and 1919G.

For example, the amino acid is selected from the group consisting of any one of (a) to (e):-
a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; or
b) 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; or
c) 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (Here X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) , 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (where X is an amino acid other than K) Amino acids other than W); or
d) 395K, 1200L and 1235L; or
e) 422D, 490N, 943L, 1002L, 1161W and 1919G.
Amino acids (a)-(d) are considered in Drenth & Waxman to be associated with pain status (either an increase or decrease in undesired pain). Thus, in one embodiment, the disease or condition is a pain disease or condition, and the amino acid is selected from (a), (b), (c) or (d). Thus, in one embodiment, the disease or condition is pruritus disease or condition, and the amino acid is selected from (a), (b), (c) or (d). In one example, the disease is PE and the amino acid is selected from (a). In one example, the disease is PEPD and the amino acids are selected from (b). In one example, the disease is CIP and the amino acid is selected from (c) or (d). In some examples, the disease or condition is a pain or pruritus disease or condition, and the amino acids are selected from (e).

In one embodiment, this pain is chronic pain.

In one embodiment, the pain is neuropathic pain, eg, chronic neuropathic pain. For example, this pain is painful diabetic neuropathy (PDN), postherpetic neuralgia (PHN) or trigeminal neuralgia (TN).

In some examples, this pain is spinal cord injury pain, multiple sclerosis pain, phantom limb pain, post-stroke pain, chronic back pain, osteoarthritis pain, cancer-related pain or HIV-related pain.

In one embodiment, the pain is inflammatory pain, eg, chronic inflammatory pain.

In one embodiment, the disease or condition is a channel disorder or is associated with a channel disorder.

In one embodiment, the disease or condition is selected from the group consisting of primary acute pain (PE), paroxysmal extreme pain (PEPD) and channel disorder associated painlessness (CIP).

Optionally, the NAV-mediated disease or condition is selected from one of the following:
Neuropathic or neurogenic pain (eg, painful diabetic neuropathy (PDN), post-herpes neuropathy (PHN), central neuropathy, peripheral neuropathy, trigeminal neuropathy (TN), painful Desensitization, spinal cord injury, multiple sclerosis, phantom limb pain, hyperalgesia, hyperpathia, dysesthesia, psychogenic pain, post-stroke pain and HIV-related pain, back pain, chronic back pain , Degenerative arthritis, cancer, breakthrough pain, apical erythema [eg, primary apical erythema], paroxysmal 30 apical pain disorder, nerve compression and / or strangulation [eg, carpal canal syndrome, foot Radical syndrome, ulnar nerve strangulation, compression nerve root disorder, nerve root lumbar pain, spinal cord nerve root lesion, spinal cord compression, lumbar spinal canal stenosis, sciatic nerve compression, intercostal nerve pain], neuritis, chemotherapy pain, congenital Sexual deficiencies / channel disorders [eg, resulting from channel disorder-related pain and congenital pain] or chronic alcohol dependence [alcoholic polyneuropathy]);
b. Inflammatory pain (eg, osteoarthritis pain, chronic back pain, rheumatoid arthritis pain, cancer pain, breakthrough pain, burn pain, encephalitis pain, fracture pain, neuritis pain, autoimmune disease pain, surgery Post-pain, toothache, bacterial infection-related pain, radiation therapy-related pain, gout-related pain and irritable bowel syndrome pain);
c. Pain resulting from trauma (eg, lacerations, incisions, burns, foreign or bullet and / or shot damage, spinal cord injury, arm neuropathy, nerve crush and / or strangulation (eg, carpal tunnel syndrome, etc.) Carpal tunnel syndrome, ulnar nerve strangulation, compression radiculopathy, radiculopathy, spinal cord lesions, spinal cord compression, lumbar spinal canal stenosis, sciatic nerve compression, intercostal nerve pain), nerve crossing, postoperative pain, Derived from toothache and toxic exposure)
d. Infection-induced pain [eg, postherpetic neuropathy (PHN), HIV-related pain, smallpox infection, encephalitis, herpes infection, and bacterial infection];
e. Pain from malignant tumors (eg, cancer pain, breakthrough pain, and nerve compression pain);
f. Visceral pain (eg, renal or urinary colic, irritable bowel syndrome, angina or heart pain, cardiac arrhythmia, dysmenorrhea, interstitial cystitis, rectal pain, diarrhea-related pain, appendicitis, cholecystitis And pancreatitis);
g. Metabolic or chronic disease pain (eg, multiple sclerosis-related pain, cancer pain, breakthrough pain, gout-related pain, peripheral diabetic neuropathy, chronic alcohol-dependent pain [alcoholic multiple neuropathy] ], Urotoxicity pain, hypothyroidism-related pain, and vitamin deficiency-related pain);
h. Headaches (eg, tension headaches, migraine headaches and cluster headaches);
i. Idiopathic pain (eg, trigeminal neuralgia, complex regional pain syndrome [eg, type I complex regional pain syndrome and / or type II complex regional pain syndrome], allodynia or fibromyalgia);
j. Respiratory pain (eg, asthma-related pain, airway hyperresponsiveness in asthma, chronic cough, eg asthma and / or chronic obstructive lung disease); or
k. Other pain (eg, pain associated with hormone therapy, diabetes, hypothyroidism, epilepsy, ataxia, periodic paralysis, acute pruritus or chronic pruritus).

In some cases, NAV-mediated disorders or conditions include painful diabetic neuropathy, post-herpes neuropathy, trigeminal neuralgia, osteoarthritis, chronic back pain, nerve compression pain (eg, sciatic nerve compression) or It is selected from pain pain; or from migraine, postoperative pain and fibromyalgia.

In one example, the ligand of the invention comprises an anti-human Nav1.7 binding site, where the binding site is a human or humanized binding site, eg, this binding site is human or humanized. Contains or consists of an antibody variable domain or multiple variable domains (eg, human VH / VL binding sites). Further, or instead, the ligand comprises one or more human antibody constant regions [eg, human antibodies CH1, CH2, CH3 (or all of them) or Fc]. In one example, this ligand is an antibody that contains a human or humanized variable region and a human constant region (eg, carrying one or more mutations that enhance or weaken Fc function in a human patient). is there.

In one example, a ligand (eg, an amino acid or an amino acid fragment) for treating or reducing the risk of a Nav1.7-mediated disease or condition in a human in need thereof is provided and the method is directed to said human. Including the step of administering the ligand, wherein the ligand is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K. , 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S) Where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R), Amino acids other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is W) Amino acids other than), 422D, 490N, 943L, 1002L, 1161W and 1919G specifically bind to human Nav1.7 proteins containing amino acids selected from the group. The humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R). ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (Here X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, Includes a nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group consisting of 1002L, 1161W and 1919G.

In one example, the invention is a method of targeting Nav1.7 in humans, with respect to said humans: 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) , 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R) ), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) ), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G specifically binds to human Nav1.7 proteins containing amino acids selected from the group. A method comprising the step of administering a ligand (eg, an amino acid or an amino acid fragment) is provided. The humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R). ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (Here X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, Includes a nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group consisting of 1002L, 1161W and 1919G. In some cases, this human has or is at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

In one example, a ligand (eg, an antibody or antibody fragment) for targeting Nav1.7 in humans is also provided, the method comprising administering the ligand to said human, wherein. This ligand is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R). ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (Here X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, It specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of 1002L, 1161W and 1919G. The humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R). ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (Here X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, Includes a nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group consisting of 1002L, 1161W and 1919G. In some cases, this human has or is at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

In certain embodiments, (i) the antibody or fragment comprises a VH domain derived from the recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment comprising Framework 1 of SEQ ID NO: 40. Encoding and where the human comprises a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40. And here (ii) the humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (here). And X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) ), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Includes a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of), 422D, 490N, 943L, 1002L, 1161W and 1919G.

Furthermore, or instead, in one example, (i) this antibody or fragment contains a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Containing and, here, said human comprises the IGHG4 * 01 human heavy chain constant region gene segment, or this human is at position 189 Leu found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Amino Acids Containing Human Gamma-4 Heavy Chain Constant Regions Containing; And Where (ii) The Humans Are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D , 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) Amino acids other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R) Yes), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) ), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and a nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group consisting of 1919G. Including.

In some examples, the ligand, antibody or fragment is to treat or reduce the risk of pain or pruritus (or treat or reduce that risk), or optionally the antibody is a humanized mouse or It is a camelid (eg, llama or camel) antibody.

In certain embodiments, this anti-Nav 1.7 ligand, antibody or fragment also specifically binds to another Nav selected from Nav 1.1-1.9, eg, it is specifically Nav 1.8 or 1.9. Join.

In certain embodiments, the anti-Nav1.7 ligand, antibody or fragment of the invention comprises an Fc region, wherein the Fc region is numbered with an EU index as shown in Kabat, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y , 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y , 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D , 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D , 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A , 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V , 331I, 331C, 331Y, 331H, 331R , 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W Contains at least one unnatural amino acid residue selected from the group consisting of. Optionally, this Fc region may contain additional and / or alternative non-natural amino acid residues known to those of skill in the art. (See, for example, U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT International Publication Nos. WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO 05/040217. ).

Ligands, antibodies or fragments according to the invention are incorporated herein by reference, for example, WO 2011/051351 (disclosure of the disease or condition is incorporated herein by reference for possible inclusion in one or more claims. To treat or prevent (or treat, prevent or reduce) the risk of any disease or condition disclosed in. Guidance on obtaining and testing antibodies can also be found in the PCT application.

Further included by the present invention is the use of ligands, antibodies or fragments in the manufacture of medicaments for use in attenuating or inhibiting Nav1.7-mediated diseases or disorders in humans. Nav1.7-mediated or related disorders treated with the ligands, antibodies or fragments of the invention include, for example, pain or pruritus disease or condition.

Therefore, the ligands, antibodies or fragments of the present invention are useful as therapeutic agents in the treatment of conditions involved in Nav1.7 expression and / or activity. One embodiment is a method of treatment comprising administering, among other things, an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein Nav1.7 activation is functional. The consequences are reduced. Another embodiment is, among other things, (i) identifying a patient exhibiting Nav1.7 expression or activity, and (ii) administering to this patient an effective amount of a ligand, antibody or fragment of the invention. A method of treatment involving steps, where the functional consequences of Nav1.7 activation are diminished. An effective amount according to the invention is the severity of at least one symptom of the particular disease or disorder being treated by modulating (eg, reducing) the functional consequences of activation of Nav1.7. Is an amount that modulates (eg, decreases or weakens) the disease, but does not necessarily have to cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, for a patient in need thereof. , Is an effective amount of one or more ligands, antibodies or fragments of the invention known in the art alone or in the art so as to reduce the severity of at least one symptom of any disorder? Alternatively, it comprises the step of administering in a therapeutic regimen combined with another suitable drug described herein. Another embodiment of the invention is, among other things, a method of antagonizing at least one effect of Nav1.7, wherein the at least one effect of Nav1.7 (eg, Nav1.7 is an ion channel such as a sodium channel). Includes the step of contacting or administering to an effective amount of one or more ligands, antibodies or fragments of the invention such that the ability to form) is antagonized.

Tailor Made Antibodies to Rare Nav1.7 Variant Profiles As outlined herein (eg, in the context of PCSK9 in Example 1), the present invention allows the TOI type of variant to meet the selection criteria of the present invention. It further includes the possibility of tailor-making human treatment by selecting antibody-based ligands with variable and / or constant domains based on the genetic segments found in many humans of the ethnic populations found. This also applies mutatis mutandis when the TOI is human Nav 1.7, as in this example. Accordingly, all disclosures herein relating to tailor-making variable domains and / or constant domains apply to this example with respect to Nav 1.7 and are combined with use in one or more claims herein. It is possible.

As described in Example 1, in some examples, a ligand comprising an antibody VH domain from a recombination of a human IGHV gene segment comprising a nucleotide selected at a position in HCDR1 or FW3 that is variable in human is provided. (That is, SNPs are present in humans here).

Further information is provided in Table 4, which also shows variations at these locations and variant distributions across the 1000 Genomes Project database associated with many human populations.

In other embodiments, as described in more detail above, the invention is based on an alternative human VH gene segment, or V _K , V _λ or constant region gene segment, and the genotype of the human recipient and /. Alternatively, provide tailor-made ligands for the phenotype (see further Table 9 for representative variants).

In a further example, therefore there is:-
(i) Here, the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a human JH segment, the human VH segment having SEQ ID NO: 40 Framework 1 is encoded, and where the human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human is the Framework 1 of SEQ ID NO: 40. Express the VH domain containing.
(ii) The ligand comprises a human VH segment IGHV3-7 * 01, a human D gene segment and a recombination-derived VH domain of a human JH segment, and the human contains the IGHV3-7 * 01 VH gene segment or is human. Expresses the VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment.
(iii) The ligand comprises the human Vκ segment IGKV1-12 * 01 and the recombinantly derived Vκ domain of the human Jκ segment, and the human comprises the IGKV1-12 * 01 Vκ gene segment, or the human contains the human Vκ segment. It expresses the Vκ domain derived from recombination of IGKV1-12 * 01 and human Jκ segment.
(iv) The ligand comprises the Vκ domain from the recombination of the human Vκ segment and the human Jκ segment, and the human Vκ segment encodes (i) CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, said human. Does it contain the Vκ gene segment encoding CDR3 containing Pro at position 7 shown in SEQ ID NO: 36, or does human express the Vκ domain containing CDR3 containing Pro at position 7 shown in SEQ ID NO: 36? Or (ii) the Vκ gene segment encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38 and the human encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38, or Humans express the Vκ domain containing FW3, which contains Ser at position 15 shown in SEQ ID NO: 38.
(v) The ligand contains a human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human is (i) IGHG1 * 01 human. An antibody containing a heavy chain constant region gene segment or containing a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4 is expressed. ..
(vi) The ligands are Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 and Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala at position 257 shown in SEQ ID NO: 6, and whether the human contains (i) IGHG2 * 01 human heavy chain constant region gene segment. , Or human, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Val or the antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown in SEQ ID NO: 6.
The (vii) ligand comprises a human kappa chain constant region containing Val at position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16, and the human is (i) IGKC1 * 01 human kappa chain constant region. An antibody containing a gene segment or containing a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16 is expressed.
(viii) the ligand comprises a human IGLC1 * 01 lambda chain constant region, said human containing (i) a human IGLC1 * 01 lambda chain constant region gene segment, or a human containing a human IGLC1 * 01 lambda chain constant region. Expresses an antibody containing.
(ix) The ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human. An antibody comprising a heavy chain constant region gene segment or containing a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73 is expressed. ..
The (x) ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3 * 01) constant region gene segment, wherein the human is (i) said the first constant region gene. An antibody comprising a segment (eg, IGHG3 * 01) or containing a human gamma-3 heavy chain constant region encoded by the first human IGHG3 (eg, IGHG3 * 01) constant region gene segment is expressed. ..
(xi) The ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human epsilon heavy chain constant region encoded by the first constant region gene segment.
(xii) The ligand comprises the human mu heavy chain constant region encoded by the first human mu heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expresses an antibody comprising the human mu heavy chain constant region encoded by the first constant region gene segment.
(xiii) The ligand comprises the human alpha heavy chain constant region encoded by the first human alpha heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human alpha heavy chain constant region encoded by the first constant region gene segment.
The (xiv) ligand comprises the human delta heavy chain constant region encoded by the first human delta heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expressing an antibody comprising the human delta heavy chain constant region encoded by the first constant region gene segment.
The (xv) ligand comprises the human kappa light chain constant region encoded by the first human kappa light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human kappa light chain constant region encoded by the first constant region gene segment.
The (xvi) ligand comprises the human lambda light chain constant region encoded by the first human lambda light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human lambda light chain constant region encoded by the first constant region gene segment.

These examples are described in the context of the Nav 1.7 Examples of the present invention, but are applicable to the present invention as they relate to any other TOI herein and are therefore applicable to any TOI. It can be used in combination in the claims of the present specification. In any of these examples, the ligand is optionally an antibody, eg, a human or humanized antibody [eg, a humanized mouse, rat or Camelid antibody].
ADCC efficacy for the human constant region is as follows: IgGl ≥ IgG3 >> IgG4 ≥ IgG2
The effectiveness of CDC for the human constant region is as follows: IgG3 ≥ IgGl >> IgG2 ≒ IgG4
Thus, this may be advantageous to the Nav1.7 ligand for ligands containing the gamma-4 or gamma-2 constant region (eg, as in the previous embodiment). For example, gamma-4 is IgG4PE (ie, a gamma-4 constant region with 228Pro and 235Glu). For example, gamma-2 is IgG2Δa.

For example, as in Example (ix), the inventor identified the possibility of addressing the IGH-gamma-4 variation and identified the usefulness for variations 189L and 289R individually or in combination. This is because these residues are part of the CH3 domain and, as such, they form part of the antibody Fc region. Therefore, these CH3 variations and patient compatibility are particularly beneficial for the reasons discussed above. Thus, in this example, the ligand of the invention comprises or comprises an antibody comprising a human gamma-4 heavy chain constant region comprising Leu corresponding to position 189 of SEQ ID NO: 73 or Arg corresponding to position 289 of SEQ ID NO: 73. Consisting of that antibody, and where this human genome contains a gamma-4 heavy chain constant region nucleotide sequence encoding such Leu and / or Arg, or this human is such Leu and / Alternatively, it expresses an antibody containing the human gamma-4 constant region containing Arg.

Determination of specific binding of the ligand of the invention to the NAV1.7 variant
Specific binding of the ligand of the present invention to the NAV1.7 variant can be performed using the SPR method described in Example 1.

Embodiments are provided as follows:-

How to use VH's TaylorMade
1. A method of treating or reducing the risk of a Nav1.7-mediated disease or condition in humans in need thereof, for said humans 136I, 216S, 241T, 395K, 848T, 858H, 858F. , 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y) , 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) Where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (here X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and human Nav1 containing amino acids selected from the group consisting of 1919G. .7 Including the step of administering a ligand that specifically binds to a protein (eg, an amino acid or an amino acid fragment);
Here (i) this ligand contains a VH domain derived from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40. And here, said human contains a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here. (ii) The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In one alternative, Clause 1 provides:-
A method of targeting Nav1.7 in humans, with respect to the humans: 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V. , 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S) ), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (here X is an amino acid other than R) In X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (here X is an amino acid other than K) X is a ligand (eg, antibody or antibody) that specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of amino acids other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Including the step of administering (fragment);
Here (i) this ligand contains a VH domain derived from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40. And here, said human contains a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here. (ii) The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In some cases, this human has or is at risk for a Nav1.7-mediated disease or condition.

In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

2. The amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is a pain disease or condition. The method of any of the aforementioned provisions.

3. The amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the method of any of the aforementioned provisions where the disease or condition is a pain disease or condition. ..

4. The amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S). Yes), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) Where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (here X is an amino acid other than K) In X is an amino acid other than W) selected from the group; optionally the method of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition.

5. The method of any of the aforementioned provisions, wherein the VH gene segment contained in the human is a germline VH gene segment.

6. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

7. Humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R) ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L , 1002L, 1161W and a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) May include Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of any of the aforementioned provisions determined.

8. Humans (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is R) Amino acids other than), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L , 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, A nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 490N, 943L, 1002L, 1161W and 1919G, and / or (b) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) In X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (here X is an amino acid other than R) X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of any of the aforementioned provisions, comprising the step of determining to include the Nav1.7 protein comprising, optionally, this determining step is performed prior to administration of this antibody to this human.

9. The process to decide is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R) 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G. The method of Clause 8 comprising assaying a human-derived biological sample for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G.

10. The steps to be assayed are with biological samples 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group, or said contiguous. Contains the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V. , 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (Here X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) , 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), Contains a nucleotide sequence encoding the amino acid selected from the group consisting of amino acids other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. , Thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. If at least one nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from is present, the step of forming the complex and
The step of detecting the presence or absence of this complex, wherein the step of detecting the presence of this complex is that this human has 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Contains Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of Clause 9, including the process of deciding.

11. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 9 in which this assaying step is performed in a multiplex format.

12. The method of Clause 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). Amino acids), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L , 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, It has been shown to be heterozygous for a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 943L, 1002L, 1161W and 1919G, and optionally the human being of SEQ ID NO: 76. Further indicated to contain a nucleotide sequence, or said human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X. (Here X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is other than W) (Amino acids of), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Any of the aforementioned provisions shown to be homozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of amino acids), 422D, 490N, 943L, 1002L, 1161W and 1919G. Method.

14. The method of any of the aforementioned provisions, wherein the human being is substantially or is further determined to be substantially resistant to the treatment of pain or pruritus.

15. The method of any of the aforementioned provisions, wherein the person has been or has been treated for pain or pruritus, or has been less responsive to the treatment for pain or pruritus.

16. The method of any of the aforementioned provisions, wherein the disease or condition is a pain or pruritus disease or condition.

17. Whether the disease or condition is channel disorder or is associated with channel disorder; or primary congenital insensitivity (PE), paroxysmal extreme pain (PEPD) and channel disorder-related painlessness (CIP) The method of any of the above clauses selected from the group consisting of).

18. The method of any of the aforementioned provisions in which the person has been diagnosed with at least one of the conditions listed in claim 16 or 17.

19. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment treats or reduces the risk in said human of the condition set forth in claim 16 or 17.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rsl3402180 and rsl2478318.

21. The method of any of the aforementioned provisions, wherein the ligand (eg, antibody or antibody fragment) is administered by inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. The method of any of the aforementioned clauses, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 69.

Preferably, here the Nav1.7 mutation is 1161W (some publications, eg, Reimann et al. [Proc Natl Acad Sci US A. March 16, 2010; 107 (ll): 5148-53. Doi. 10.1073 / pnas.0913181107. Epub March 8, 2010; "Pain perception is altered by nucleotide polymorphism in SCN9A"; also known as 1150W by Reimann F et al.]. Here, 1150W in normal humans (1150W). That is, the relationship between 1161W) and increased pain sensation (lower threshold) is described, although the SNP rs6746030 encoding 1161W has a cumulative frequency of 11% in all human populations in the 1000 Genome Phase I database. It has been shown to exceed the average cumulative frequency in the AFR, AMR, EUR, ASW, YRI, CLM, CEU, GBR, IBS and TSI populations. Therefore, in one embodiment, this human is AFR, AMR, EUR, ASW. , YRI, CLM, CEU, GBR, IBS or TSI pedigree, this ligand specifically binds to human Nav1.7 containing the mutant R1161W, and this human contains the mutant R1161W Nav1. Containing 7, or this person contains SNP rs6746030. In one example, this person is of AFR pedigree. In some cases, this person is of AMR pedigree. In some cases, this person is EUR. Pedigree. In some cases this person is of ASW pedigree. In some cases this person is of YRI pedigree. In some cases this person is of CLM pedigree. In some cases this person is of CLM pedigree. In some cases, this person is of GBR pedigree. In some cases, this person is of IBS pedigree. In some cases, this person is of TSI pedigree. In some cases, this person is of TSI pedigree. Humans are of pedigree where SNP rs6746030 and constant or variable region SNPs (mutations) exceed the average cumulative frequency.

Ligand using VH tailor made
1. A ligand (eg, an antibody or antibody fragment) for use in a method that treats or reduces the risk of a Nav1.7-mediated disease or condition (eg, asthma) in humans in need thereof. 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (Here X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (here X is an amino acid other than E) X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is X) Is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and It specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of 1919G;
Here (i) this ligand contains a VH domain derived from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40. And here, said human contains a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here. (ii) The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G, a ligand comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In one alternative, paragraph 1 provides:-
In humans, ligands (eg, antibodies or amino acid fragments) for use in methods of targeting Nav1.7, 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F. , 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) Is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R) Amino acids), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) ), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G specifically binds to human Nav1.7 proteins containing amino acids selected from the group. A;
Here (i) this ligand contains a VH domain derived from the recombination of the human VH segment, the human D gene segment and the human JH segment, and this human VH segment encodes Framework 1 of SEQ ID NO: 40. And here, said human contains a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40; and here. (ii) The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G, a ligand comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In some cases, this human has or is at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

2. The amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is a pain disease or condition. , Ligand of any of the aforementioned clauses.

3. The amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the ligand of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition. ..

4. The amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S). Yes), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) Where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (here X is an amino acid other than K) X is an amino acid other than W); optionally the ligand of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition.

5. The ligand of any of the aforementioned provisions, wherein the VH gene segment contained in the human is a germline VH gene segment.

6. Of any of the aforementioned provisions, wherein the method comprises the step of selecting a human (where this human is the human of Clause 1) comprising the nucleotide sequence of (ii) prior to the step of administration. Ligand.

7. Humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R) ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L , 1002L, 1161W and a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) May include Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Determined, ligand of any of the aforementioned clauses.

8. Humans are (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is R) Amino acids other than), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L , 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, A nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 490N, 943L, 1002L, 1161W and 1919G, and / or (b) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) In X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R) X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. A ligand of any of the aforementioned provisions, wherein the method comprises a step of determining to include a Nav1.7 protein comprising, optionally, this determining step is performed prior to administration of this antibody to this human.

9. The process to decide is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R) 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G, comprising the step of assaying a human-derived biological sample for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group.

10. The steps to be assayed are with biological samples 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group, or said contiguous. Contains the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V. , 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (Here X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) , 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), Contains a nucleotide sequence encoding the amino acid selected from the group consisting of amino acids other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. , Thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. If at least one nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from is present, the step of forming the complex and
The step of detecting the presence or absence of this complex, wherein the step of detecting the presence of this complex is that this human has 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Contains Nav1.7 protein containing said amino acid selected from the group consisting of (amino acid), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Clause 9 ligand, including the step of determining.

11. The assaying step comprises or / or comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay is performed in a multiplex format, clause 9 ligand.

12. Ligand of Clause 9 or 11, wherein the biological sample comprises said human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). Amino acids), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L , 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, It has been shown to be heterozygous for a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 943L, 1002L, 1161W and 1919G, and optionally the human being of SEQ ID NO: 76. Further indicated to contain a nucleotide sequence, or said human, is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X. (Here X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is other than W) (Amino acids of), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Any of the aforementioned provisions shown to be homozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of (amino acids), 422D, 490N, 943L, 1002L, 1161W and 1919G. Lagent.

14. A ligand of any of the aforementioned provisions, wherein the human is substantially resistant to, or is further determined to be, substantially resistant to the treatment of pain or pruritus.

15. A ligand of any of the aforementioned provisions, wherein the human has been or has been treated for pain or pruritus, or has been less responsive to the treatment for pain or pruritus.

16. A ligand of any of the aforementioned provisions, wherein the disease or condition is a pain or pruritus disease or condition.

17. Whether the disease or condition is channel disorder or is associated with channel disorder; or primary congenital insensitivity (PE), paroxysmal extreme pain (PEPD) and channel disorder-related painlessness (CIP) ), The ligand of any of the above clauses.

18. The ligand of any of the aforementioned provisions, wherein the human has been diagnosed with at least one of the conditions listed in claim 16 or 17.

19. A ligand of any of the aforementioned provisions, wherein the antibody or antibody fragment treats or reduces the risk in said human of the condition set forth in claim 16 or 17.

20. A ligand of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rsl3402180 and rsl2478318.

21. The ligand of any of the aforementioned provisions, wherein the ligand (eg, antibody or antibody fragment) is for inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. The ligand of any of the aforementioned clauses, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 69.

Gamma-4 stationary region tailor-made method
1. A method of treating or reducing the risk of a Nav1.7-mediated disease or condition in humans in need thereof, for said humans 136I, 216S, 241T, 395K, 848T, 858H, 858F. , 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y) , 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) Where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (here X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and human Nav1 containing amino acids selected from the group consisting of 1919G. .7 Including the step of administering a ligand that specifically binds to a protein (eg, an amino acid or an amino acid fragment);
Here (i) this ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, wherein said human. (i) IGHG4 * 01 Human gamma-4 heavy chain determination containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. It expresses an amino acid containing a normal region; and here (ii) the human is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I. , 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (here X is an amino acid other than R) Is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than K) , An amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In one alternative, Clause 1 provides:-
A method of targeting Nav1.7 in humans, with respect to the humans: 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V. , 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S) ), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (here X is an amino acid other than R) In X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (here X is an amino acid other than K) X is a ligand (eg, antibody or antibody) that specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of amino acids other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Including the step of administering (fragment);
Here (i) this ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, wherein the human is i) IGHG4 * 01 Human gamma-4 heavy chain constant containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Amino acids containing the region are expressed; and where (ii) the human is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (Here X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) , 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than K), A method comprising a nucleotide sequence encoding the Nav1.7 protein comprising said the amino acid selected from the group consisting of), 422D, 490N, 943L, 1002L, 1161W and 1919G (which is an amino acid other than W).

In some cases, this human has or is at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

2. The amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is a pain disease or condition. , Any of the aforementioned clause methods.

3. The amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the method of any of the aforementioned provisions where the disease or condition is a pain disease or condition. ..

4. The amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S). Yes), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) Where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (here X is an amino acid other than K) In X is an amino acid other than W) selected from the group; optionally the method of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition.

5. The method of any of the aforementioned provisions, wherein the constant region gene segment contained in the human is a germline gene segment.

6. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

7. Humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R) ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L , 1002L, 1161W and a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) May include Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of any of the aforementioned provisions determined.

8. Humans (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is R) Amino acids other than), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L , 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, A nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 490N, 943L, 1002L, 1161W and 1919G, and / or (b) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) In X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (here X is an amino acid other than R) X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of any of the aforementioned provisions, comprising the step of determining to include the Nav1.7 protein comprising, optionally, this determining step is performed prior to administration of this antibody to this human.

9. The process to decide is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R) 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G. The method of Clause 8 comprising assaying a human-derived biological sample for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G.

10. The steps to be assayed are with biological samples 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group, or said contiguous. Contains the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V. , 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (Here X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) , 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), Contains a nucleotide sequence encoding the amino acid selected from the group consisting of amino acids other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. , Thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. If at least one nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from is present, the step of forming the complex and
The step of detecting the presence or absence of this complex, wherein the step of detecting the presence of this complex is that this human has 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Contains Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of Clause 9, including the process of deciding.

11. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 9 in which this assaying step is performed in a multiplex format.

12. The method of Clause 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). Amino acids), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L , 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, It has been shown to be heterozygous for a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 943L, 1002L, 1161W and 1919G, and optionally the human being of SEQ ID NO: 76. Further indicated to contain a nucleotide sequence, or said human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X. (Here X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is other than W) (Amino acids of), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Any of the aforementioned provisions shown to be homozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of amino acids), 422D, 490N, 943L, 1002L, 1161W and 1919G. Method.

14. The method of any of the aforementioned provisions, wherein the human being is substantially or is further determined to be substantially resistant to the treatment of pain or pruritus.

15. The method of any of the aforementioned provisions, wherein the person has been or has been treated for pain or pruritus, or has been less responsive to the treatment for pain or pruritus.

16. The method of any of the aforementioned provisions, wherein the disease or condition is a pain or pruritus disease or condition.

17. Whether the disease or condition is channel disorder or is associated with channel disorder; or primary congenital insensitivity (PE), paroxysmal extreme pain (PEPD) and channel disorder-related painlessness (CIP) The method of any of the above clauses selected from the group consisting of).

18. The method of any of the aforementioned provisions in which the person has been diagnosed with at least one of the conditions listed in claim 16 or 17.

19. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment treats or reduces the risk in said human of the condition set forth in claim 16 or 17.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rsl3402180 and rsl2478318.

21. The method of any of the aforementioned provisions, wherein the ligand (eg, antibody or antibody fragment) is administered by inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. The method of any of the aforementioned provisions, wherein the human gamma-4 heavy chain constant region of the ligand comprises the amino acid sequence of SEQ ID NO: 73 or the ADCC inactivated version thereof.

23. The method of any of the aforementioned provisions, wherein the human gamma-4 heavy chain constant region comprises 228P and 235E.

Gamma-4 constant region tailor-made ligand
1. A ligand (eg, antibody or antibody fragment) for use in methods that treat or reduce the risk of a Nav1.7-mediated disease or condition (eg, pain) in humans in need thereof, 136I , 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X ( Where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (here X is an amino acid other than E) Is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than W) , 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G It specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of;
Here (i) this ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, where said human is ( i) IGHG4 * 01 Human gamma-4 heavy chain constant containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Amino acids containing the region are expressed; and where (ii) the human is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (Here X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) , 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than K), A ligand comprising a nucleotide sequence encoding the Nav1.7 protein comprising said the amino acid selected from the group consisting of), 422D, 490N, 943L, 1002L, 1161W and 1919G (which is an amino acid other than W).

In one alternative, paragraph 1 provides:-
In humans, ligands (eg, antibodies or amino acid fragments) for use in methods of targeting Nav1.7, 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F. , 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) Is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R) Amino acids), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) ), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G specifically binds to human Nav1.7 proteins containing amino acids selected from the group. A;
Here (i) this ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, and where said human (i). ) IGHG4 * 01 Human gamma-4 heavy chain constant region containing the human heavy chain constant region gene segment, or this human containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. And where (ii) the humans expressed 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K. , 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S) Where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R), Amino acids other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is W) Amino acid comprising), 422D, 490N, 943L, 1002L, 1161W and 1919G comprising the nucleotide sequence encoding the Nav1.7 protein comprising the amino acid selected from the group.

In some examples, this human is at or at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

2. The amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is a pain disease or condition. , Ligand of any of the aforementioned clauses.

3. The amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the ligand of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition. ..

4. The amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S). Yes), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) Where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (here X is an amino acid other than K) In X is an amino acid other than W) selected from the group; optionally said the disease or condition is a pain disease or condition, a ligand of any of the aforementioned provisions.

5. The ligand of any of the aforementioned provisions, wherein the VH gene segment contained in the human is a germline VH gene segment.

6. Of any of the aforementioned provisions, wherein the method comprises the step of selecting a human (where this human is the human of Clause 1) comprising the nucleotide sequence of (ii) prior to the step of administration. Ligand.

7. Humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R) ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L , 1002L, 1161W and a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) May include Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Determined, ligand of any of the aforementioned clauses.

8. Humans are (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is R) Amino acids other than), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L , 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, A nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 490N, 943L, 1002L, 1161W and 1919G, and / or (b) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) In X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R) X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. A ligand of any of the aforementioned provisions, wherein the method comprises a step of determining to include a Nav1.7 protein comprising, optionally, this determining step is performed prior to administration of this antibody to this human.

9. The process to decide is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R) 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G, comprising the step of assaying a human-derived biological sample for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group.

10. The steps to be assayed are with biological samples 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group, or said contiguous. Contains the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V. , 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (Here X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) , 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), Contains a nucleotide sequence encoding the amino acid selected from the group consisting of amino acids other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. , Thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. If at least one nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from is present, the step of forming the complex and
The step of detecting the presence or absence of this complex, wherein the step of detecting the presence of this complex is that this human has 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Contains Nav1.7 protein containing said amino acid selected from the group consisting of (amino acid), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Clause 9 ligand, including the step of determining.

11. The assaying step comprises or / or comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay is performed in a multiplex format, clause 9 ligand.

12. Ligand of Clause 9 or 11, wherein the biological sample comprises said human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). Amino acids), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L , 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, It has been shown to be heterozygous for a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 943L, 1002L, 1161W and 1919G, and optionally the human being of SEQ ID NO: 76. Further indicated to include a nucleotide sequence, or said human, is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X. (Here X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is other than W) (Amino acids of), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Any of the aforementioned provisions shown to be homozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of (amino acids), 422D, 490N, 943L, 1002L, 1161W and 1919G. Lagent.

14. A ligand of any of the aforementioned provisions, wherein the human is substantially resistant to, or is further determined to be, substantially resistant to the treatment of pain or pruritus.

15. A ligand of any of the aforementioned provisions, wherein the human has been or has been treated for pain or pruritus, or has been less responsive to the treatment for pain or pruritus.

16. A ligand of any of the aforementioned provisions, wherein the disease or condition is a pain or pruritus disease or condition.

17. Whether the disease or condition is channel disorder or is associated with channel disorder; or primary congenital insensitivity (PE), paroxysmal extreme pain (PEPD) and channel disorder-related painlessness (CIP) ), The ligand of any of the above clauses.

18. The ligand of any of the aforementioned provisions, wherein the human has been diagnosed with at least one of the conditions listed in claim 16 or 17.

19. A ligand of any of the aforementioned provisions, wherein the antibody or antibody fragment treats or reduces the risk in said human of the condition set forth in claim 16 or 17.

20. A ligand of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rsl3402180 and rsl2478318.

21. The ligand of any of the aforementioned provisions, wherein the ligand (eg, antibody or antibody fragment) is for inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. A ligand of any of the aforementioned provisions, wherein the human gamma-4 heavy chain constant region of the ligand comprises the amino acid sequence of SEQ ID NO: 73 or its ADCC inactivated version.

23. Human gamma-4 Ligand of any of the aforementioned clauses, wherein the heavy chain constant region comprises 228P and 235E.

Gamma-2 Steady Region Tailor Made Method
1. A method of treating or reducing the risk of a Nav1.7-mediated disease or condition in humans in need thereof, for said humans 136I, 216S, 241T, 395K, 848T, 858H, 858F. , 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y) , 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) Where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (here X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and human Nav1 containing amino acids selected from the group consisting of 1919G. .7 Including the step of administering a ligand that specifically binds to a protein (eg, an amino acid or an amino acid fragment);
Here (i) this ligand is Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Contains a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val and Ala at position 257 set forth in SEQ ID NO: 6, where the human is (i) IGHG2 * 01 human heavy chain determination. Containing a normal region gene segment, or this human being, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6. It expresses an antibody containing a human gamma-2 heavy chain constant region containing Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6;
Here (ii) the human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is, Amino acids other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) Amino acids), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D , 490N, 943L, 1002L, 1161W and 1919G comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In one alternative, Clause 1 provides:-
A method of targeting Nav1.7 in humans, with respect to the humans: 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V. , 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S) ), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (here X is an amino acid other than R) In X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (here X is an amino acid other than K) X is a ligand (eg, antibody or antibody) that specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of amino acids other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Including the step of administering (fragment);
Here, (i) this ligand is Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Contains a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val and Ala at position 257 set forth in SEQ ID NO: 6, where the human is (i) IGHG2 * 01 human heavy chain determination. Containing a normal region gene segment, or this human being, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6. It expresses an antibody containing a human gamma-2 heavy chain constant region containing Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6; and where (ii) the human is 136I, 216S. , 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than R), 328X X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E), Amino acids other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is R) Consists of 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. A method comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from the group.

In some examples, this human has or is at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

2. The amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is a pain disease or condition. , Any of the aforementioned clause methods.

3. The amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the method of any of the aforementioned provisions where the disease or condition is a pain disease or condition. ..

4. The amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S). Yes), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) Where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (here X is an amino acid other than K) In X is an amino acid other than W) selected from the group; optionally the method of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition.

5. The method of any of the aforementioned provisions, wherein the constant region gene segment contained in the human is a germline gene segment.

6. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

7. Humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R) ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L , 1002L, 1161W and a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) May include Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of any of the aforementioned provisions determined.

8. Humans (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is R) Amino acids other than), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L , 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, A nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 490N, 943L, 1002L, 1161W and 1919G, and / or (b) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) In X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (here X is an amino acid other than R) X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of any of the aforementioned provisions, comprising the step of determining to include the Nav1.7 protein comprising, optionally, this determining step is performed prior to administration of this antibody to this human.

9. The process to decide is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R) 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G. The method of Clause 8 comprising assaying a human-derived biological sample for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G.

10. The steps to be assayed are with biological samples 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group, or said contiguous. Contains the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V. , 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (Here X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) , 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), Contains a nucleotide sequence encoding the amino acid selected from the group consisting of amino acids other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. , Thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. When at least one nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from is present, the steps of forming a complex, and
The step of detecting the presence or absence of this complex, wherein the step of detecting the presence of this complex is that this human has 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Contains Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. The method of Clause 9, including the process of deciding.

11. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 9 in which this assaying step is performed in a multiplex format.

12. The method of Clause 9 or 11, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). Amino acids), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L , 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, It has been shown to be heterozygous for a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 943L, 1002L, 1161W and 1919G, and optionally the human being of SEQ ID NO: 76. Further indicated to contain a nucleotide sequence, or said human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X. (Here X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is other than W) (Amino acids of), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Any of the aforementioned provisions shown to be homozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of amino acids), 422D, 490N, 943L, 1002L, 1161W and 1919G. Method.

14. The method of any of the aforementioned provisions, wherein the human being is substantially or is further determined to be substantially resistant to the treatment of pain or pruritus.

15. The method of any of the aforementioned provisions, wherein the person has been or has been treated for pain or pruritus, or has been less responsive to the treatment for pain or pruritus.

16. The method of any of the aforementioned provisions, wherein the disease or condition is a pain or pruritus disease or condition.

17. Whether the disease or condition is channel disorder or is associated with channel disorder; or primary congenital insensitivity (PE), paroxysmal extreme pain (PEPD) and channel disorder-related painlessness (CIP) The method of any of the above clauses selected from the group consisting of).

18. The method of any of the aforementioned provisions in which the person has been diagnosed with at least one of the conditions listed in claim 16 or 17.

19. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment treats or reduces the risk in said human of the condition set forth in claim 16 or 17.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rsl3402180 and rsl2478318.

21. The method of any of the aforementioned provisions, wherein the ligand (eg, antibody or antibody fragment) is administered by inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. The method of any of the aforementioned provisions, wherein the human gamma-2 heavy chain constant region of the ligand comprises the IGHG2 * 01 amino acid sequence or its ADCC inactivated version.

Gamma-2 constant region tailor-made ligand
1. A ligand (eg, antibody or antibody fragment) for use in methods that treat or reduce the risk of a Nav1.7-mediated disease or condition (eg, pain) in humans in need thereof, 136I , 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X ( Where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (here X is an amino acid other than E) Is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than W) , 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G It specifically binds to a human Nav1.7 protein containing an amino acid selected from the group consisting of;
Here, (i) this ligand is Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Contains a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val and Ala at position 257 set forth in SEQ ID NO: 6, where the human is (i) IGHG2 * 01 human heavy chain determination. Containing a normal region gene segment, or this human being, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6. An antibody containing a human gamma-2 heavy chain constant region containing Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6 is expressed; and where (ii) said human is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. A ligand comprising a nucleotide sequence encoding the Nav1.7 protein comprising said amino acid selected from.

In one alternative, paragraph 1 provides:-
In humans, ligands (eg, antibodies or amino acid fragments) for use in methods of targeting Nav1.7, 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F. , 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) Is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R) Amino acids), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) ), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G specifically binds to human Nav1.7 proteins containing amino acids selected from the group. A;
Here, (i) this ligand is Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Contains a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val and Ala at position 257 set forth in SEQ ID NO: 6, where the human is (i) IGHG2 * 01 human heavy chain determination. It contains a normal region gene segment, or this human is the selected Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, and Phe at position 76 shown in SEQ ID NO: 6. , Expressing an antibody comprising a human gamma-2 heavy chain constant region containing Val at position 161 set forth in SEQ ID NO: 6 or Ala at position 257 set forth in SEQ ID NO: 6; and where (ii) said human was 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (here And X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) , 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than W), From 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G A ligand (eg, an antibody or antibody fragment) comprising a nucleotide sequence encoding the Nav1.7 protein containing the amino acid selected from the group.

In some examples, this human is at or at risk for a Nav1.7-mediated disease or condition. In some examples, this method treats or reduces the risk of Nav1.7-mediated disease or condition in humans.

2. The amino acid is selected from the group consisting of any one of 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P and 1449V; optionally the disease or condition is a pain disease or condition. , Ligand of any of the aforementioned clauses.

3. The amino acid is selected from the group consisting of 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I and 1627K; optionally the ligand of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition. ..

4. The amino acids are 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S). Yes), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than R) Where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) and 1689X (here X is an amino acid other than K) X is an amino acid other than W); optionally the ligand of any of the aforementioned clauses, wherein the disease or condition is a pain disease or condition.

5. The ligand of any of the aforementioned provisions, wherein the VH gene segment contained in the human is a germline VH gene segment.

6. Of any of the aforementioned provisions, wherein the method comprises the step of selecting a human (where this human is the human of Clause 1) comprising the nucleotide sequence of (ii) prior to the step of administration. Ligand.

7. Humans are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R) ), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) , 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L , 1002L, 1161W and a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 1919G, and / or 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (here X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) May include Nav1.7 proteins containing said amino acids selected from the group consisting of amino acids), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Determined, ligand of any of the aforementioned clauses.

8. Humans are (a) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is R) Amino acids other than), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L , 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, A nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of 490N, 943L, 1002L, 1161W and 1919G, and / or (b) 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (here X is an amino acid other than I) In X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R) X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. A ligand of any of the aforementioned provisions, wherein the method comprises a step of determining to include a Nav1.7 protein comprising, optionally, this determining step is performed prior to administration of this antibody to this human.

9. The process to decide is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R) 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N , 943L, 1002L, 1161W and 1919G, comprising the step of assaying a human-derived biological sample for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group.

10. The steps to be assayed are with biological samples 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than S), Amino acids other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W) (Yes), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) ), 422D, 490N, 943L, 1002L, 1161W and 1919G containing at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group, or said contiguous. Contains the step of contacting with at least one oligonucleotide probe comprising the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V. , 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (Here X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than I) , 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), 1659X (where X is an amino acid other than R), Contains a nucleotide sequence encoding the amino acid selected from the group consisting of amino acids other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. , Thereby 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is X) Is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than E) (Amino acids other than), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is other than R) 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. If at least one nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from is present, the step of forming the complex and
The step of detecting the presence or absence of this complex, wherein the step of detecting the presence of this complex is that this human has 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than Y) X is an amino acid other than S), 693X (where X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is other than R) 897X (where X is an amino acid other than W), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K) Contains Nav1.7 protein containing said amino acid selected from the group consisting of (amino acid), 1689X (where X is an amino acid other than W), 422D, 490N, 943L, 1002L, 1161W and 1919G. Clause 9 ligand, including the step of determining.

11. The assaying step comprises or / or comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay is performed in a multiplex format, clause 9 ligand.

12. Ligand of Clause 9 or 11, wherein the biological sample comprises said human serum, blood, feces, tissue, cells, urine and / or saliva.

13. The human beings are 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X (where X is other than R). Amino acids), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (where X is an amino acid other than E) ), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is an amino acid other than W), 1200L, 1235L , 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W), 422D, 490N, It has been shown to be heterozygous for a nucleotide sequence encoding the Nav1.7 protein containing said amino acid selected from the group consisting of 943L, 1002L, 1161W and 1919G, and optionally the human being of SEQ ID NO: 76. Further indicated to contain a nucleotide sequence, or said human, is 136I, 216S, 241T, 395K, 848T, 858H, 858F, 863P, 1449V, 996C, 1298F, 1298D, 1299F, 1461T, 1462V, 1464I, 1627K, 277X. (Here X is an amino acid other than R), 328X (where X is an amino acid other than Y), 395K, 459X (where X is an amino acid other than S), 693X (here X is an amino acid other than S) X is an amino acid other than E), 767X (where X is an amino acid other than I), 830X (where X is an amino acid other than R), 897X (where X is other than W) (Amino acids of), 1200L, 1235L, 1488X (where X is an amino acid other than R), 1659X (where X is an amino acid other than K), 1689X (where X is an amino acid other than W) Any of the aforementioned provisions shown to be homozygous for a nucleotide sequence encoding a Nav1.7 protein containing said amino acid selected from the group consisting of (amino acids), 422D, 490N, 943L, 1002L, 1161W and 1919G. Lagent.

14. A ligand of any of the aforementioned provisions, wherein the human is substantially resistant to, or is further determined to be, substantially resistant to the treatment of pain or pruritus.

15. A ligand of any of the aforementioned provisions, wherein the human has been or has been treated for pain or pruritus, or has been less responsive to the treatment for pain or pruritus.

16. A ligand of any of the aforementioned provisions, wherein the disease or condition is a pain or pruritus disease or condition.

17. Whether the disease or condition is channel disorder or is associated with channel disorder; or primary congenital insensitivity (PE), paroxysmal extreme pain (PEPD) and channel disorder-related painlessness (CIP) ), The ligand of any of the above clauses.

18. The ligand of any of the aforementioned provisions, wherein the human has been diagnosed with at least one of the conditions listed in claim 16 or 17.

19. A ligand of any of the aforementioned provisions, wherein the antibody or antibody fragment treats or reduces the risk in said human of the condition set forth in claim 16 or 17.

20. A ligand of any of the aforementioned provisions, wherein the nucleotide sequence comprises one or more SNPs selected from the group consisting of rs6746030, rs3750904, rs58022607, rs4369876, rsl3402180 and rsl2478318.

21. The ligand of any of the aforementioned provisions, wherein the ligand (eg, antibody or antibody fragment) is for inhalation, intravenous or subcutaneous administration and / or is included in an inhalation or injection preparation.

22. A ligand of any of the above clauses, wherein the human gamma-2 heavy chain constant region of the ligand comprises the IGHG2 * 01 amino acid sequence or its ADCC inactivated version.

(Example 4)
The rare IL6R variant interleukin-6 (IL-6) is a complex cytokine that plays an important role in the regulation of the inflammatory response. Interleukin-6 (IL-6) is a ligand-binding IL-6 receptor (IL-6R; also known as CD126) chain and a common signaling chain glycoprotein 130 (also known as gpl30; CD130) [ It also engages receptors specific for IL-11, IL-27, leukin inhibitors, oncostatin M (OSM), hairy neurotrophic factor (CNTF) and cardiotrophin 1 (CT1). Is signaled through]. Soluble IL-6R (sIL-6R) can be produced by proteolytic cleavage of mIL-6R by metalloproteinase TNFa converting enzyme (TACE; also known as ADAM17) and ADAM10, or spliced mRNA. The IL-6 response is a high-affinity tetramer complex consisting of IL-6, IL-6R and two gpl30 molecules (or six consisting of two IL-6, two mIL-6R and two gpl30 molecules). It can be classically induced in cells expressing mIL-6R through a complex of molecules). Alternatively, the SIL-6-IL-6R complex binds directly to gpl30 in cells lacking mIL-6R in a process named transsignaling and signals through it. High levels of IL-6 and sIL-6R have been reported in some chronic inflammatory diseases and in cancer. Several drugs targeting different components of the IL-6 and IL-6R systems have been described, including IL-6-specific monoclonal antibodies (mAbs) (eg, CNTO 328), IL-6R. Specific mAbs (eg tocilizumab) and soluble gpl30-Fc, IL-6R transsignaling antagonists can be mentioned. "Averting inflammation by targeting the cytokine environment", Manfred Kopf, Martin F. Bachmann & Benjamin J. Marsland, Nature Reviews Drug Discovery 9, pp. 703-718 (September 2010), doi: 10.1038 / nrd2805 See the review in (incorporated in the book).

Genetic variations in the IL-6 receptor gene are associated with the risk of several human diseases with inflammatory components, including coronary heart disease, rheumatoid arthritis, and asthma. A non-synonymous variant in the IL-6 receptor gene (IL6R Asp358Ala; rs2228145 A> C) is associated with increased susceptibility to asthma. The variant is between sIL6R (produced through cleavage of cell surface receptors and by alternative splicing of soluble IL6R isoforms) and membrane-bound IL-6R (involved in classical IL6R signaling). It is thought that the functional mechanism is exerted by adjusting the balance of. Ferreira et al. Collaborated that a small allele of this non-synonymous variant (Ala358) directly regulates the surface level of IL-6R on individual immune cells, and that these differences at the protein level are functional in IL6R signaling. It was shown for the first time as a report that it was translated into various disorders.

Ferreira et al. Note that classical IL6R signaling appears to be responsible for regulatory T cell suppression, but IL6R signaling (via sIL6R) appears to promote T-helper 2-cell polarization in the lung. did. IL6R has been shown to be expressed in human airway epithelium, smooth muscle and vascular endothelium, as well as in bronchoalveolar lavage fluid (BALF) macrophages and granulocytes. Soluble IL6R levels in BALF are elevated in asthmatics compared to controls, and are further elevated during allergen challenge, which plays an important role in sIL6R in lung and related tissue conditions. Shown. Consistent with these findings, 358 Ala is also associated with increased asthma severity, as noted above. The authors also provide evidence for the association of this non-synonymous variant at risk for type 1 diabetes (T1D) in two independent populations, and that rs2228145 has a concentration of soluble IL6R (sIL6R) levels in the circulation. Confirm that it is the major determinant (reportedly, a 34.6% increase in sIL6R per copy of the minor allele 358Ala; rs2228145 [C]). The authors provided evidence that the non-synonymous variant rs2228145 regulates surface-sIL6R equilibrium and also affects immune cell responsiveness to IL6 stimulation. This mechanism demonstrates the effect of 358 Ala on the IL-6 / IL-6R pathway.

Revez et al. Commented that the major genetic determinant of soluble interleukin-6 receptor levels is the variant rs2228145, which maps to the cleavage site of IL6R. Reportedly, for each Ala allele, serum levels of sIL6R increased up to about 20 ng ml, and asthma risk increased up to 1.09 fold. However, the authors conclude that this variant does not explain total hereditary potential for sIL6R levels. Therefore, additional independent variants in IL6R can contribute to variations in IL6R levels and affect asthma risk. The authors input 471 variants of IL6R and tested these for association with serum levels of sIL6R in 360 individuals. The intron variant (rsl2083537) was associated with IL6R levels independently of rs4129267, a surrogate single nucleotide polymorphism for rs2228145 (P = 0.0005). A significant and consistent association for rsl2083537 was observed in the replication panel of 354 individuals (P = 0.033). Each rsl2083537: A allele increased serum levels of sIL6R to 2.4 ng ml. Analysis of mRNA levels in the two cohorts did not identify a significant association between rsl2083537 and IL6R transcription levels. On the other hand, results from 16705 asthma patients and 30809 controls showed that the rsl2083537: A allele increased asthma risk by a factor of 1.04 (P = 0.0419).

J C Galicia et al. Investigated the association of IL6R gene polymorphisms with soluble IL6 receptors at serum levels. In total, 115 healthy volunteers were genotyped and 70 of them were analyzed for sIL6R levels. Two important polymorphic sites were selected for genotyping using the PCR / RFLP method (48892A / C (D358A) in exon 9 and -183G / A in the promoter region). In exon 9, carriers of the C allele have higher sIL6R levels (P <0.0001), thereby this sequence variation in response to the proteolytic cleavage site of IL-6R alpha is serum sIL6R. It has been shown to have a strong effect on levels. In the promoter region, carriers of the G allele had lower sIL6R levels compared to carriers of the A allele (P <0.0082).

Esparza-Gordillo et al. Examined the results of all genome-wide related studies from public storage and selected 318 genetic markers that were significantly associated with any inflammatory trait. These markers were examined and tested for association with AD in a three-step approach using seven study populations, including 7130 patients with atopic dermatitis (AD) and 9253 control subjects. .. Reportedly, functional amino acid changes at the IL-6 receptor (IL-6R Asp358Ala; rs2228145) were significantly associated with AD. A study of the birth cohort based on two independent populations showed that IL-6R 358Ala was particularly susceptible to persistent AD. Carriers of 358Ala have increased serum levels of soluble IL6R, and homozygous carriers show a 2-fold increase. In addition, the authors reported that sIL6R levels were higher in patients with AD than in control subjects. The authors conclude that this study supports the importance of genetic variants that affect inflammation in the etiology of AD. In addition, they identified a functional genetic variant in IL6R that affects disease prognosis and is particularly susceptible to persistent AD.

Antibodies to human IL6R are described in US Pat. Nos. 8043617, 5670373, 5817790, European Patent No. 40907, and their publications in Table 13 below. Treatment methods are described in US Pat. Nos. 5888510, 8043617, 6723319 and their publications in Table 13 below. Actemra ™ (tocilizumab) is an example of humanization of an anti-IL-6R drug that blocks both classical and transsignal transduction. When it comes to antibodies, the invention instead relates to antibodies that have fully human (non-humanized) variable regions (and optionally human constant regions as well).

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial tissue that leads to the destruction of joint structures. Cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) can play a role in joint inflammation and cartilage damage observed in RA. Be recognized. IL-6 is a multifaceted cytokine that has biological effects on many cell types. It is often considered downstream of TNF or IL-1 in the inflammatory cytokine cascade and can therefore indicate a common pathway factor in a wide range of inflammatory processes. Thus, blocking IL-6 signaling provides the ability to alleviate multiple pathogenic features of RA and other inflammatory diseases.

Treatment methods using IL-6R antagonists are referred to in US Pat. No. 5,888,510; US Pat. No. 6,723,319; and US Patent Application Publication No. 2001/0001663. Exemplary anti-IL-6R antibodies are described in US Pat. Nos. 7,582,298; 6,410,691; 5,817,790; 5,795,695; 6,670,373; and 7,582,298.

In certain embodiments, the IL6R polypeptide is a terminal residue, eg, a leader sequence residue, a targeting residue, an amino-terminal methionine residue, a lysine residue, a tag residue and / or Contains fusion protein residues. "IL6R" was also called interleukin-6 receptor subunit alpha, CD126; IL-6R-1; IL-6RA; IL6Q; IL6RA; IL6RQ; and gp80.

The present invention provides anti-IL6R ligands; as well as IL6R binding or targeting ligands as described herein. This ligand has various usefulness. Some of these ligands have other IL6R activity in affinity purification of IL6R (particularly human IL6R or its ligands) to genotype or phenotype humans, for example in specific binding assays. It is useful in screening assays to identify antagonists. Some ligands of the invention are useful for inhibiting the binding of IL6R to IL6 and / or gp130, or for inhibiting IL6R-mediated activity.

Anti-IL6R ligands (eg, antibodies and antisense RNA) have been developed based on the targeting and neutralization of so-called "wild" human IL6R, which is a commonly present type (eg, SEQ ID NO: 78). See). Although such treatments are useful for human patients carrying this type of human IL6R, we have targeted a rare but also naturally occurring type of IL6R in the human population. Was considered useful for investigating the possibility of. In this way, the inventor is a useful target for human treatment, prevention and diagnosis (protein or nucleic acid level) that is mediated by IL6R activity or is relevant for diseases and conditions associated therewith. We have reached an insight into the natural appearance and distribution of rare human IL6R types that may function. This provides tailor-made treatment, prevention and diagnosis, especially in humans who lack the common IL6R gene or protein.

Those skilled in the art are aware that SNPs or other changes that translate into amino acid variations can result in variability in the activity and / or higher-order structure of the human target to be targeted. This has led to great interest in personalized medicine that uses knowledge of genotyping and protein and nucleotide variability to more effectively tailor-make patient care and diagnosis. Accordingly, the present invention provides tailor-made pharmaceuticals and studies specifically directed to the rare IL6R polymorphic variant. Such forms or "alleles" (at the nucleotide level) contain one or more changes at the nucleotide and amino acid levels from the corresponding common types of nucleotide and amino acid sequences, i.e., protein targets in humans. There are one or more non-synonymous changes at the nucleotide level that translate into one or more corresponding changes in.

Moreover, the inventor surprisingly found that rare natural forms are present in humans much less frequently than common forms, but are nevertheless shown in large and ethnically diverse human populations. We have noticed that there are usually many human examples per ethnic group shown. Thus, by targeting such rare types, the inventor provides effective treatment, prevention or diagnosis across many human ethnic groups, thereby expanding the usefulness of the invention. I noticed.

With this recognition, the inventor is concerned with guiding the selection of anti-IL6R ligands for administration to human patients for the treatment and / or prevention of IL6R-mediated or related diseases or conditions. I noticed that there are various industrial and medical applications. In this method, the patient receives tailor-made drugs and ligands for their needs as determined by the composition of the patient's genes or phenotypes. Along with that, the present invention provides patient genotyping and / or phenotyping associated with such treatments, thereby allowing the drug to be appropriately adapted to the patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment (eg, poor efficacy and / or side effects) with drugs or ligands that are not suitable for the patient, and avoids drug prescribing mistakes and waste. ..

To develop this idea, in this non-limiting example, the inventor decided to determine a set of human IL6R variants based on the following criteria, but these criteria were noticed by the inventor. Provides medical drugs and diagnostics useful for tailor-made needs in the human population. We have selected variants that have at least three of the following four criteria:
Naturally occurring human IL6R variations with a cumulative human allele frequency of 35% or less;
Naturally occurring human IL6R variations with a total human genotype frequency of about 50% or less;
Naturally occurring human IL6R variations found in many different human populations (using standard categorization of the 1000 Genomes Project; see Table 14 below); and.
A naturally occurring human IL6R variation found in many individuals distributed across many such different ethnic groups.

Based on these criteria, the inventor identified the variants listed in Table 13 below. The inventor's choice was to consider nucleotide variations (ie, non-synonymous variations) that produced amino acid variations in the corresponding IL6R types, as opposed to silent variations that did not alter the amino acid residues in the target protein. Included a selection of.

Tailor-Made Antibodies to Rare IL6R Variant Profiles As outlined above, the invention is a gene segment found in many humans of the ethnic population in which the variant IL6R type is found to meet the selection criteria of the invention. Includes the possibility of further tailoring human treatment by selecting antibody-based ligands with variable and / or constant domains based on. In one example, a ligand comprising the VH domain of a recombinantly derived antibody of a human IGHV gene segment comprising a nucleotide selected at a position in HCDR1 or FW3 that is variable in human (ie, SNP occurs in human) is provided. Will be done.

The inventor has analyzed human IGHV variations and used them to adapt to the genotype and / or phenotype of the human recipient based on the human IGHV allele containing the selected nucleotide. The ligand was selected for. The present inventor (i) contains a CDR1 encoding Phe at position 4 set forth in SEQ ID NO: 109 (where the human contains a VH gene segment encoding CDR1 containing Phe at position 4 set forth in SEQ ID NO: 109? , Or this human expresses a VH gene containing CDR1 containing Phe at position 4 set forth in SEQ ID NO: 109); or (ii) FW3 containing Thr at position 33 set forth in SEQ ID NO: 111 (where described above). The human contains the VH gene segment encoding FW3 containing Thr at position 33 shown in SEQ ID NO: 111, or this human expresses the VH domain containing FW3 containing Thr at position 33 shown in SEQ ID NO: 111. The VH gene segment encoding (to) was identified for its usefulness in using the gene. Further information is provided in Table 14 (Table 15), which shows variations at these locations, as well as variant distributions across the 1000 Genomes Project database involving many human populations.

In other embodiments, as will be further described in detail above, the present invention provides an alternative to human VH gene segment, or V _K, genotype of a human recipient based on Vλ or constant region gene segments and / Alternatively, provide tailor-made ligands for the phenotype [see further Table 16 for representative variants].

In some examples, according to this guidance, the selected ligand may be salilumab.

Therefore, a further example is:-
(i) Here, this ligand comprises a human VH segment IGHV3-7 * 01, a VH domain derived from the recombination of the human D gene segment and the human JH segment, and where the human is IGHV3-7 * 01. Contains the VH gene segment, or this human expresses the VH domain from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment.
(ii) Here, this ligand contains the human V _K segment IG KV1-12 * 01 and the V _K domain derived from the recombination of the human J _K segment, wherein the human is the IGK V1-12 * 01 V _K gene. Includes a segment, or this human expresses the V _K domain from the recombination of the human V _K segment IGK V1-12 * 01 and the human J _K segment.
(iii) Here, this ligand contains the V _K domain derived from the recombination of the human V _K segment and the human J _K segment, and this human V _K segment has Pro at position 7 shown in (i) SEQ ID NO: 113. Containing CDR3 (and where said human contains a V _K gene segment encoding CDR3 containing Pro at position 7 set forth in SEQ ID NO: 113, or this human contains Pro at position 7 set forth in SEQ ID NO: 113. Expresses a V _K domain containing CDR3 containing); or (ii) FW3 containing Ser at position 15 shown in SEQ ID NO: 115 (and where the human has Ser at position 15 shown in SEQ ID NO: 115). It contains a V _K gene segment that encodes FW3 that contains, or this human expresses a V _K domain that contains FW3 with Ser at position 15 set forth in SEQ ID NO: 115).
(iv) Here, this ligand comprises a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 81 or Leu at position 206 set forth in SEQ ID NO: 81, wherein said human. (i) IGHG1 * 01 Human gamma-1 heavy chain determination containing the human heavy chain constant region gene segment or containing Asp at position 204 shown in SEQ ID NO: 81 or Leu at position 206 shown in SEQ ID NO: 81. It expresses an antibody containing a normal region.
(v) Here, this ligand is Pro at position 72 shown in SEQ ID NO: 83, Asn at position 75 shown in SEQ ID NO: 83, Phe at position 76 shown in SEQ ID NO: 83, and position 161 shown in SEQ ID NO: 83. Contains a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val and Ala at position 257 set forth in SEQ ID NO: 83, wherein the human is (i) IGHG2 * 01 human heavy chain determination. This human contains the normal region gene segment, or this human is the selected Pro at position 72 shown in SEQ ID NO: 83, Asn at position 75 shown in SEQ ID NO: 83, and Phe at position 76 shown in SEQ ID NO: 83. , Val at position 161 shown in SEQ ID NO: 83 or Ala at position 257 shown in SEQ ID NO: 83 to express an antibody containing a human gamma-2 heavy chain constant region.
(vi) Here, the ligand comprises a human kappa chain constant region containing Val at position 84 set forth in SEQ ID NO: 93 or Cys at position 87 set forth in SEQ ID NO: 93, wherein the human is (i). IGKC1 * 01 An antibody containing a human kappa chain constant region gene segment, or this human contains a Val corresponding to position 84 shown in SEQ ID NO: 93 or a human kappa chain constant region containing Cys at position 87 shown in SEQ ID NO: 93. Is expressed.
(vii) where this ligand comprises a human IGLC1 * 01 lambda chain constant region, where the human comprises (i) a human IGLC1 * 01 lambda chain constant region gene segment, or this human is , Human IGLC1 * 01 Expresses an antibody containing a lambda chain constant region.

For example, as in Example (iv), the inventor tackles the rare IGH-gamma-1 SNP 204D (observing a cumulative frequency of 0.296) and 206L (observing a cumulative frequency of 0.283) individually or in combination. Identified the possibility. These residues are part of the CH3 domain and, as such, form part of the antibody Fc region. Therefore, these CH3 variations and patient compatibility are particularly beneficial for the reasons discussed above. Thus, in this example, the ligand of the invention comprises or comprises an antibody comprising a human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 81 or Leu corresponding to position 206 of SEQ ID NO: 81. Consisting of those antibodies, and where the human genome contains a gamma-1 heavy chain constant region nucleotide sequence encoding such Asp or Leu, or this human has such Asp or Leu. Human Gamma-Contains antibodies containing the constant region. An example of such a ligand is salilumab.

In another example, as in Example (v), the inventor identified the possibility of addressing the SNP of IGH-gamma-2. This included the examination of Fc region variations, and in this regard, the present inventor focused on positions 161 and 257 in the Fc region. Therefore, in this example, the ligands of the present invention are Pro corresponding to position 72 of SEQ ID NO: 83, Asn corresponding to position 75 of SEQ ID NO: 83, Phe corresponding to position 76 of SEQ ID NO: 83, and 161 of SEQ ID NO: 83. Human gamma containing an amino acid selected from the group consisting of Val corresponding to the position Val and Ala corresponding to the 257th position of SEQ ID NO: 83-Containing or consisting of an antibody containing a heavy chain constant region; In this human genome contains a gamma-2 heavy chain constant region nucleotide sequence encoding such a selected amino acid, or this human contains a human gamma-2 constant region containing such a selected amino acid. Express the containing antibody.

In another example, as in Example (vi), the inventor worked on the human kappa constant region variation. Thus, in this example, the ligand of the invention comprises or comprises an antibody comprising a human kappa light chain constant region comprising Val corresponding to position 84 of SEQ ID NO: 93 or Cys corresponding to position 87 of SEQ ID NO: 93. Consisting of antibodies; and where this human genome contains a kappa light chain constant region nucleotide sequence encoding such Val or Cys, or this human is a human kappa light chain constant containing such Val or Cys. It expresses an antibody containing the normal region. An example of such a ligand is salilumab.

In another example, as in Example (vii), the inventor worked on a human lambda constant region variation. Thus, in this example, the ligand of the invention comprises or consists of antibodies comprising the human IGLC2 * 01 light chain constant region; and where the human genome comprises the human IGLC2 * 01 nucleotide sequence. Or, this human expresses an antibody containing the human light chain IGLC2 * 01 constant region.

Determination of specific binding of the ligand of the invention to the IL6R variant
Specific binding of the ligand of the present invention to the IL6R variant may be carried out using the following method.

How to determine SPR for binding
Binding of antibodies to the IL6R variant is performed by SPR using the ProteOn XPR36 ™ Array system (BioRad). Anti-human IgG surfaces (Jackson Labs 109-005-008) were made on GLC Biosensor chips by primary amine coupling. The test antibody is captured as a ligand on this surface. The IL6R variant is used as an analyte and passed over the captured antibody at 256 nM, 64 nM, 16 nM, 4 nM and 1 nM. The binding curve is doubly referenced using buffer infusion (ie 0 nM) to eliminate baseline drift and infusion artifacts. Regeneration of the capture surface uses 100 mM phosphate, which removes the captured antibody, allowing another cycle of capture and binding. The generated coupling sensorgram is analyzed using a 1: 1 model specific to the ProteOn XPR36 Array system analysis software. The assay is performed at 25 ° C. using 1 × HBS-EP (Teknova) as a running buffer.

In certain embodiments of the invention, methods such as the following provisions are provided.
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Including the step of administering a ligand that binds to (eg, an antibody or antibody fragment).
Here, this ligand contains the VH domain derived from the recombination of the human VH segment, the human D gene segment, and the human JH segment, and this human VH segment contains (i) Phe at the 4-position shown in SEQ ID NO: 109. (And here, said human contains a VH gene segment encoding CDR1 containing Phe at position 4 set forth in SEQ ID NO: 109, or this human contains CDR1 containing Phe at position 4 set forth in SEQ ID NO: 109. (I express a VH domain containing); or (ii) FW3 containing Thr at position 33 shown in SEQ ID NO: 111 (and here, the human has FW3 containing Thr at position 33 shown in SEQ ID NO: 111). The VH gene segment encoding, or the human expresses a VH domain comprising FW3 with Thr at position 33 set forth in SEQ ID NO: 111; and where the human expresses the Asp358Ala or A method of encoding (including a nucleotide sequence encoding the IL6R protein, including Val385Ile).

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

In one example, the IL6R protein comprises the mutant Asp358Ala in SEQ ID NO: 78.

In one example, the IL6R protein comprises the mutant Val385Ile in SEQ ID NO: 78.

2. A method of treating or reducing the risk of an IL6R-mediated disease or condition in humans in need thereof, specific for the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment); where this ligand comprises a recombinantly derived VH domain of a human VH gene segment, a human D gene segment and a human JH segment, the human being. The VH gene segment is (a) the T of nucleotide number 86 set forth in SEQ ID NO: 84 (and where the human comprises the VH gene segment containing the T of nucleotide number 86 set forth in SEQ ID NO: 84); or ( b) C of nucleotide number 272 set forth in SEQ ID NO: 84 (and where said human comprises a VH gene segment containing C of nucleotide number 272 set forth in SEQ ID NO: 84; and where said human is the sequence. The method comprising the nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile at number 78).

3. The method of any of the aforementioned provisions, wherein each said human VH gene segment comprises SEQ ID NO: 108.

4. The method of any of the aforementioned provisions, wherein each said human VH gene segment comprises SEQ ID NO: 110.

5. The method of any of the aforementioned provisions, wherein the VH domain is derived from recombination of human VH segment IGHV3-9 * 01, human D gene segment and human JH segment.

6. The method of any of the aforementioned clauses, wherein the VH domain comprises the FW3 sequence of SEQ ID NO: 111.

7. The method of any of the aforementioned provisions, wherein the VH gene segment contained in the human is a germline VH gene segment.

8. The ligand contains the human gamma-1 heavy chain containing the VH domain and the human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 81 and Leu at position 206 shown in SEQ ID NO: 81, and Here, the method of any of the aforementioned provisions, wherein the human comprises an IGHG1 * 01 human heavy chain constant region gene segment.

9. The method of any of the aforementioned provisions, wherein the antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

10. The method of any of the aforementioned provisions in which the human is a human of AMR, ASN or EUR pedigree.
For example, this human is a human of CHB, CHS, JPT, CEU or FIN pedigree. These pedigrees have a very high cumulative frequency (> 85%, eg,> 90%, or even 100%) for SNPs (FW3 SNPs), so the ligands of the invention are human in such humans. Well suited for treating or preventing IL6R-mediated diseases or conditions.

11. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

12. It is determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

13. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The method of any of the aforementioned provisions, wherein this determining step is performed prior to administration of this antibody to this human.

14. The method of Clause 13, wherein the determination step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

15. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contact with at least one oligonucleotide probe containing the antisense sequence of the nucleotides, wherein the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. Thus, the method of Clause 14, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

16. The step to assay comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of clause 14 in which this assaying step is performed in a multiplex format.

17. The method of clause 14 or 16, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

18. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

19. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.
The method of any of the aforementioned provisions, wherein the human has received or has received anti-TNF alpha treatment or has had reduced responsiveness to anti-TNF alpha treatment.

20. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

21. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

22. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

23. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

24. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

25. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

26. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or contained in an injectable preparation.

In certain embodiments of the invention, ligands according to the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It specifically binds to the IL6R protein and contains the VH domain from the recombination of the human VH segment, human D gene segment and human JH segment, and this human VH segment is located at position 4 shown in (i) a SEQ ID NO: 109. CDR1 containing Phe (and where said human contains the VH gene segment encoding CDR1 containing Phe at position 4 set forth in SEQ ID NO: 109, or this human is located at position 4 set forth in SEQ ID NO: 109. (Expressing a VH gene containing CDR1 containing Phe); or (ii) FW3 containing Thr at position 33 set forth in SEQ ID NO: 111 (and where the human has Thr at position 33 set forth in SEQ ID NO: 111). Contains a VH gene segment encoding a FW3 containing, or this human expresses a VH domain containing a FW3 containing Thr at position 33 set forth in SEQ ID NO: 111); and where said human An anti-IL6R ligand (eg, an antibody or fragment) comprising the nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It specifically binds to the IL6R protein and contains the VH domain derived from the recombination of the human VH gene segment, the human D gene segment and the human JH segment, and this human VH gene segment is (a) the nucleotide number shown in SEQ ID NO: 84. 86 T (and where said human comprises the VH gene segment containing T of nucleotide number 86 set forth in SEQ ID NO: 84); or (b) C of nucleotide number 272 set forth in SEQ ID NO: 84 (and here). The human comprises the VH gene segment comprising C of nucleotide number 272 set forth in SEQ ID NO: 84; and where the human comprises the Asp358Ala or Val385Ile in SEQ ID NO: 78. An anti-IL6R ligand (eg, an antibody or fragment) that contains a nucleotide sequence that encodes a protein.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

3. The ligand of any of the aforementioned paragraphs, wherein each said human VH gene segment comprises SEQ ID NO: 108.

4. The ligand of any of the aforementioned paragraphs, wherein each said human VH gene segment comprises SEQ ID NO: 110.

5. The ligand of any of the aforementioned paragraphs, wherein the VH domain is derived from the recombination of human VH segment IGHV3-9 * 01, human D gene segment and human JH segment.

6. The ligand of any of the aforementioned paragraphs, wherein the VH domain comprises the FW3 sequence of SEQ ID NO: 111.

7. The ligand of any of the above paragraphs, wherein the VH gene segment contained in the human is a germline VH gene segment.

8. The ligand contains the human gamma-1 heavy chain containing the VH domain and the human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 81 and Leu at position 206 shown in SEQ ID NO: 81, and Here, the ligand of any of the aforementioned paragraphs, wherein the human comprises an IGHG1 * 01 human heavy chain constant region gene segment.

9. Ligand of any of the above paragraphs, wherein the antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

10. The ligand of any of the above paragraphs, where the human is a human of AMR, ASN or EUR lineage.
For example, this human is a human of CHB, CHS, JPT, CEU or FIN pedigree.

11. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

12. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The ligand of any of the aforementioned paragraphs, in which this determining step is performed prior to administration of this antibody to this human.

13. The ligand of paragraph 12, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

The step of assay comprises a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, or said contiguous nucleotides. Contact with at least one oligonucleotide probe containing the antisense sequence of, wherein the sequence of contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 13 comprising the step of detecting the presence of this complex, wherein the human comprises the IL6R protein comprising the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

14. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay in paragraph 13 is performed in a multiplex format.

15. The ligand of paragraph 13 or 15, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

16. The ligand of any of the aforementioned paragraphs, the human being shown to be heterozygous for the nucleotide sequence encoding the IL6R protein, including the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, is optionally shown. The human is further shown to contain the nucleotide sequence of SEQ ID NO: 79, or the human is homozygous for the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. Shown, ligand.

17. Ligand of any of the preceding paragraphs for administration to humans who are substantially resistant to anti-TNF alpha treatment or who are further determined to be substantially resistant.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

18. Ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who have reduced responsiveness to anti-TNF alpha treatment.

19. Ligand in any of the preceding paragraphs for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

20. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

21. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected. In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

22. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

23. The ligands are inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease. The ligand of any of the aforementioned paragraphs that treats or reduces risk in said human in at least one condition.

24. Ligand of any of the preceding paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

25. The ligand of any of the aforementioned paragraphs, wherein the ligand is for intravenous or subcutaneous administration and / or is included in the injectable preparation.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment);
Whether this ligand comprises a human VH segment IGHV3-7 * 01, a human D gene segment and a recombinantly derived VH domain of a human JH segment, and where the human comprises the IGHV3-7 * 01 VH gene segment. , Or this human expresses a VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment; and where the human expresses the Asp358Ala or Val385Ile in SEQ ID NO: 78. A method comprising a nucleotide sequence encoding said IL6R protein comprising.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The ligand contains the human gamma-1 heavy chain containing the VH domain and the human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 81 and Leu at position 206 shown in SEQ ID NO: 81, and Here, the method of Clause 1, wherein the human comprises an IGHG1 * 01 human heavy chain constant region gene segment.

3. The method of any of the aforementioned provisions, wherein the antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

4. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

5. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

6. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The method of any of the aforementioned provisions, wherein this determining step is performed prior to administration of this antibody to this human.

7. The method of Clause 6, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

8. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contact with at least one oligonucleotide probe containing the antisense sequence of the nucleotides, wherein the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. Thus, the method of Clause 7, wherein the step of detecting the presence of this complex comprises determining that the human comprises the IL6R protein comprising the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

9. The step of the assay comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 7 in which this assaying step is performed in a multiplex format.

10. The method of clause 7 or 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

12. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is optional, with or without methotrexate, infliximab [eg, Remicade®], adalimumab [eg, adalimumab]. , Humira®], certolizumab pegol [eg Cimzia®], golimumab [eg Simoni®], and etanercept [eg Enbrel (Registered Trademark)] Selected from the group consisting of treatments.

13. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

14. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.
The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

15. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected. In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

16. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma. , Adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition in said human.

17. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.
The method of any of the aforementioned provisions, wherein said antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is included in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, including the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It specifically binds to the IL6R protein and contains the human VH segment IGHV3-7 * 01, the human D gene segment and the VH domain derived from the recombination of the human JH segment, where the human is the IGHV3-7 * 01VH gene. Contains a segment, or the human expresses a VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment; and where the human expresses the VH domain in SEQ ID NO: 78. A ligand comprising a nucleotide sequence encoding the IL6R protein, including Asp358Ala or Val385Ile.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. Contains the human gamma-1 heavy chain containing the VH domain and the human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 81 and Leu at position 206 shown in SEQ ID NO: 81, and here. The ligand according to paragraph 1, wherein the human comprises an IGHG1 * 01 human heavy chain constant region gene segment.

3. Ligand of any of the above paragraphs, wherein the antibody or fragment comprises the IGHG1 * 01 human heavy chain constant region.

4. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

5. Optional, including the step of determining that a human comprises a nucleotide sequence encoding an IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile. In any of the above-mentioned paragraphs, this determining step is performed prior to administration of this antibody to this human.

6. Ligand of paragraph 5, wherein the step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

7. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contact with at least one oligonucleotide probe containing the antisense sequence of the resulting nucleotide, wherein the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 6 comprising the step of detecting the presence of this complex, wherein the human comprises the IL6R protein comprising the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

8. The step of the assay comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay in paragraph 6 is performed in a multiplex format.

9. The ligand of paragraph 6 or 8, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

10. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The ligand of any of the aforementioned paragraphs, wherein said human is homozygous for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

11. Ligand in any of the preceding paragraphs for administration to humans who are substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.

In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

Ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who have reduced responsiveness to anti-TNF alpha treatment.

12. Ligand in any of the preceding paragraphs for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

13. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

14. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected. In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

15. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

16. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

17. Ligand of any of the preceding paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

18. Ligand of any of the preceding paragraphs for intravenous or subcutaneous administration and / or contained in an injectable preparation.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment);
Wherein the ligand comprises a human V _K segment IGKV1-12 * 01 and V _K domains from recombinant human J _K segments, and wherein said human comprises a IGKV1-12 * 01 V _K gene segment Or, this human expresses the V _K domain from the recombination of the human V _K segment IGKV 1-12 * 01 and the human J _K segment; where the human comprises the Asp358Ala or Val385Ile in SEQ ID NO: 78. A method comprising a nucleotide sequence encoding the IL6R protein.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The ligand comprises the human kappa chain containing the V _K domain and the human kappa chain constant region containing the Val at position 84 set forth in SEQ ID NO: 93 and the Cys at position 87 set forth in SEQ ID NO: 93, and where the human However, the method of Clause 1 comprising the IGKC * 01 human kappa chain constant region gene segment.

3. The method of any of the aforementioned provisions, wherein the antibody or fragment comprises the IGKC * 01 human kappa chain constant region.

4. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

5. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

6. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The method of any of the aforementioned provisions, wherein this determining step is performed prior to administration of this antibody to this human.

7. The method of Clause 6, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

8. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby thereby. When at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 7, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

9. The step of the assay comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 7 in which this assaying step is performed in a multiplex format.

10. The method of clause 7 or 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein said human is homozygous for a nucleotide sequence encoding an IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

12. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.

In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

13. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

14. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

15. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

16. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected. In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, adult breathing At least selected from the group consisting of distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. The method of any of the aforementioned provisions diagnosed as one condition.

17. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

18. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

19. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is contained in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, including the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. specifically bind to IL6R protein comprises a human V _K segment IGKV1-12 * 01 and V _K domains from recombinant human J _K segments, and wherein said human, IGKV1-12 * 01V _K gene segment , Or this human expresses the V _K domain derived from the recombination of the human V _K segment IGK V1-12 * 01 and the human J _K segment; and where the human expresses the Asp358Ala or in SEQ ID NO: 78. An anti-IL6R ligand (eg, antibody or fragment) comprising a nucleotide sequence encoding the IL6R protein, including Val385Ile.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The human kappa chain containing the V _K domain and the human kappa chain constant region containing the Val at position 84 shown in SEQ ID NO: 93 and the Cys at position 87 shown in SEQ ID NO: 93, where the human is IGKC. * 01 Ligand of paragraph 1 containing the human kappa chain constant region gene segment.

3. Ligand of any of the preceding paragraphs, wherein the antibody or fragment comprises the IGKC * 01 human kappa chain constant region.

4. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

5. Optional, including the step of determining that a human comprises a nucleotide sequence encoding an IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile. In any of the above-mentioned paragraphs, this determining step is performed prior to administration of this antibody to this human.

6. Ligand of paragraph 5, wherein the step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.
The step of assay comprises a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, or said contiguous nucleotides. Contacting with at least one oligonucleotide probe containing the antisense sequence of, the sequence of contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby the SEQ ID NO: In 78, if at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present, a step of forming a complex; and a step of detecting the presence or absence of this complex, wherein , The ligand of paragraph 6, wherein the step of detecting the presence of this complex comprises the step of determining that the human comprises the IL6R protein comprising the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

7. The step to be assayed comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. The step of this assay in paragraph 6 is performed in multiplex format.

8. The ligand of paragraph 6 or 8, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

9. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. Or the ligand of any of the aforementioned paragraphs, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

10. Ligand of any of the preceding paragraphs for administration to humans who are substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

11. Ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who are less responsive to anti-TNF alpha treatment.

12. Ligand in any of the preceding paragraphs for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

13. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

14. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected.

In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

15. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

16. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

17. Ligand of any of the preceding paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

18. Ligand of any of the preceding paragraphs for intravenous or subcutaneous administration and / or contained in an injectable preparation.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment);
Here, this ligand contains the V _K domain derived from the recombination of the human V _K segment and the human J _K segment, and this human V _K segment contains Pro at position 7 shown in (i) SEQ ID NO: 113. (And here, the human comprises the V _K gene segment encoding CDR3 with Pro at position 7 set forth in SEQ ID NO: 113, or the human comprises Pro at position 7 set forth in SEQ ID NO: 113. (Expressing the V _K domain containing CDR3); or (ii) FW3 containing Ser at position 15 shown in SEQ ID NO: 115 (and where the human is FW3 containing Ser at position 15 shown in SEQ ID NO: 115). The V _K gene segment encoding, or this human expresses a V _K domain containing FW3 containing Ser at position 15 set forth in SEQ ID NO: 115); and where said human expresses the sequence. A method comprising a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile at number 78.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment);
Here, this ligand contains a V _K domain derived from the recombination of the human V _K gene segment and the human J _K segment, and this human V _K gene segment is (a) T (a) of nucleotide number 199 shown in SEQ ID NO: 98. And here, the human comprises the V _K gene segment containing T of nucleotide number 199 set forth in SEQ ID NO: 98; or (b) C of nucleotide number 284 set forth in SEQ ID NO: 98 (and where said human). Includes the V _K gene segment containing T at nucleotide number 284 set forth in SEQ ID NO: 98; and where the human encodes the nucleotide sequence encoding said IL6R protein containing said Asp358Ala or Val385Ile in SEQ ID NO: 78. Including, method.

3. The method of any of the aforementioned provisions, wherein each said human _VK gene segment comprises SEQ ID NO: 112.

4. The method of any of the aforementioned provisions, wherein each said human _VK gene segment comprises SEQ ID NO: 114.

5. The V _K domain is recombinant-derived human V _K segment IGKV3-11 * 01 and human JK segments, any of the methods of the aforementioned provisions.

6. The V _K domain comprises a FW3 sequences of SEQ ID NO: 115, any method of the aforementioned provisions.

7. The method of any of the aforementioned provisions, wherein the V _K gene segment contained in the human is a germline V _K gene segment.

8. The ligand comprises the human kappa chain comprising the V _K domain and the human kappa chain constant region comprising the Val at position 84 set forth in SEQ ID NO: 93 and the Cys at position 87 set forth in SEQ ID NO: 93, where said human. However, the method of any of the aforementioned provisions comprising the IGKC * 01 human kappa chain constant region gene segment.

9. The method of any of the above clauses, wherein the antibody or fragment comprises the IGKC * 01 human heavy chain constant region.

10. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

11. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

12. Optional, including the step of determining that the human contains a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The method of any of the aforementioned provisions, wherein this determining step is performed prior to administration of this antibody to this human.

13. The method of Clause 12, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

14. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 13, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

15. The step to be assayed comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. The method of Clause 13 in which this assaying step is performed in a multiplex format.

16. The method of clause 13 or 15, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

17. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein, including the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

18. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

19. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

20. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

21. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

22. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, implant rejection, allogeneic implant rejection, implant-to-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of choice for any of the aforementioned provisions. In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

23. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

24. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

25. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

26. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or contained in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It specifically binds to the IL6R protein and
Here, this ligand contains the V _K domain derived from the recombination of the human V _K segment and the human J _K segment, and this human V _K segment contains Pro at position 7 shown in (i) SEQ ID NO: 113. (and here, CDR3 comprises or a V _K gene segment encoding a CDR3 comprising Pro at the 7-position of the person is shown in SEQ ID NO: 113, or the human, including the Pro in position 7 of SEQ ID NO: 113 expressing V _K domain containing); or (ii) FW3 including Ser at position 15 of SEQ ID NO: 115 (and where encoding FW3 comprising Ser at position 15 of the person is shown in SEQ ID NO: 115 Contains a V _K gene segment, or this human expresses a V _K domain containing FW3 containing Ser at position 15 set forth in SEQ ID NO: 115; and where said human is sequenced. An anti-IL6R ligand (eg, antibody or fragment) comprising the nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile at number 78.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It specifically binds to the IL6R protein and
Here, this ligand contains a V _K domain derived from the recombination of the human V _K gene segment and the human J _K segment, and this human V _K gene segment is (a) T of nucleotide number 199 shown in SEQ ID NO: 98. (And here, said human comprises the V _K gene segment containing T of nucleotide number 199 set forth in SEQ ID NO: 98); or (b) C of nucleotide number 284 set forth in SEQ ID NO: 98 (and here, The human comprises a V _K gene segment comprising T of nucleotide number 284 set forth in SEQ ID NO: 98; and where the human encodes the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78. An anti-IL6R ligand (eg, an antibody or fragment) comprising a nucleotide sequence.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

3. The ligand of paragraph 1 or 2, wherein each said human VH gene segment comprises a monobasic polymorphism rsl82958807 and / or rsl91612627.

4. The ligand of any of the aforementioned paragraphs, wherein each said human _VK gene segment comprises SEQ ID NO: 112.

5. The ligand of any of the aforementioned paragraphs, wherein each said human _VK gene segment comprises SEQ ID NO: 114.

6. Ligand of any of the aforementioned paragraphs, wherein the V _K domain is derived from the recombination of human V _K segment IGK V3-11 * 01 and human J _K segment.

7. The ligand of any of the aforementioned paragraphs, wherein the V _K domain comprises the FW3 sequence of SEQ ID NO: 115.

8. The ligand of any of the aforementioned paragraphs, wherein the V _K gene segment contained in the human is a germline V _K gene segment.

9. The ligand comprises the human kappa chain containing the V _K domain and the human kappa chain constant region containing the Val at position 84 set forth in SEQ ID NO: 93 and the Cys at position 87 set forth in SEQ ID NO: 93, and where the human Is a ligand of any of the above paragraphs, comprising the IGKC * 01 human kappa chain constant region gene segment.

10. The ligand of any of the above paragraphs, wherein the antibody or fragment comprises the IGKC * 01 human heavy chain constant region.

11. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

12. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The ligand of any of the aforementioned paragraphs, in which this determining step is performed prior to administration of this antibody to this human.

13. The ligand of paragraph 12, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

14. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 14, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

15. The step to be assayed comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. This assaying step is performed in multiplex format, paragraph 13 ligand.

16. The ligand of paragraph 13 or 14, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

17. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. Or the ligand of any of the aforementioned paragraphs, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

18. Religants in any of the preceding paragraphs for administration to humans who are substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any of the embodiments herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg, Remicade®], adalimumab [eg, Humila (eg, Humila). Registered Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel® )] Selected from the group consisting of treatments.

19. Ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who have reduced responsiveness to anti-TNF alpha treatment.

20. The ligands of any of the aforementioned paragraphs, for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

21. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

22. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

23. The human being has inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, implant rejection, allogeneic implant rejection, implant-to-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

24. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, transplant rejection, allogeneic transplant rejection, transplant-to-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), septic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

25. Ligand of any of the aforementioned paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

26. The ligand of any of the aforementioned paragraphs, wherein the ligand is for intravenous or subcutaneous administration and / or is included in the preparation for injection.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment), wherein the ligand contains Asp at position 204 set forth in SEQ ID NO: 81 or Leu at position 206 set forth in SEQ ID NO: 81. -Contains a heavy chain constant region, wherein the human is (i) IGHG1 * 01 human heavy chain constant region gene segment (or this human is Asp or SEQ ID NO: 81 at position 204 set forth in SEQ ID NO: 81. A method comprising a human gamma-1 heavy chain constant region containing an antibody containing Leu at position 206) and (ii) a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78.

In one example, the ligand, antibody or antibody fragment was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The method of Clause 1, wherein the ligand comprises a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 81 and Leu at position 206 set forth in SEQ ID NO: 81.

3. The ligand comprises a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 81 and Leu at position 206 set forth in SEQ ID NO: 81, where the human is (i) IGHG1. * 01 The method of any of the aforementioned provisions comprising a human heavy chain constant region gene segment and (ii) a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

4. The method of Clause 1, 2 or 3, wherein the antibody or fragment comprises an IGHG1 * 01 human heavy chain constant region.

5. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

6. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

7. Optional, including the step of determining that the human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The method of any one of clauses 1-6, wherein this determining step is performed prior to administration of this antibody to this human.

8. The method of Clause 7, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

9. The step to be assayed comprises a biological sample and at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, or said contiguous. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. When at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 8, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

10. The step of the assay comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. The method of Clause 8 in which this assaying step is performed in a multiplex format.

11. The method of Clause 8 or 10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

12. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

13. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

14. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

15. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

16. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

17. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

18. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

19. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

21. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is contained in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, where this ligand is mutated in SEQ ID NO: 78. A human gamma-1 heavy chain constant that specifically binds to an IL6R protein containing Asp358Ala or Val385Ile, where this ligand contains Asp at position 204 set forth in SEQ ID NO: 81 or Leu at position 206 set forth in SEQ ID NO: 81. It comprises a region, wherein the human is (i) IGHG1 * 01 human heavy chain constant region gene segment (or this human is at position 204 shown in SEQ ID NO: 81 as Asp or position 206 shown in SEQ ID NO: 81. An anti-IL6R ligand (eg, an antibody) comprising a nucleotide sequence encoding the IL6R protein comprising the Asp358Ala or Val385Ile in SEQ ID NO: 78) and (ii) expressing an antibody comprising a human gamma-1 heavy chain constant region comprising Leu. Or fragment).

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The ligand of paragraph 1 comprising the human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 81 and Leu at position 206 set forth in SEQ ID NO: 81.

3. A human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 81 and Leu at position 206 shown in SEQ ID NO: 81, wherein the human is (i) IGHG1 * 01 human. A ligand of any of the aforementioned paragraphs comprising a heavy chain constant region gene segment and (ii) a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

4. Ligand of paragraph 1, 2 or 3 containing the human heavy chain constant region of IGHG1 * 01.

5. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

6. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. , The ligand of any one of paragraphs 1-5, wherein this determining step is performed prior to administration of this antibody to this human.

7. The ligand of paragraph 6 which comprises the step of assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

8. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 7, wherein the step of detecting the presence of this complex comprises determining that the human comprises the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

9. The step of the assay comprises or / or comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. This assaying step is performed in multiplex format, paragraph 7 ligand.

10. The ligand of paragraph 7 or 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. Or the ligand of any of the aforementioned paragraphs, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

12. Ligand in any of the preceding paragraphs for administration to humans who are substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any of the embodiments herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg, Remicade®], adalimumab [eg, Humila (eg, Humila). Registered Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel® )] Selected from the group consisting of treatments.

13. A ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who have reduced responsiveness to anti-TNF alpha treatment.

14. The ligands of any of the aforementioned paragraphs, for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

15. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

16. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

17. The humans have inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, implant rejection, allogeneic implant rejection, implant-to-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

18. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, transplant rejection, allogeneic transplant rejection, transplant-to-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), septic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

19. The ligand of any of the aforementioned paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

20. Ligands from any of the aforementioned paragraphs for intravenous or subcutaneous administration and / or contained in an injectable preparation.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of an IL6R-mediated disease or condition in a person in need thereof, specific to the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment), wherein the ligand is Pro at position 72 shown in SEQ ID NO: 83, Asn at position 75 shown in SEQ ID NO: 83, SEQ ID NO: Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Phe at position 76 shown in 83, Val at position 161 shown in SEQ ID NO: 83 and Ala at position 257 shown in SEQ ID NO: 83. And here, the human is (i) IGHG2 * 01 human heavy chain constant region gene segment (or this human is Pro at position 72 shown in the selected SEQ ID NO: 83, position 75 shown in SEQ ID NO: 83. Asn, Phe at position 76 shown in SEQ ID NO: 83, Val at position 161 shown in SEQ ID NO: 83, or Ala at position 257 shown in SEQ ID NO: 83 to express an antibody containing a human gamma-2 heavy chain constant region. ), And (ii) a method comprising a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The ligands are (i) Pro at position 72 shown in SEQ ID NO: 83, Asn at position 75 shown in SEQ ID NO: 83, Phe at position 76 shown in SEQ ID NO: 83, and (ii) SEQ ID NO: optionally. Includes a human gamma-2 heavy chain constant region containing Val at position 161 shown in 83 and / or Ala at position 257 shown in SEQ ID NO: 83; and where this human genome is such that of (i). The method of Clause 1 comprising a gamma-2 heavy chain constant region nucleotide sequence encoding an amino acid, or in which the human expresses an antibody comprising a human gamma-2 constant region comprising such an amino acid in (i).
In one example, the ligand, antibody or antibody fragment was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

3. The ligands are (i) Val at position 161 shown in SEQ ID NO: 83, Ala at position 257 shown in SEQ ID NO: 83, and optionally (ii) Pro at position 72 shown in SEQ ID NO: 83, SEQ ID NO: It contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Asn at position 75 shown in 83 and Phe at position 76 shown in SEQ ID NO: 83; and here the human genome is (1). A gamma-2 heavy chain constant region nucleotide sequence encoding such an amino acid in i) is contained, or the human expresses an antibody comprising a human gamma-2 constant region containing such an amino acid in (i). The method of any of the aforementioned provisions.

4. The method of Clause 1, 2 or 3, wherein the antibody or fragment comprises an IGHG2 * 01 human heavy chain constant region.

5. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

6. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

7. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. , Any one method of clauses 1-6, wherein this determining step is performed prior to administration of this antibody to this human.

8. The method of Clause 7, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

9. The step to be assayed comprises a biological sample and at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, or said contiguous. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. When at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 8, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

10. The step of the assay comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. The method of Clause 8 in which this assaying step is performed in a multiplex format.

11. The method of Clause 8 or 10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

12. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

13. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

14. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

15. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

16. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

17. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

18. The humans have inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, implant rejection, allogeneic implant rejection, implant-to-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

19. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, implant rejection, allogeneic implant rejection, implant-to-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

21. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or contained in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. A human gamma-1 heavy chain constant region that specifically binds to the IL6R protein and contains Asp at position 204 set forth in SEQ ID NO: 81 or Leu at position 206 set forth in SEQ ID NO: 81, where said human is ( i) IGHG1 * 01 human heavy chain constant region gene segment (or this human contains a human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 81 or Leu at position 206 shown in SEQ ID NO: 81. An anti-IL6R ligand (eg, an antibody or fragment) comprising (ii) a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78.

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. (i) Pro at position 72 shown in SEQ ID NO: 83, Asn at position 75 shown in SEQ ID NO: 83, Phe at position 76 shown in SEQ ID NO: 83, and optionally (ii) shown in SEQ ID NO: 83. Contains a human gamma-2 heavy chain constant region containing Val at position 161 and / or Ala at position 257 set forth in SEQ ID NO: 83; and where the human genome encodes such an amino acid of (i). The ligand of paragraph 1 comprising the gamma-2 heavy chain constant region nucleotide sequence, or in which the human expresses an antibody comprising a human gamma-2 constant region comprising such an amino acid of (i).

3. Human gamma-2 heavy chain constant region, which includes (i) Val at position 161 shown in SEQ ID NO: 83 and Ala at position 257 shown in SEQ ID NO: 83, and optionally (ii) SEQ ID NO: 83. Includes a human gamma-2 heavy chain constant region containing amino acids selected from the group consisting of Pro at position 72 shown in, Asn at position 75 shown in SEQ ID NO: 83 and Phe at position 76 shown in SEQ ID NO: 83; , Where this human genome contains a gamma-2 heavy chain constant region nucleotide sequence encoding such an amino acid in (i), or a human gamma in which this human contains such an amino acid in (i)- A ligand of any of the aforementioned paragraphs that expresses an antibody containing two constant regions.

4. Ligand of paragraph 1, 2 or 3 containing the human heavy chain constant region of IGHG2 * 01.

5. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

6. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The ligand of any one of paragraphs 1-5, wherein this determining step is performed prior to administration of this antibody to this human.

7. Ligand of paragraph 6, wherein the step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

8. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 7, wherein the step of detecting the presence of this complex comprises determining that the human comprises the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

9. The assaying step comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay in paragraph 7 is performed in a multiplex format.

10. The ligand of paragraph 7 or 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The ligand of any of the aforementioned paragraphs, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

12. Ligand in any of the preceding paragraphs for administration to humans who are substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any of the embodiments herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg, Remicade®], adalimumab [eg, Humila (eg, Humila). Registered Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel® )] Selected from the group consisting of treatments.

13. Ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who have reduced responsiveness to anti-TNF alpha treatment.

14. Ligand in any of the preceding paragraphs for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

15. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

16. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

17. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

18. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

19. The ligand of any of the aforementioned paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

20. The ligand of any of the aforementioned paragraphs, wherein the ligand is for intravenous or subcutaneous administration and / or is included in the injectable preparation.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of an IL6R-mediated disease or condition in humans in need thereof, specific for the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment), wherein the ligand comprises Val at position 84 set forth in SEQ ID NO: 93 or Cys at position 87 set forth in SEQ ID NO: 93. Containing a chain constant region, wherein the human is (i) IGKC1 * 01 human heavy chain constant region gene segment (or this human is on Val or SEQ ID NO: 93 corresponding to position 84 set forth in SEQ ID NO: 93). A method comprising the nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78) and (ii) expressing an antibody comprising a human kappa chain constant region comprising Cys at position 87 shown.

In one example, the ligand, antibody or antibody fragment was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The method of Clause 1, wherein the ligand comprises a human kappa chain constant region comprising Val at position 84 set forth in SEQ ID NO: 93 and Cys at position 87 set forth in SEQ ID NO: 93.

3. The ligand comprises a human kappa chain constant region containing Val at position 84 set forth in SEQ ID NO: 93 and Cys at position 87 set forth in SEQ ID NO: 93, wherein the human is (i) IGKC * 01 human kappa. The method of any of the aforementioned provisions comprising a chain constant region gene segment and (ii) a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

4. The method of Clause 1, 2 or 3 where the ligand comprises the IGKC1 * 01 human kappa chain constant region.

5. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

6. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

7. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. , Any one method of clauses 1-6, wherein this determining step is performed prior to administration of this antibody to this human.

8. The method of Clause 7, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

9. The step to be assayed comprises a biological sample and at least 10 contiguous nucleotide sequences of the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, or said contiguous. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. When at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 8, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

10. The step of the assay comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. The method of Clause 8 in which this assaying step is performed in a multiplex format.

11. The method of Clause 8 or 10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

12. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

13. The method of the aforementioned clause, wherein the human is substantially resistant to, or is further determined to be, substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

14. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

15. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

16. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

17. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

18. The humans have inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, implant rejection, allogeneic implant rejection, implant-to-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

19. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

20. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

21. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered by intravenous or subcutaneous administration and / or is contained in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It comprises a human kappa chain constant region that specifically binds to the IL6R protein and comprises Val at position 84 set forth in SEQ ID NO: 93 or Cys at position 87 set forth in SEQ ID NO: 93, wherein the human is (i). IGKC1 * 01 Human heavy chain constant region gene segment (or this human expresses an antibody containing a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 93 or Cys at position 87 shown in SEQ ID NO: 93. And (ii) an anti-IL6R ligand (eg, antibody or fragment) comprising the nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78.
In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The ligand of paragraph 1 comprising the human kappa chain constant region containing Val at position 84 set forth in SEQ ID NO: 93 and Cys at position 87 set forth in SEQ ID NO: 93.

3. Contains a human kappa chain constant region containing Val at position 84 shown in SEQ ID NO: 93 and Cys at position 87 shown in SEQ ID NO: 93, wherein the human is (i) IGKC * 01 human kappa chain constant region. A ligand of any of the aforementioned paragraphs comprising a gene segment and (ii) a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

4. IGKC1 * 01 Ligand of paragraph 1, 2 or 3 containing the human kappa chain constant region.

5. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of any of the aforementioned paragraphs.

6. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. , This determination step is performed prior to administration of this antibody to this human, any one of paragraphs 1-5 ligand.

7. The ligand of paragraph 6 comprising the step of assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

8. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 7, wherein the step of detecting the presence of this complex comprises determining that the human comprises the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

9. The step of the assay comprises or / or comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allelic gene-specific amplification. This assaying step is performed in multiplex format, paragraph 7 ligand.

10. The ligand of paragraph 7 or 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

11. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. Or the ligand of any of the aforementioned paragraphs, wherein the human is shown to be homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

12. Ligand in any of the preceding paragraphs for administration to humans who are substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any of the embodiments herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg, Remicade®], adalimumab [eg, Humila (eg, Humila). Registered Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel® )] Selected from the group consisting of treatments.

13. A ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who have reduced responsiveness to anti-TNF alpha treatment.

14. The ligands of any of the aforementioned paragraphs, for administration to humans separately or concurrently with methotrexate or anti-TNF alpha treatment.

15. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

16. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

17. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

18. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, transplant rejection, allogeneic transplant rejection, transplant-to-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), septic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

19. The ligand of any of the aforementioned paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

20. The ligand of any of the aforementioned paragraphs, wherein the ligand is for intravenous or subcutaneous administration and / or is included in the injectable preparation.

In certain embodiments of the invention, methods are provided as per the following provisions:-
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, specific for the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 for said human. Includes the step of administering a ligand that binds to (eg, an antibody or antibody fragment), wherein the ligand comprises a human IGLC1 * 01 lambda chain constant region, wherein the human is (i) human IGLC1. * 01 The lambda chain constant region gene segment (or this human expresses an antibody containing the human IGLC1 * 01 lambda chain constant region), and (ii) encode the IL6R protein containing the Asp358Ala or Val385Ile in SEQ ID NO: 78. A method comprising a nucleotide sequence.

In one example, the ligand, antibody or antibody fragment was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. The method of any of the aforementioned clauses, comprising the step of selecting a human (where this human is the human of Clause 1) comprising said nucleotide sequence of (ii) prior to the step of administration.

3. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The method of any of the aforementioned provisions made.

4. Optional, including the step of determining that a human comprises a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The method of any one of clauses 1-3, wherein this determining step is performed prior to administration of this antibody to this human.

5. The method of Clause 4, wherein the determining step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing said mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

6. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. When at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 5, wherein the step of detecting the presence of this complex comprises determining that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

7. The step to assay comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 5 in which this assaying step is performed in a multiplex format.

8. The method of clause 5 or 7, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

9. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any of the aforementioned provisions, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

10. The method of any of the aforementioned provisions, wherein the human is substantially resistant to or further determined to be substantially resistant to anti-TNF alpha treatment.
In any embodiment herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg Remicade®], adalimumab [eg Humila (registration). Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel®. ] Selected from the group consisting of treatments.

11. The method of any of the aforementioned provisions, wherein the person was or was receiving anti-TNF alpha treatment or was less responsive to anti-TNF alpha treatment.

12. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

13. The method of any of the aforementioned provisions, wherein the disease or condition is an inflammatory disease or condition.

14. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of any of the aforementioned clauses selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

15. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any method of the above clause that has been diagnosed with at least one condition.

16. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease The method of any of the aforementioned provisions that treats or reduces the risk of at least one condition selected from in said human.

17. The method of any of the aforementioned provisions, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

18. The method of any of the aforementioned provisions, wherein the antibody or antibody fragment is administered intravenously or subcutaneously and / or is contained in an injectable preparation.

In certain embodiments of the invention, the ligands provided in the following paragraphs are provided:-
1. An anti-IL6R ligand (eg, antibody or fragment) for treating or reducing the risk of IL6R-mediated disease or condition in humans in need thereof, comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. It specifically binds to the IL6R protein, where the ligand comprises a human IGLC1 * 01 lambda chain constant region, wherein said human is (i) a human IGLC1 * 01 lambda chain constant region gene segment (or This human expresses an antibody comprising a human IGLC1 * 01 lambda chain constant region), and (ii) an anti-IL6R ligand (eg,) comprising a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile in SEQ ID NO: 78. , Antibodies or fragments).

In one example, the ligand was determined to specifically bind to an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

2. It has been determined that humans contain a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. The ligand of paragraph 1.

3. Optional, including the step of determining that a human contains a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile and / or the IL6R protein containing the mutant Asp358Ala and / or Val385Ile. The ligand of paragraph 1 or 2, wherein this determining step is performed prior to administration of this antibody to this human.

4. The ligand of paragraph 3 wherein the determination step comprises assaying a human-derived biological sample for a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile at SEQ ID NO: 78.

5. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. In the presence of at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The ligand of paragraph 4, wherein the step of detecting the presence of this complex comprises determining that the human comprises the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

6. The step of assaying comprises or / or includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization, and allele-specific amplification. The step of this assay in paragraph 4 is performed in a multiplex format.

7. The ligand of paragraph 4 or 6, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

8. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The ligand of any of the aforementioned paragraphs, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

9. Ligand of any of the preceding paragraphs for administration to humans who are substantially resistant to anti-TNF alpha treatment or who are further determined to be substantially resistant.
In any of the embodiments herein, "anti-TNF alpha treatment" is with or without methotrexate, infliximab [eg, Remicade®], adalimumab [eg, Humila (eg, Humila). Registered Trademarks], certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], and etanercept [eg, Enbrel® )] Selected from the group consisting of treatments.

10. Ligand in any of the preceding paragraphs for administration to humans who have received or have received anti-TNF alpha treatment or who are less responsive to anti-TNF alpha treatment.

11. Ligand in any of the preceding paragraphs for administration to humans separately or simultaneously with methotrexate or anti-TNF alpha treatment.

12. The ligand of any of the aforementioned paragraphs, wherein the disease or condition is an inflammatory disease or condition.

13. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), From the group consisting of asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The ligand of any of the aforementioned paragraphs selected.
In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

14. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. A ligand in any of the aforementioned paragraphs that has been diagnosed with at least one condition.

15. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome At least one selected from the group consisting of (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease. A ligand of any of the aforementioned paragraphs that treats or reduces the risk of the condition in said human.

16. The ligand of any of the preceding paragraphs, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

17. The ligand of any of the aforementioned paragraphs, wherein the ligand is for intravenous or subcutaneous administration and / or is included in the injectable preparation.

Regimen
A: The present invention further provides the following regimens, ligands and kits.
1. Rare variant A method for treating a human IL6R-mediated disease or condition in humans by targeting human IL6R, which can specifically bind to the IL6R variant. Includes the step of administering the determined ligand (eg, antibody or fragment); where the human expresses the IL6R variant or the human genome comprises a nucleotide sequence encoding the IL6R variant. A method in which the human is treated here for the disease or condition.

This variant IL6R may be, for example, an IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. In one example, this IL6R comprises the mutation Asp358Ala in SEQ ID NO: 78. In one example, this IL6R comprises the mutation Val385Ile in SEQ ID NO: 78.

In one example, this IL6R is human soluble IL6R.

In one example, this IL6R is a human cell surface IL6R.

In certain embodiments, the determination of the specific binding is by reference, for example, binding assay data as determined using SPR or ELISA. The determination may refer to the information in the printed publication, eg, using knowledge of the data presented in the present invention or another patent application, or in a bibliographic article. Once such information is available (eg, without further testing of binding), one of ordinary skill in the art will identify relevant humans whose genotype or phenotype is compatible with the binding specificity of the ligand (instructions of the invention). Can be treated (by).
Antibodies or fragments may be in accordance with any of the configurations, examples, embodiments, embodiments, clauses or paragraphs herein.

In certain embodiments, the method is the step of selecting a human containing the nucleotide sequence encoding IL6R prior to the administration step [where this human is said human in clause 1 (eg, 1a). ]including.

2. The method of Clause 1, comprising the step of determining that the ligand specifically binds to the IL6R, eg, using SPR or ELISA prior to the administration step.

3. The method of Clause 1 or 2, wherein the specific binding to IL6R is a binding with a dissociation constant (Kd) of 1 nM or less, eg, 100, 10 or 1 pM or less.

4. The method of clauses 1-3 (eg, clause 1a), wherein the disease or condition is an inflammatory disease or condition.

5. The disease or condition is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma , Adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease The method of Clause 4 (eg, independent of Clause 1a).

In any embodiment herein, the arthritis is moderate to severe rheumatoid arthritis or systemic juvenile idiopathic arthritis.

6. It has been determined that humans contain a nucleotide sequence encoding IL6R containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 and / or the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, Clauses 1-5. Any one of the methods (eg, independent of clause 1a).

7. Optional, including the step of determining that a human comprises a nucleotide sequence encoding IL6R comprising a mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein comprising a mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. The method of any one of clauses 1-6 (eg, independent of clause 1a), wherein this determining step is performed prior to administration of this antibody to this human.

8. The method of Clause 7, wherein the determination step comprises assaying a human-derived biological sample for a nucleotide sequence encoding IL6R encoding IL6R comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

9. The step of assay comprises a biological sample and a sequence of at least 10 contiguous nucleotides of a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, or said above. Contacting with at least one oligonucleotide probe containing an antisense sequence of contiguous nucleotides, the sequence of contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. , If at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. Thus, the method of Clause 8 comprising determining that the step of detecting the presence of this complex comprises the IL6R protein comprising the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78.

10. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of Clause 8 or 9, wherein this assaying step is performed in a multiplex format.

11. The method of any one of Clauses 1-10, wherein the human is substantially resistant to anti-TNF alpha treatment or further determined to be substantially resistant.

12. The method of any one of Clauses 1-11, wherein the human has received or has received anti-TNF alpha treatment or has had reduced responsiveness to anti-TNF alpha treatment.

13. The method of Clause 11 or 12, wherein the antibody or antibody fragment is administered to humans separately or simultaneously with the anti-TNF alpha treatment.

14. The method of any one of clauses 8-10, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

15. Any one of Clauses 1-14, wherein the human is shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein, including the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, optionally. The human is further shown to contain the nucleotide sequence of SEQ ID NO: 79, or the human is homozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. One of the methods of Clauses 1-14 shown.

16. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any one method of clauses 1-15 that has been diagnosed with at least one condition.

17. The antibody or antibody fragment is inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, dermatitis, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD). , Asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease Any one method of Clauses 1-16 that treats or reduces the risk of at least one condition selected from in said human.

18. The method of any one of any clauses 1-17, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

19. The method of any one of Clauses 1-18, wherein the antibody or antibody fragment is administered intravenously or subcutaneously and / or is contained in an injectable preparation.

20. A ligand (eg, antibody or fragment) for use in any one of Clauses 1-19, which specifically binds to IL6R.

21. A kit containing the ligand of Clause 20 and instructions for performing any one of Clauses 1-19.

B: The present invention further provides the following regimens, ligands and kits.
1. A method of treating or reducing the risk of IL6R-mediated disease or condition in humans in need of it:-
The step of performing the initial treatment of the human during the initial treatment period by administering an anti-human IL6R ligand (eg, an antibody or fragment) to the human, wherein (i) this ligand It was determined in SEQ ID NO: 78 that it specifically binds to an IL6R protein containing the mutant Asp358Ala or Val385Ile; (ii) this human expresses said IL6R or the human genome encodes said IL6R. Containing a nucleotide sequence and (iii) this human has received or has undergone anti-TNF alpha treatment to treat or reduce the risk of IL6R-mediated disease or condition; where this initial treatment is this A step comprising administering a single or multiple doses of a ligand to a human;
b. (i) End anti-TNF alpha treatment or (ii) refrain from anti-TNF alpha treatment in humans; or (iii) determine to reduce anti-TNF alpha treatment after the initial treatment period; and
c. The step of continuing to administer a ligand to the patient after the expiration of the period, thereby treating or reducing the risk of an IL6R-mediated disease or condition in the human.
Including methods.

In certain embodiments, the determination of the specific binding is by reference to binding assay data, eg, using SPR or ELISA. The determination may refer to the information in the printed publication, eg, using knowledge of the data presented in the present invention or another patent application, or in a bibliographic article. Once such information is available (eg, without further testing of binding), one of ordinary skill in the art will identify relevant humans whose genotype or phenotype is compatible with the binding specificity of the ligand (instructions of the invention). Can be treated (by).

Antibodies or fragments may be in accordance with any of the configurations, examples, embodiments, embodiments, clauses or paragraphs herein.

A pharmaceutically effective amount of the ligand is administered.

In certain embodiments, the method comprises selecting a human comprising the nucleotide sequence encoding IL6R, which is the human listed in Clause 1, prior to the administration step.

In some cases, the initial treatment period is 7 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months or 1 year. Is.

2. The method of Clause 1 in which the human has or suffers from anti-TNF alpha treatment-related side effects.

Examples of side effects include hepatitis B infection of the viral carrier, allergic reaction, nervous system condition, blood condition, heart failure, immune response (eg, lupus erythematosus syndrome), liver condition, and new or worsening. Selected from the group consisting of psoriasis. In one example, the treatment is adalimumab treatment.

3. The method of Clause 1 or 2, comprising the step of determining that a human has or suffers from anti-TNF alpha treatment-related side effects prior to said initial treatment.

4. The method of Clause 2 or 3, which comprises the step of determining that the side effect is diminished after step (b) (eg, during step (c)).

5. The method of any one of clauses 1-4, wherein the person is over 40 years old (eg, 50 years old or older, 55 years old or older, 60 years old or older, 65 years old or older, or 70 years old or older).

6. Clause 1 comprising step (c) determining to increase the dose of the ligand to be administered after the initial treatment period and administering the increased dose to the human. Any one method from ~ 5.

7. The method of any one of Clauses 1-6, wherein step (b) comprises determining that a human is intolerant or refractory to treatment with anti-TNF alpha treatment.

8. Initial treatment with or without methotrexate for humans in addition to ligands, infliximab [eg Remicade®], adalimumab [eg Humila®], Certolizumab Administration of certolizumab pegol [eg, Cimzia®], golimumab [eg, Simponi®], or etanercept [eg, Enbrel®] treatment One of the methods of clauses 1-7, including.

9. Step (b) is infliximab during step (c) [eg Remicade®], adalimumab [eg Humila®], certolizumab pegol) [eg, Cimzia®], golimumab [eg, Simponi®], or etanercept [eg, Enbrel®] steps to end or reduce the procedure. One method of any one of clauses 1-8, including.

10. Any one method of Clauses 1-9 that includes step (ie, increasing relative to the initial treatment dose) of the ligand dose during step (c).

11. The human has received a high dose of anti-TNF alpha treatment prior to the initial treatment, where step (c) is in addition to the ligand at a lower (eg, medium or low dose) dose. Any one method of Clauses 1-10, including the step of applying anti-TNF alpha treatment.
Those skilled in the art are familiar with the implications of high, medium and low dose treatments (and methods determined by the weight of each patient, eg, patient).

12. Humans have undergone a medium dose of anti-TNF alpha treatment prior to initial treatment, where step (c) provides a lower (eg, lower) dose of statin treatment in addition to the ligand. Any one method of clauses 1-10 that includes the process or is free of anti-TNF alpha.

13. Humans have undergone a low dose of anti-TNF alpha treatment prior to initial treatment, and step (c) here comprises administering anti-TNF alpha in addition to said ligand. Any one method from ~ 10.

14. The method of any one of clauses 1-13, comprising the step of determining that the ligand specifically binds to the IL6R prior to the initial treatment, eg, using SPR or ELISA.

15. The method of any one of clauses 1-14, wherein the specific binding to the IL6R is a binding with a dissociation constant (Kd) of 1 nM or less, eg, 100, 10 or 1 pM or less.

16. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic lupus erythematosus and coronary heart disease. Any method of clauses 1-15 that is at risk of or suffers from a condition.

17. The method of Clause 16, wherein step (c) treats or reduces the risk of the condition in humans.

18. It has been determined that the human comprises a nucleotide sequence encoding IL6R comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein comprising the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, Clause 1-. Any one of the 17 methods.

19. Optional, including the step of determining that a human comprises a nucleotide sequence encoding IL6R comprising a mutant Asp358Ala or Val385Ile in SEQ ID NO: 78 and / or an IL6R protein comprising a mutant Asp358Ala or Val385Ile in SEQ ID NO: 78. The method of any one of Clauses 1-18, wherein this determining step is performed prior to administration of this antibody to this human.

20. The method of Clause 19, wherein the step of determining comprises assaying a human-derived biological sample for a nucleotide sequence encoding IL6R containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

21. The step to be assayed comprises or said a sequence of at least 10 contiguous nucleotides of a biological sample and a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78. Contacting with at least one oligonucleotide probe containing the antisense sequence of the nucleotide, the sequence of the contiguous nucleotides comprises the nucleotide sequence encoding the mutant Asp358Ala and / or Val385Ile in SEQ ID NO: 78, thereby. When at least one nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala and / or Val385Ile is present in SEQ ID NO: 78, a step of forming a complex; and a step of detecting the presence or absence of this complex. The method of Clause 20, wherein the step of detecting the presence of this complex comprises determining in SEQ ID NO: 78 that the human comprises an IL6R protein comprising said mutant Asp358Ala and / or Val385Ile.

22. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization, and allele-specific amplification. The method of clause 20 or 21, wherein this assaying step is performed in a multiplex format.

23. Any of Clauses 1-22 that the human was either substantially resistant to anti-TNF alpha (eg, adalimumab, infliximab or etanercept) treatment or was further determined to be substantially resistant. One way.

24. The method of any one of clauses 20-22, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

25. The human has been shown to be heterozygous with respect to the nucleotide sequence encoding the IL6R protein containing the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78, and optionally the human further comprises the nucleotide sequence of SEQ ID NO: 79. The method of any one of clauses 1-24, wherein said human is homozygous with respect to a nucleotide sequence encoding the IL6R protein comprising said mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

26. Inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, psoriasis, bronchitis, gingival inflammation, graft rejection, allogeneic graft rejection, graft-versus-host disease (GvHD), asthma, Selected from the group consisting of adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Schegren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, systemic erythematosus and coronary heart disease. Any one method of clauses 1-25 that has been diagnosed with at least one condition.

27. The method of any one of any clause 1-26, wherein the nucleotide sequence comprises SNP rs2228145 and / or SNP rs28730736.

28. The method of any one of Clauses 1-27, wherein the ligand (eg, antibody or antibody fragment) is administered by intravenous or subcutaneous administration and / or is included in an injectable preparation.

29. A ligand (eg, antibody or fragment) for use in any one of Clauses 1-28, which specifically binds to IL6R.

30. A kit containing the ligand of Clause 29 and instructions for performing any one of Clauses 1-28.

In an example of any aspect of the invention, the ligand (eg, antibody or fragment, eg, salilumab) is administered to humans at a 2-week dose of 75-200 mg (eg, once or twice over 2 weeks, 150). ~ 1200 mg, eg 150 mg or 200 mg once a week, or once every other week). In one example, this ligand is for such administration to humans.

rs12083537
With reference to any of the configurations, embodiments, embodiments or examples of the invention described herein, in certain embodiments, the human comprises an IL6R nucleotide sequence comprising mutation 2913A> G (SEQ ID NO: 79). (Ie, here, this human contains SNP rs12083537). In one example, this human has been genotyped for the mutation. In one example, the method comprises the step of genotyping a human for said mutation (eg, prior to administration of the ligand). In some cases, this human has asthma or is at risk of developing asthma. In one example, this ligand or method is to treat, prevent or reduce the risk of asthma in humans. In some examples, this ligand or method treats or reduces the risk of asthma in humans.

Thus, in one alternative to any of the configurations, embodiments, embodiments or examples of the invention herein, "where the human is a nucleotide sequence encoding the IL6R protein comprising said Asp358Ala or Val385Ile at SEQ ID NO: 78". Instead of the phrase "contains", this means "where the human comprises a nucleotide sequence encoding an IL6R protein, where this nucleotide sequence comprises mutation 2913A> G in SEQ ID NO: 79" or "here. In, the human contains a nucleotide sequence encoding an IL6R protein, wherein the nucleotide sequence comprises SNP rs12083537 "or" where the human genome comprises SNP rs12083537 ". Further, in one example, the IL6R protein comprises the mutant Asp358Ala or Val385Ile in SEQ ID NO: 78.

SNP rs2228145 [encoding 358A; see Table 13] is present in humans with an average cumulative frequency of 32% according to the 1000 Genomes Phase I database, but AMR, ASN, EUR, CLM, MXL, Higher in the PUR, CHB, CHS, JPT, CEU, GBR, IBS and TSI populations; therefore in certain embodiments, this human has an AMR, ASN or EUR pedigree, eg, CLM, MXL, PUR, CHB, CHS, JPT. , CEU, GBR, IBS and TSI pedigree humans. Thus, in certain embodiments, where the ligand (eg, antibody or fragment) specifically binds to an IL6R protein containing the mutant Asp358Ala, the human comprises SNP rs2228145; and optionally this Humans are humans of AMR, ASN or EUR pedigree, such as CLM, MXL, PUR, CHB, CHS, JPT, CEU, GBR, IBS and TSI pedigree. For example, this human is of AMR pedigree. For example, this human is of ASN pedigree. For example, this human is of EUR pedigree. For example, this human is of CLM pedigree. For example, this human is of MXL pedigree. For example, this human is of PUR pedigree. For example, this human is of CHB pedigree. For example, this human is of JPT pedigree. For example, this human is of CEU pedigree. For example, this human is of GBR pedigree. For example, this human is of IBS pedigree. For example, this human is of TSI pedigree.

SNP rs28730736 [encoding 385I; see Table 13] is present in humans with an average cumulative frequency of 5% by the 1000 Genomes Phase I database, but more in the AFR, ASW, LWK and YRI populations. High; therefore in certain embodiments, this human is of AFR pedigree, eg, ASW, LWK and YRI pedigree. Thus, in certain embodiments, where the ligand (eg, antibody or fragment) specifically binds to an IL6R protein containing the mutant Val385Ile, the human comprises SNP rs28730736; and optionally this human. Is an AFR pedigree, eg, a human of ASW, LWK and YRI pedigree. For example, this human is of AFR pedigree. For example, this human is of ASW pedigree. For example, this human is of LWK pedigree. For example, this human is of YRI pedigree.

References:
The cited references cited herein are hereby incorporated by reference in their entirety.
1. Ferreira et al (PLoS Genet. 2013 Apr; 9 (4): e1003444. Doi: 10.1371 / journal.pgen.1003444. Epub 2013 Apr 4, "Functional IL6R 358Ala allele impairs classical IL-6 receptor signaling [sic] and influences risk of diverse in flammatory diseases "; Rantala A et al, Hum Immunol. 2011 Jan; 72 (1): 63-8. doi: 10.1016 / j.humimm.2010.10.010. Epub 2010 Oct 15," Association of IL -6 and IL-6R gene polymorphisms with susceptibility to respiratory tract infections in young Finnish men ";
2. Zhang HY et al, Oral Dis. 2014 Jan; 20 (1): 69-75. Doi: 10.1111 / odi.12075. Epub 2013 Feb 24, "The association of IL-6 and IL-6R gene polymorphisms with chronic periodontitis in a Chinese population ";
3. JC Galicia et al, Genes and Immunity (2004) 5, 513-516. Doi: 10.1038 / sj.gene.6364120 Published online 12 August 2004, "Polymorphisms in the IL-6 receptor (IL-6R) gene: strong evidence that serum levels of soluble IL-6R are genetically influenced ";
4. Esparza-Gordillo J et al, J Allergy Clin Immunol. 2013 Aug; 132 (2): 371-7. doi: 10.1016 / j.jaci.2013.01.057. Epub 2013 Apr 9, "A functional IL-6 receptor (IL6R) variant is a risk factor for persistent atopic dermatitis ";
Revez et al, Genes and Immunity (2013) 14, 441-446; doi: 10.1038 / gene.2013.38; published online 15 August 2013, "A new regulatory variant in the interleukin-6 receptor gene associates with asthma risk".

(Example 5)
VEGF-A, PDGF-B pathway and ANG-2
VEGF-A is also referred to as VEGF, Vascular Endothelial Growth Factor A, MVCD1, VEGF-A, Vascular Endothelial Growth Factor, Vascular Permeability Factor and VPF. Human VEGF-A binds to human FLT1 / VEGFR1 and KDR / VEGFR2 receptors. Human isoforms (including VEGF-165 and VEGF-145) have been observed.

Vascular Endothelial Growth Factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are produced as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular weight and in their biological properties such as their ability to bind cell surface heparamp roteoglycans sulfate. VEGF expression is enhanced by activated oncogenes and by various cytokines in response to hypoxia. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo, VEGF induces angiogenesis and vascular permeability and plays a central role in the regulation of angiogenesis. Disordered VEGF expression contributes to the development of solid tumors and the etiology of some additional diseases characterized by abnormal angiogenesis by promoting tumor angiogenesis. As a result, inhibition of VEGF signaling suppresses the development of a wide variety of tumors. Various types of VEGF bind to two human tyrosine-kinase receptors, VEGFR-1 (fit-1) and VEGFR-2 (KDR / flk-1), which are expressed almost exclusively in endothelial cells. .. Endothelial cells are expressed in addition to the neuropilin-1 and neuropilin-2 co-receptors, which selectively bind to the 165 amino acid form of VEGF (VEGF165).

VEGF is an important regulator of angioplasty, and its expression in producing cells is regulated by a number of external factors. Cytokines, growth factors and gonadotropins that do not directly stimulate angiogenesis can modulate angiogenesis by modulating VEGF expression in specific cell types, thereby indirect angiogenesis or inhibition of angiogenesis. It can be effective. Factors that can enhance VEGF production include fibroblast growth factor 4 (FGF-4), PDGF, tumor necrosis factor α, transforming growth factor β (TGF-β), keratinocyte growth factor (KGF), and IGF-I. , Interleukin 1β (IL-1β), and IL-6.

Angioplasty has a causative role in many diseases, including age-related macular degeneration (AMD) with new blood vessels. Identification of key regulators of angiogenesis, including vascular endothelial growth factor (VEGF), fibroblast growth factor 2, pigment epithelial-derived growth factor, angiopoietin and extracellular matrix molecules, is the underlying pathological angiogenesis process. It facilitated the development of new therapeutic agents that target the disease. Of these, VEGF acts as a "master switch" for many ocular neovascular conditions through endothelial cell proliferation and its promotion of survival, vascular permeability and eye inflammation. Anti-VEGF agents are currently available, including, for example, aflibercept [EYLEA ™], bevacizumab [AVASTIN ™], ranibizumab [LUCENTIS ™] and pegaptanib sodium [MACUGEN ( Trademark)] (aptamer for neovascular AMD). Unlike bevacizumab, which binds to all VEGF isoforms, pegaptanib targets only VEGF165, the isoform responsible for pathological neoangiogenesis of the eye.

Flt-1 (VEGFR-1; Unitprot ID: P17948) is one of two human receptors for vascular endothelial cell growth factor. The other is KDR (VEGFR-2; Uniprot ID: P35968). The flt-1 protein consists of an external domain containing seven immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic region containing a tyrosine kinase domain. In contrast to other members of the receptor tyrosine kinase family, the kinase domain of flt-1 is in two segments with an intervening sequence of about 70 amino acids. The biology of VEGF receptors has been reviewed [Neufeld et al. (1999) FASEB Journal. 13: 11-22; Zachary (1998) Experimental Nephrology. 6: 480-487], and the tyrosine phosphorylation site. It has been identified [Ito et al. (1998) J. Biol. Chem. 273: 23410-23418].

Although flt-1 may be important in regulating tissue construction in the developing vasculature, the second VEGF receptor (KDR, VEGFR-2) has a VEGF mitotic effect on endothelial cells. And mediates the angiogenesis effect. Evidence supporting this theory derives from knockout studies in mice [Fong et al., (1995) Nature. 376: 66-70]. VEGF and its receptors are overexpressed in many tumor types, blocking VEGF function inhibits angiogenesis and suppresses tumor growth, whereas overexpression of VEGF causes angiogenesis and tumor growth. Enhance [Skobe et al., (1997) Nature Medicine 3: 1222-1227].

The flt-1 cDNA (EMBL accession number X51602, 7680 bp) encodes a mature protein of 1338 amino acids. The structure of the mouse flt-1 gene has been determined [Kondo et al. (1998) Gene 208: 297-305] and has been used to predict intron / exon boundaries within human genes. There is. The promoter region of the human gene has been characterized [Ikeda et al., (1996) Growth Factors. 13: 151-162; Morishita et al. (1995) J Biol Chem 270: 27948-27953; EMBL Accession No. D64016. , 1745bp]. The flt-1 gene, which is organized into 30 exons, is localized on chromosome 13q12 [Rosnet et al. (1993) Oncogene 8: 73-179]. See EP1130123A2.

Diabetic retinopathy (DR)
Diabetic retinopathy (DR), a microvascular complication of diabetes, is a major cause of blindness in adults. It is well established that DR is determined by both genetic and environmental factors. Long-term diabetes, poor glycemic control, and elevated blood pressure are major risk factors in the development of DR. However, genetic factors also play an important role in the pathogenicity of DR. Over the last three decades, the number of people with diabetes mellitus has more than doubled worldwide, making it one of the most important public health challenges in all countries. DR occurs in 75% of patients with type 2 diabetes and in almost all patients with type 1 diabetes within 15 years of signs of diabetes. Evidence supports an important role in genetics in determining the risk of DR.

VEGF-A has been regarded as a major contributor to the development of DR. VEGF-A can induce the earliest changes in DR, including leukostasis, blood-retinal barrier disruption, and macular edema and neovascularization in the progression of DR. The VEGF-A gene is located on the human chromosome 6p21.3 and consists of 8 exons. It is suggested that genetic variants in the VEGF-A gene affect the level of VEGF-A protein expression. To date, many studies have examined the association between polymorphisms in the human VEGF-A gene and DR. High linkage with -634G / C (+ 405G / C or rs2010963) gene polymorphism in 5 untranslated regions and -2578C / A (-2549I / D [-2578C / A] in the promoter region of the VEGF-A gene In an imbalance] or rs699947) genetic polymorphisms have been investigated.

Age-related macular degeneration (AMD)
Age-related macular degeneration (AMD) is a neurodegenerative disease that leads to visual impairment and accounts for half of all registered cases of blindness in Westerners over the age of 65. Approximately 8 million people in the United States have early or intermediate symptoms of AMD, of which nearly 1 million develop progressive AMD within the next 5 years. AMD is estimated to affect about 50 million people worldwide, and the growing population of aging makes AMD a serious public health concern and a major focus of research effort. ing. AMD is a clinically heterogeneous, genetically complex disease that involves a number of environmental and genetic risk factors. Epidemiological studies linked tobacco smoking, alcohol consumption, exposure, diet, drugs and hypertension to AMD risk, but with familial agglomeration and two studies, genetic variation is important for disease It has been suggested that it can play a role.

Anti-VEGF therapy is currently regarded as the standard of treatment for the treatment of neovascular AMD. Two of the most widely used treatments are ranibizumab and bevacizumab. Ranibizumab [Lucentis®, Genentech, CA, USA] is a US FDA-approved anti-VEGF treatment for AMD, a recombinant, fragmented monoclonal antibody that binds to VEGF. On the other hand, bevacizumab [Avastin®, Genentech] is a full-length monoclonal antibody that has not been approved by the FDA for eye treatment but has been reported to have similar functions to ranibizumab. Bevacizumab has been approved by the FDA for metastatic colon cancer and has been administered as an unapproved treatment for ocular angioplasty. Genetic variation by individual can affect the treatment response in both of these drugs.

Polypoid choroidal angiopathy (PCV)
A subset of wet AMD, named polypoid choroidal angiopathy (PCV), has been identified as a clinical entity. PCV is important because it can be a major cause of AMD-induced visual impairment in certain Asian and black populations.

Unique clinical features of PCV include one or more hemorrhagic retinal pigment epithelial detachment (PED), excess exudate and extramacular disease, including peripheral. There is a preference for "polyps" around the nipple. The patient is also characteristically young.

There is increasing evidence that Asian patients with neovascular AMD have a variant of AMD, polypoid choroidal angiopathy (PCV), which is characterized by polypoid lesions with medial choroidal vascular abnormalities. PCV is more prevalent in Asians, reporting about 50% of neovascular AMD, compared to 10% in Europeans. Studies have shown that the combination of anti-VEGF therapy (ranibizumab) and photodynamic therapy (PDT) has more favorable outcomes compared to ranibizumab monotherapy.

Retinal hemangiomatous growth (RAP)
Another variant of AMD in the occult CNV spectrum is retinal hemangiomatosis (RAP), which corresponds to about 12% to 15% of neovascular AMD. It is accompanied by proliferation of intraretinal capillaries with retinal anastomosis or CNV.

Diabetic macular edema (DME)
Diabetic macular edema (DME) is the most common cause of visual impairment in subjects with diabetes, and it can occur at any stage of DR and is therefore assessed independently of the DR spectrum. It is another classification. The pathogenicity of DR and DME is thought to be associated with loss of peripheral cells, thickening of the basement membrane and loss of endothelial cells, leading to microaneurysms, blood-retinal barrier destruction, and increased inflammation and vascular leakage. There are several modes of treatment in DME. The goal of laser treatment with DME is to reduce disease progression by targeting areas of retinal leakage. Intravitreal triamcinolone acetonide (IVTA) has also been shown to significantly reduce DME, with maximal action lasting 3 to 6 months per week.

Retinal vein occlusion (RVO)
Retinal vein occlusion (RVO) is the most common retinal angiopathy after diabetic retinopathy in the elderly and systemic disorders such as hypertension, hyperlipidemia, diabetic and / or atherosclerotic blood vessels. Often related to disability. It is classified as branch retinal vein occlusion (BRVO) or central retinal vein occlusion (CRVO), depending on the site of occlusion, and is further divided into ischemic (non-perfused) or non-ischemic (perfused) RVO. , Each with different prognosis and treatment. Macular edema and retinal neovascularization are the two most common causes of visual impairment; therefore, by laser photocoagulation, intravitreal injection of glucocorticoids or anti-VEGF agents, and other surgical or systemic treatments. Eye management has focused on these two sequelae.

Retinopathy of prematurity (ROP)
ROP remains one of the leading causes of blindness in children. In the late stages of ROP, abnormal or pathological neovascularization of blood vessels results from the immature retina, resulting in retinal traction, detachment, hemorrhage and funnel-like structures, ultimately leading to vision loss .. Neoangiogenesis is primarily driven by VEGF and is now the recommended treatment for type 1 ROP is laser peripheral ablation.

Angioplasty and angiogenesis inhibitors Angioplasty is the formation of new blood vessels. This process involves the migration, proliferation and differentiation of endothelial cells that line the lining of blood vessels. The process of angioplasty is controlled by the body's chemical signals. These signals can stimulate both the repair of damaged blood vessels and the formation of new blood vessels. Other chemical signals (called angioplasty inhibitors) interfere with angioplasty. Normally, the stimulating and inhibitory effects of these chemical signals are balanced so that blood vessels form only when and where they are needed. Neoangioplasty is the formation of a functional microvascular network by erythrocyte perfusion. Neoangiogenesis differs from angioplasty in that angioplasty is characterized primarily by the protrusion and growth of capillaries, as well as sprouting from existing blood vessels.

Angioplasty plays an important role in the growth and spread of cancer. Blood supply is essential for tumors that grow larger than 2-3 millimeters in size. Tumors can generate this blood supply by emitting chemical signals that stimulate angioplasty. Tumors can also stimulate adjacent normal cells to give rise to angioplasty signaling molecules. The resulting new blood vessels "supply" oxygen and nutrients to the growing tumor, allowing cancer cells to invade adjacent tissues, migrate throughout the body, and create new colonies of cancer cells called metastases. It becomes possible to form. Tumors cannot grow or spread beyond a certain size without a blood supply, so find a way to block tumor angioplasty by the idea that angioplasty inhibitors prevent or slow the growth of cancer. Research is focused on trying things out. Similarly, eye diseases and conditions, such as AMD and DR, are associated with angiogenesis, and inhibitors are also useful in treating or preventing them.

Thus, the anti-VEGF (ie, VEGF-A) ligands and antibodies of the invention are intended to treat, prevent or reduce the risk of one or more of such eye conditions and cancers (eg, solid tumors). It is useful.

Angioplasty requires the binding of signaling molecules such as vascular endothelial growth factor (VEGF) to receptors on the surface of normal endothelial cells. When VEGF and other endothelial cell growth factors bind to their receptors on endothelial cells, signals within these cells are initiated to promote the growth and survival of new blood vessels. Angioplasty inhibitors interfere with various steps in this process. For example, bevacizumab [Avastin®] is an anti-VEGF monoclonal antibody that specifically recognizes and binds to VEGF. Aflibercept [Eylea®] is a VEGF receptor-Fc fusion that binds to VEGF. Other angioplasty inhibitors, including sorafenib and sunitinib, block their activity by binding to receptors on the surface of endothelial cells or to other proteins within downstream signaling pathways.

In addition to or alternatively targeting human VEGF-A to address eye conditions or cancer (or angioplasty or generally neoangiogenesis), platelet-derived growth factor pathways (eg, PDGF-B and) / Or targeting PDGFR-B), or targeting the angiopoietin-2 pathway may be useful. Further details are shown below.

In one example, the invention provides a method of treating or reducing the risk of a VEGF-A-mediated disease or condition in a human in need thereof, the method of which is to the said human VEGF-A. It comprises the step of administering a ligand that specifically binds to the protein (eg, an antibody or antibody fragment). The present invention also provides a corresponding ligand.

In one example, the invention provides a method of treating or reducing the risk of PDGF-B (ie, platelet-derived growth factor beta) -mediated disease or condition in humans in need thereof. The step includes administering to the human a ligand (eg, an antibody or antibody fragment) that specifically binds to human PDGF-B protein. The present invention also provides a corresponding ligand.

The present invention provides anti-VEGF-A ligands; as well as VEGF-A binding or targeting ligands as described herein. This ligand has various usefulness. Some of these ligands have been used in affinity purification of VEGF-A (particularly human VEGF-A or its ligands) to genotype or phenotype humans, for example in specific binding assays. -Useful in screening assays to identify other antagonists with A activity. Some ligands of the invention are useful for inhibiting VEGF-A mediated activity.

In one example, the invention provides a method of treating or reducing the risk of PDGFR-B (ie, platelet-derived growth factor beta receptor) -mediated disease or condition in humans in need thereof. Includes the step of administering to said human a ligand (eg, an antibody or antibody fragment) that specifically binds to human PDGFR-B protein. The present invention also provides a corresponding ligand.

The present invention provides anti-PDGF-B ligands; and PDGF-B binding or targeting ligands (described herein). This ligand has various usefulness. Some of these ligands are, for example, in a particular binding assay, PDGF-B, specifically human PDGF-B or affinity purification of its ligands, to genotype or phenotype humans. And in screening assays to identify other antagonists of PDGF-B activity. Some of the ligands of the invention are useful for inhibiting PDGF-B mediated activity.

The present invention provides anti-VEGF-A ligands; and VEGF-A binding or targeting ligands (described herein). This ligand has various usefulness. Some of these ligands are, for example, in a particular binding assay, for affinity purification of VEGF-A, specifically human VEGF-A or its ligands, to genotype or phenotype humans. And in screening assays to identify other antagonists of VEGF-A activity. Some of the ligands of the invention are useful for inhibiting VEGF-A mediated activity.

The present invention provides anti-PDGFR-B ligands; and PDGFR-B binding or targeting ligands (described herein). This ligand has various usefulness. Some of these ligands are, for example, in a particular binding assay, PDGFR-B, specifically, human PDGFR-B or affinity purification of its ligands to genotype or phenotype humans. And in screening assays to identify other antagonists of PDGFR-B activity. Some of the ligands of the invention are useful for inhibiting PDGFR-B mediated activity.

Anti-VEGF-A ligands (eg, antibodies and antisense RNA) have been developed based on the targeting and neutralization of so-called "wild" human VEGF-A, which is a common type. Although such treatments are useful for human patients carrying this type of human VEGF-A, the inventor, although a rare type of VEGF-A in the human population, is also naturally present. It was considered useful for investigating the possibility of type targeting. In this way, the inventor is a useful target for human treatment, prevention and diagnosis (protein or) that is mediated by VEGF-A activity or appropriate for diseases and conditions associated thereto. We have reached an insight into the natural appearance and distribution of rare human VEGF-A types that may function (at the nucleic acid level). This provides tailor-made treatment, prevention and diagnosis, especially in humans who lack the common VEGF-A gene or protein.

Those skilled in the art are aware that SNPs or other changes that translate into amino acid variations can result in variability in the activity and / or higher-order structure of the human target to be targeted. This has led to great interest in personalized medicine that uses knowledge of genotyping and protein and nucleotide variability to more effectively tailor-make patient care and diagnosis. Accordingly, the present invention provides tailor-made medicines and studies specifically directed to the rare VEGF-A polymorphic variant. Such forms or "alleles" (at the nucleotide level) contain one or more changes at the nucleotide and amino acid levels from the corresponding common types of nucleotide and amino acid sequences, ie, protein targets in humans. There are one or more non-synonymous changes (also known as "missenses") at the nucleotide level that translate into one or more corresponding changes in.

Moreover, the inventor surprisingly found that rare natural forms are present in humans much less frequently than common forms, but are nevertheless shown in large and ethnically diverse human populations. We have noticed that there are usually many human examples per ethnic group shown. Thus, by targeting such rare types, the inventor provides effective treatment, prevention or diagnosis across many human ethnic groups, thereby expanding the usefulness of the invention. I noticed. Specifically, human VEGF-A variations correlate with VEGF-A-mediated diseases or conditions, such as eye conditions or increased frequency or risk of cancer. Additional or alternative, VEGF-A variations correlate with improved post-treatment responses for eye conditions. The inventor analyzed such variations and devised a collection of variants in Tables 18-20 (Tables 19-21). As shown in those tables, the inventor also correlates with a disease or condition, such as an eye condition or frequency or risk of cancer, or an improvement in post-treatment response for an eye condition in other humans. Variations in the target were also evaluated. Therefore, in this regard, the present invention provides various aspects and concepts as shown below in this example.

The present inventors have anti-TOI (VEGF-) for administration to human patients for the treatment and / or prevention of VEGF-A, PDGF-B and / or PDGFR-B mediated or related diseases or conditions. It has been understood that the present invention has important industrial and medical applications with respect to guiding the selection of A, PDGF-B and / or PDGFR-B) ligands. In this method, the patient receives tailor-made drugs and ligands for their needs as determined by the composition of the patient's genes or phenotypes. Along with that, the present invention provides patient genotyping and / or phenotyping associated with such treatments, thereby allowing the drug to be appropriately adapted to the patient. This increases the chances of medical efficacy, reduces the likelihood of inferior treatment (eg, poor efficacy and / or side effects) with drugs or ligands that are not suitable for the patient, and avoids drug prescribing mistakes and waste. ..

To develop this idea, in this non-limiting embodiment, the inventor decided to determine a series of human VEGF-A, PDGF-B and PDGFR-B variants based on the following criteria: However, these criteria, which the inventor has noticed, provide medical drugs and diagnostics useful for tailor-made needs in the human population. We have selected variants that have at least three of the following four criteria:
Naturally occurring human TOI (VEGF-A, PDGF-B or PDGFR-B) variations with a cumulative human allele frequency of 35% or less;
Naturally occurring human TOI variations with a total human genotype frequency of about 50% or less;
Naturally occurring human TOI variations found in many different human populations (using the standard classification of the 1000 Genomes Project; see Table 2 below); and distributed across many such different populations. A naturally occurring human TOI variation found in many individuals.

Based on these criteria, the inventor has identified the variants listed below.

In addition, the inventor has identified useful variations in the following human TOIs: factor H (CFH), complement component 2 (C2), prothrombin, methylenetetrahydrofolate reductase (MTHFR), factor V. , ARMS2, VEGFR2 and HTRA1. As shown in the titles of Tables 18-20 (Tables 19-21), the examiner found that such variants had a useful correlation.

In one example, the ligand of the invention comprises an anti-TOI (eg, anti-human VEGF-A) binding site, where the binding site is a human or humanized binding site, eg, this binding site Contains or consists of a human or humanized antibody variable domain or multiple variable domains (eg, human VH / VL binding sites). Additionally or optionally, this ligand comprises one or more human antibody constant regions [eg, human antibodies CH1, CH2, CH3 (or all of them) or Fc]. In one example, this ligand is an antibody that comprises a human or humanized variable region and a human constant region (eg, carrying one or more mutations to enhance or weaken Fc function in a human patient). ..

In one example, the invention provides a method of targeting VEGF-A in humans, the method comprising human VEGF containing VEGF-A SNP as described herein for said human. It comprises the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to the human VEGF-A protein encoded by the -A nucleotide. In some cases, this human has or is at risk for a VEGF-A-mediated disease or condition. In some examples, this method treats or reduces the risk of VEGF-A-mediated disease or condition in humans. In some examples, this condition involves angioplasty or neovascularization.

In one example, the invention provides a method of targeting PDGF-B in humans, the method comprising a human PDGF containing PDGF-B SNP as described herein for said human. It comprises administering a ligand (eg, an antibody or antibody fragment) that specifically binds to the human PDGF-B protein encoded by the -B nucleotide. In some cases, this human has or is at risk for a PDGF-B-mediated disease or condition. In some examples, this method treats or reduces the risk of PDGF-B-mediated disease or condition in humans. In some examples, this condition involves angioplasty or neovascularization.

In one example, the invention provides a method of targeting PDGFR-B in humans, the method comprising human PDGFR-B SNP as described herein for said human PDGFR. It comprises administering a ligand (eg, an antibody or antibody fragment) that specifically binds to the human PDGFR-B protein encoded by the -B nucleotide. In some cases, this human has or is at risk for a PDGFR-B-mediated disease or condition. In some examples, this method treats or reduces the risk of PDGFR-B-mediated disease or condition in humans. In some examples, this condition involves angioplasty or neovascularization.

In certain embodiments, (i) the antibody or fragment comprises a VH domain derived from recombination of a human VH segment, a human D gene segment and a human JH segment, the human VH segment comprising Framework 1 of SEQ ID NO: 40. And here the human comprises a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human comprises a VH domain comprising Framework 1 of SEQ ID NO: 40. Expressed; and here (ii) the human comprises the nucleotide sequence encoding said VEGF-A, PDGF-B or PDGFR-B.

Additional or alternative, in certain embodiments, (i) this antibody or fragment is a human gamma-4 fold containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Containing a chain constant region, wherein the human comprises an IGHG4 * 01 human heavy chain constant region gene segment, or this human is found in Leu at position 189 or SEQ ID NO: 73 found in SEQ ID NO: 73. An antibody containing a human gamma-4 heavy chain constant region containing Arg at position 289 is expressed; and where (ii) the human has the nucleotide sequence encoding the VEGF-A, PDGF-B or PDGFR-B. Including.

In certain embodiments, the anti-VEGF-A, PDGF-B or PDGFR-B ligands, antibodies or fragments of the invention comprise an Fc region, where the Fc region is numbered with an EU index represented by Kabat. 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F , 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 33 1C, 331Y, 331H, 331R, 331N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, Contains at least one unnatural amino acid residue selected from the group consisting of 419H, 421K, 440Y and 434W. Optionally, this Fc region may contain additional and / or alternative non-natural amino acid residues known to those of skill in the art (eg, US Pat. No. 5,624,821; 6,277,375; 6,737,056). See PCT Patent Publication WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752 and WO05 / 040217).

Ligands, antibodies or fragments according to the invention (eg, anti-VEGF-A ligands, antibodies or fragments) are subject to disclosure of their disease and condition for potential inclusion in one or more claims herein. To treat, prevent, or reduce, for example, the risk of any disease or condition disclosed in U.S. Patent Application Publication No. 2012064621A1 or U.S. Pat. No. 7,704,500B2, incorporated herein by reference. Treat or prevent or reduce risk). Guidance for obtaining and testing antibodies can also be found in the PCT application. Suitable anti-VEGF-A ligands, antibodies or fragments for use in the present invention are disclosed in U.S. Patent Application Publication No. 2012064621A1 or U.S. Pat. No. 7,704,500B2, the disclosure of which (including its sequences) is herein. Incorporated herein by reference for potential inclusion in one or more claims in the specification.

Further included by the present invention is an anti-TOI (eg, VEGF-A) in the manufacture of a medicament for use in reducing or inhibiting a TOI (eg, VEGF-A) -mediated disease or disorder in humans. ) Use of ligands, antibodies or fragments. VEGF-A-mediated disorders or related disorders treated with the ligands, antibodies or fragments of the invention include, for example, the disorders described below.

Therefore, the ligands, antibodies or fragments of the present invention are useful as therapeutic agents in the treatment of conditions involved in TOI (eg, VEGF-A) expression and / or activity. In particular, one embodiment is a method of treatment comprising administering to a patient in need thereof an effective amount of a ligand, antibody or fragment of the invention, wherein the functional consequences of TOI activation are diminished. Will be done. Another embodiment comprises, among other things, (i) identifying a patient exhibiting TOI expression or activity, and (ii) administering to this patient an effective amount of a ligand, antibody or fragment of the invention. The functional consequences of TOI activation are mitigated here. Effective amounts according to the invention modulate (eg, alleviate) the functional consequences of TOI activation, thereby modulating the severity of at least one symptom of the particular disease or disorder being treated. An amount that rates (eg, decreases or decreases) but does not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, for a patient in need thereof. The step of administering an effective amount of one or more ligands, antibodies or fragments of the invention alone or in a therapeutic regimen in combination with another suitable medicament known in the art or described herein. It is a method that, as a result, reduces the severity of at least one symptom of any disorder. Another embodiment of the invention is, among other things, a method of antagonizing at least one effect of TOI, in which an effective amount of the invention is contacted with or administered to one or more ligands, antibodies or fragments. A method of antagonizing the at least one effect of the TOI, eg, the ability to promote or maintain angiogenesis or neoangiogenesis.

Tailor-made antibodies to the rarer TOI (VEGF-A, PDGF-B or PDGFR-B) variant profiles As outlined herein (eg, in the context of PCSK9 in Example 1), the invention presents the variants. Further human by selecting antibody-based ligands with variable and / or constant domains based on the genetic segments found in many humans of the ethnic population in which the TOI type is found to meet the selection criteria of the present invention. Includes the possibility of tailor-making the procedure. This also applies mutatis mutandis when the TOI is human VEGF-A, PDGF-B or PDGFR-B, as in this example. Thus, all disclosures herein regarding tailor-making of variable and / or constant domains apply to this example with respect to VEGF-A, PDGF-B or PDGFR-B, and one or one herein. Can be combined with use in multiple claims.

As described in Example 1, in one example, a ligand comprising an antibody VH domain from a recombination of a human IGHV gene segment comprising a nucleotide selected at a position in HCDR1 or FW3 that is variable in human is provided ( That is, here SNPs are present in humans).

Further information is provided in Table 4, which also shows variations at these locations and variant distributions across the 1000 Genomes Project database associated with many human populations.

In other embodiments, as described in more detail above, the invention is based on an alternative human VH gene segment, or V _K , V _λ or constant region gene segment, and the genotype of the human recipient and /. Alternatively, provide a phenotypically tailored ligand (see further Table 9 for representative variants).

In a further example, therefore there is:-
(i) The ligand comprises a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a recombinantly derived VH domain of the human JH segment, and the human VH segment is the framework 1 of SEQ ID NO: 40. The human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40.
(ii) The ligand comprises a human VH segment IGHV3-7 * 01, a human D gene segment and a recombination-derived VH domain of a human JH segment, and the human contains the IGHV3-7 * 01 VH gene segment or is human. Expresses the VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment.
(iii) The ligand comprises the human Vκ segment IGKV1-12 * 01 and the recombinantly derived Vκ domain of the human Jκ segment, and the human comprises the IGKV1-12 * 01 Vκ gene segment, or the human contains the human Vκ segment. It expresses the Vκ domain derived from recombination of IGKV1-12 * 01 and human Jκ segment.
(iv) The ligand comprises the Vκ domain from the recombination of the human Vκ segment and the human Jκ segment, and the human Vκ segment encodes (i) CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, said human. Does it contain the Vκ gene segment encoding CDR3 containing Pro at position 7 shown in SEQ ID NO: 36, or does human express the Vκ domain containing CDR3 containing Pro at position 7 shown in SEQ ID NO: 36? Or (ii) the Vκ gene segment encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38 and the human encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38, or Humans express the Vκ domain containing FW3, which contains Ser at position 15 shown in SEQ ID NO: 38.
(v) The ligand contains a human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human is (i) IGHG1 * 01 human. An antibody containing a heavy chain constant region gene segment or containing a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4 is expressed. ..
(vi) The ligands are Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 and Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala at position 257 shown in SEQ ID NO: 6, and whether the human contains (i) IGHG2 * 01 human heavy chain constant region gene segment. , Or human, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Val or the antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown in SEQ ID NO: 6.
The (vii) ligand comprises a human kappa chain constant region containing Val at position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16, and the human is (i) IGKC1 * 01 human kappa chain constant region. An antibody containing a gene segment or containing a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16 is expressed.
(viii) the ligand comprises a human IGLC1 * 01 lambda chain constant region, said human containing (i) a human IGLC1 * 01 lambda chain constant region gene segment, or a human containing a human IGLC1 * 01 lambda chain constant region. Expresses an antibody containing.
(ix) The ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human. An antibody comprising a heavy chain constant region gene segment or containing a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73 is expressed. ..
The (x) ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3 * 01) constant region gene segment, wherein the human is (i) said the first constant region gene. An antibody comprising a segment (eg, IGHG3 * 01) or containing a human gamma-3 heavy chain constant region encoded by the first human IGHG3 (eg, IGHG3 * 01) constant region gene segment is expressed. ..
(xi) The ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human epsilon heavy chain constant region encoded by the first constant region gene segment.
(xii) The ligand comprises the human mu heavy chain constant region encoded by the first human mu heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expresses an antibody comprising the human mu heavy chain constant region encoded by the first constant region gene segment.
(xiii) The ligand comprises the human alpha heavy chain constant region encoded by the first human alpha heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human alpha heavy chain constant region encoded by the first constant region gene segment.
The (xiv) ligand comprises the human delta heavy chain constant region encoded by the first human delta heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expressing an antibody comprising the human delta heavy chain constant region encoded by the first constant region gene segment.
The (xv) ligand comprises the human kappa light chain constant region encoded by the first human kappa light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human kappa light chain constant region encoded by the first constant region gene segment.
The (xvi) ligand comprises the human lambda light chain constant region encoded by the first human lambda light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human lambda light chain constant region encoded by the first constant region gene segment.

For VEGF-A, PDGF-B or PDGFR-B ligands, it is advantageous for this ligand to include a gamma-1 or gamma-2 constant region [eg, such as embodiments (i)-(xvi) above]. It is possible. For example, this ligand comprises said gamma-1 constant region, eg, ADCC or CDC enhanced constant region satisfying embodiment (v).

Determination of specific binding of the ligand of the invention to VEGF-A, PDGF-B or PDGFR-B variants
Specific binding of the ligand of the invention to the VEGF-A, PDGF-B or PDGFR-B variant can be performed using the SPR method described in Example 1.

Human gene polymorphism citations
1. PLoS One. 2013 Dec 20; 8 (12): e84069. doi: 10.1371 / journal.pone.0084069, eCollection 2013, "Association of VEGF gene polymorphisms with diabetic retinopathy: a meta-analysis", Gong JY et al. The authors reported a meta analysis which, in conclusion, reportedly demonstrated that DR is associated with VEGF gene -460T / C polymorphism (rs833061).
2. J Diabetes Res. 2014; 2014: 805801. doi: 10.1155 / 2014/805801, Epub 2014 Apr 28, "The associations between VEGF gene polymorphisms and diabetic retinopathy susceptibility: a meta-analysis of 11 case-control studies", Han L et al. A total of 11 studies fulfilling the inclusion criteria were included in this meta-analysis. A significant relationship between VEGF + 936C / T (rs3025039) polymorphism and DR was reported.
3. BMC Ophthalmol. 2013 Oct 16; 13:56. Doi: 10.1186 / 1471-2415-13-56, "Two polymorphisms (rs699947, rs2010963) in the VEGF-A gene and diabetic retinopathy: an updated meta-analysis", Lu Y et al. The authors reported meta-analysis confirmed the significant association between rs699947 polymorphism and DR after exclusion of outliers.
4. Gene. 2013 Apr 15; 518 (2): 310-5. doi: 10.1016 / j.gene.2013.01.018. Epub 2013 Jan 24.
VEGF -634G> C polymorphism and diabetic retinopathy risk: a meta-analysis.
Qiu M et al. The aim of the published study was to investigate whether VEGF -634G> C polymorphism is associated with the risk of DR in type 2 diabetes. A systematic search of electronic databases (PubMed, Embase and Web of Science) and reference lists of relevant articles was carried out until September 15, 2012. The pooled odds ratios (ORs) and their corresponding 95% confidence interval (CI) were calculated by a fixed effect model. A total of 1525 DR cases and 1422 diabetic without retinopathy ( DWR) controls in 9 independent studies were included in the meta-analysis. A significant relationship between VEGF -634G> C polymorphism and DR was found in an allelic genetic model (OR: 1.13, 95% CI: 1.01 to 1.25, P = 0.03 ) and a recessive genetic model (OR: 1.26, 95% CI: 1.02 to 1.55, P = 0.03). The authors concluded that their research confirmed the association between the VEGF -634G> C polymorphism and DR in subjects with type 2 diabetes.
5. Diabetes. 2002 May; 51 (5): 1635-9, "A common polymorphism in the 5'-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes", Awata T et al. These authors suggest that the -634G> C polymorphism in the 5'UTR of the VEGF gene is a genetic risk factor for diabetic retinopathy.
6. Molecular Vision 2010; 16: 1958-1981 <http://www.molvis.org/molvis/v16/a213>, Tong Y et al. In conclusion, according to the authors the described Human Genome Epidemiology (HuGE) systematic review presents strong evidence for an association between the HTRA1 rs11200638 G → A polymorphism, LOC387715 / ARMS2 rs10490924 G → T polymorphism, and AMD, and suggests that both of these genes play important roles in this disease. Potential gene-gene and gene-environmental interactions. and possible mechanisms of AMD are also summarized and discussed.
7. J. Pers. Med. 2013, 3, 40-69; doi: 10.3390 / jpm3010040, "Personalized Medicine in Ophthalmology: From Pharmacogenetic Biomarkers to Therapeutic and Dosage Optimization", Frank S Ong et al. This review comments that: In the case of intravitreal bevacizumab, CFH Y402H genotypes, TC and TT, show more than five-fold increased improvement compared to the CC genotype. The data shows that after treatment with bevacizumab, visual acuity of the patients improved from 20/248 to 20 / 166 (TT) and from 20/206 to 20/170 (TC), but actually decreased from 20/206 to 20/341 for the CC genotype (p = 0.016). In a prospective study with twice the number of patients, the CC genotype was confirmed to have worse outcome as measured by distance and reading visual acuity. In a similar experiment with intravitreal ranibizumab, the TC and TT genotypes for CFH showed improvement with fewer injections compared to the CC genotype. Recurrent analysis showed that patients homozygous for the CFH Y402H risk allele (CC) were 37% more likely to require additional ranibizumab injections (p = 0.04). Another study found that individuals homozygous for 69S in ARMS2 had decreased central subfield retinal thickness and no improvement in visual outcomes compared to improved visual acuity in ARMS2 rs10490924 and rs1061170 genotypes following ranibizumab treatment. Taking genetics and environmental factors together, the CFH Y402H homozygous CC genotype with BMI ≧ 30 and smoking reportedly conferred the greatest risk.
8. Expert Rev Ophthalmol. 2013 April 1; 8 (2): 12-140. doi: 10.1586 / eop.13.3, "Genetic risk, ethnic variations and pharmacogenetic biomarkers in age-related macular degeneration and polypoidal choroidal vasculopathy", Jane Z Kuo et al. This article comments that: Variants in the factor H (CFH) gene confers a 7.4-fold increased likelihood of late AMD. AMD is a common complex disorder with complex inheritance patterns due to various gene and environmental interactions. Yet Even though the most well-established and consistent association of AMD is observed, despite these challenges, AMD represents an unusual example of the "common disease common variant" theory in which several variants identified on the CFH gene have relatively large effects on disease outcome. with complement factor H (CFH) on chromosome 1, recent studies have also shown that other candidate genes in the complement pathway, ARMS2 / HTRA1 on chromosome 10q26, LIPC, CETP, and TIMP2 are also susceptible loci associ ated with AMD. In 2005, three independent research groups identified a common coding variant, Y402H (rs1061170), in the CFH gene on chromosome 1 that was strongly associated with susceptibility of AMD, with reported odds ratio (OR) ranging from 2.5 to 3.3 For all AMD and as high as 7.4 for advanced AMD. The Y402H variant results in a tyrosine-to-histidine substitution. The association of CFH in AMD implicated that the complement cascade plays a critical role in the development of AMD and is now also regarded. as a major contributor to susceptibility of AMD. In addition to CFH and the complement pathway, the region at chromosome 10q26 also harbors additional signals to AMD susceptibility, namely the age-related maculopathy susceptibility 2 (ARMS2) and the high-temperature requirement factor H (HTRA1) genes. It is also possible that susceptible variants within ARMS2 modulates the promoter activity of HTRA1, so that both ARMS2 / HTRA1 contribute to AMD susceptibility. In one European study, t he SNP rs11200638 at the promoter region of HTRA1 confers a 49.3% electrically risk and the risk allele was associated with an elevated mRNA and protein expression and level of HTRA1. The risk allele of the same SNP was highly significant in the Chinese and has 10 times The risk of developing AMD compared to subjects with the wild-type allele. Two VEGF polymorphisms, rs699947 and rs2146323, were significantly associated with PDT treatment response. The allele frequency of rs699947 for AA, AC, and CC were 14%, 39%, and 46% in non-responders versus 40%, 48%, and 12% in responders, respectively (p = 0.0008). The allele frequency of rs2146323 for AA, AC, and CC were 4%, 32%, and 64% in non-responders versus 24%, 38%, and 38% in responders, respectively (p = 0.0369). Thus, the C allele in both SNPs in VEGF were associated with a higher rate of non-responder to PDT (p = 0.0003 for) rs699947 and p = 0.0036 for rs2146323). In classic choroidal neovascularization (CNV), promoterbin (G20210A) and MTHFR ( C677A) were significantly associated with PDT responders. In occult CNV, prothrombin (G20210A) and factor V (G1691A) were significantly associated with PDT responders. Two studies have shown a worse visual improvement in subjects with the homozygous TT risk allele at ARMS2 rs10490924 ( A69S) after ranibizumab or bevacizumab injections. Other candidate gene studies found that patients with the risk genotype (CC) at rs1413711 of VEGF has a more significant visual gain after ranibizumab, the G allele at rs699946 of VEGF was significantly associated with a better visual prognosis in a study examining the cumulative effect of risk alleles at CFH (Y402H; rs1061170), ARMS2 (A69S; rs10490924), VEGF (rs699947 and rs833069), after either intravitreal bevacizumab or triple therapy (PDT with intravitreal bevacizumab and triamcinolone acetonide). (rs2071559 and rs7671745), LPR5 (rs3736228), and FZD4 (rs10898563) after ranibizumab treatment, subjects without the high-risk alleles in CFH and ARMS 2 have significantly more visual improvement compared to subjects with the high-risk alleles (p = 0.009). The mean visual acuity improvement was 10 letters in subjects without the 4-risk alleles, compared to no improvement in subjects with all 4-risk alleles By adding VEGF into the model, they found that subjects with all 6-risk alleles in CFH, ARMS2, and VEGF demonstrated a mean loss of 10 letters after ranibizumab treatment, the other allele groups all demonstrated improvement (p <0.0001), suggesting a cumulative effect of the risk alleles with a poorer response rate to treatment. A comprehensive meta-analysis found variants at ARMS2 / HTRA1 (rs10490924, OR = 2.27, p <0.00001; rs11200638, OR = 2.72, p <0.00001), CFH (rs1061170, OR = 1.72, p <0.00001; rs800292, OR = 2.10, p <0.00001) or the complement pathway (C2; rs547154, OR = 0.56, p = 0.01) were significantly associated with PCV, with higher odds ratio observed at variants in ARMS2 / HTRA1. The strong association of ARMS2 / HTRA1 to PCV was suppo rted by numerous studies of Asian populations.
9. Survey of Ophthalmology, Volume 59, Issue 1, January-February 2014, Pages 1-18, "Predictors of anti-VEGF treatment response in neovascular age-related macular degeneration", Finger et al. The authors reviewed evidence on predictors of anti-vascular endothelial growth factor (VEGF) treatment response in neovascular age-related macular degeneration. Just under half of the SNPs assessed in the CFH gene and 15% of the SNPs assessed in the VEGF gene were found to be associated with visual outcomes or the number of injections required during follow-up.
10. Ophthalmology. 2013 Jan; 120 (1): 115-21. doi: 10.1016 / j.ophtha. 2012.10.006. Epub 2012 Nov 11, "Variants in the VEGFA gene and treatment outcome after anti-VEGF treatment for neovascular age -related macular degeneration2, Abedi F et al. The authors concluded that: Pharmacogenetic association with anti-VEGF treatments may influence the visual outcomes in neovascular AMD. In patients with the T allele in tSNP rs3025000, there was a significantly better visual outcome at 6 months and a greater chance of the patients belonging to the responder group with anti-VEGF treatment at 3, 6, and 12 months. The visual acuity (VA) outcomes of patients harbouring the T allele at SNP rs3025000 were comparable with those of the pivotal clinical trials but with fewer injections.
11. Ophthalmology. 2014 Apr; 121 (4): 905-10. doi: 10.1016 / j.ophtha. 2013.10.047. Epub 2013 Dec 21, "Polymorphisms in vascular endothelial growth factor receptor 2 are associated with better response rates to ranibizumab treatment in age-related macular degeneration ", Hermann MM et al. The authors evaluated the association of single nucleotide polymorphisms (SNPs) in VEGF genes and their receptors (VEGFR) with the response rate to ranibizumab in 366 patients with neovascular AMD. Univariate analyzes of variance (ANOVAs) revealed a significant effect of SNP rs4576072 in the VEGFR2 gene on VA change after 12 months (F [1,235] = 14.05; P = 0.02). A stepwise linear regression analysis returned a model (P = 0.01) with SNPs rs4576072 and rs6828477 in the VEGFR2 gene as independent predictors for VA change after 12 months, with a mean increase in VA of 0.26 on the logarithm of the minimum angle of resolution (logMAR) scale in patients with 3 contributing minor alleles compared with a loss of 0.03 logMAR in patients with no minor allele.
12. Research and Reports in Neonatology 2011: 1 5-11; "VEGF 936C> T is predictive of threshold
retinopathy of prematurity in Japanese infants with a 30-week gestational age or less ", Yagi M et al. The authors found that VEGF 936C> T polymorphism and duration of oxygen administration were independent risk factors for threshold ROP using a multivariate logistic analysis with adjustment for gestational age and birth weight.
13. Clin Cancer Res 2009; 15 (17) September 1, 2009, pp5297-5302, DOI: 10.1158 / 1078-0432.CCR-08-2576, "The Role of Vascular Endothelial Growth Factor Genetic Variability in Cancer", Bryan P Schneider et al. The authors comment that recently, genetic variability in VEGF has been studied as a potential predictive biomarker for bevacizumab. The VEGF-1154 AA and -2578 AA genotypes predicted an improved median overall survival, the VEGF-634 CC and -1498 TT genotypes predicted protection from grade 3-4 hypertension in the pivotal trial, E2100. If validated, these finding could help direct which subgroup of patients should receive bevacizumab.

The present inventor has identified the following SNPs as the SNPs of interest for the present invention.

NB: As is known in the art, the position of SNP is expressed as chromosome number: nucleotide position, for example 6: 43768652 is nucleotide number 43768652 on human chromosome 6. ARMS2 is also known as age-related macular degeneration-sensitive protein 2, ARMD8 and ARMS2_HUMAN LOC387715.

Therefore, the present invention provides the following concepts.

Human VEGF-A SNPs associated with VEGF-A-mediated diseases & conditions
1. A method of treating or reducing the risk of a disease or condition mediated by VEGF-A in humans, the method for which the humans are rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, An anti-VEGF-A ligand that specifically binds to human VEGF-A expressed by a VEGF-A nucleotide sequence containing an SNP selected from the group consisting of rs833068, rs833069, rs3025000 and rs1570360 (eg, anti-VEGF-A). A step of administering a trap, antibody or antibody fragment), wherein said human comprises a VEGF-A nucleotide sequence containing said selected SNP.

Here, specific binding is performed using a conventional method known to those skilled in the art, such as ELISA or SPR, and sample VEGF-A (eg, using a method as described herein). It may be tested. For example, VEGF-A is provided by said human or another human-derived sample, such as a serum or blood sample. In some examples, this ligand comprises an antibody constant region (eg, an antibody Fc region). Optionally, this ligand comprises a human gamma-1 heavy chain constant region comprising an amino acid selected from the group consisting of Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42. Here, the human comprises an IGHG1 * 01 human heavy chain constant region gene segment, or the human expresses an antibody comprising a human gamma-1 heavy chain constant region comprising the selected amino acid. Optionally, this constant region is contained in the Fc region of the ligand.

In one example, this ligand comprises an anti-VEGF-A binding site that specifically binds to human VEGF-A. Optionally, this binding site comprises one or more antibody variable domains, or one or more human VEGF-A receptor domains. In some examples, the specific binding is Kd less than or equal to 1 mm (ie, 1 mM or less), eg, less than 1 nM; or less than 100 pM; or less than 10 pM. Specific binding can be determined, for example, by assessing the binding of a ligand to VEGF-A in a sample from said human or another human (including said VEGF-A). In some examples, this sample comprises said human serum, blood, feces, tissue, cells, urine and / or saliva.

Optionally, VEGF-A is a VEGF-A-165 isoform.

In one example, this ligand is an anti-VEGF-A trap, eg, aflibercept. In one example, this ligand is EYLEA ™.

In one example, the ligand is an anti-VEGF-A antibody or antibody fragment, such as ESBA1008, bevacizumab or ranizumab. In one example, this ligand is AVASTIN ™ or LUCENTIS ™.

In one example, this ligand is an anti-VEGF ankyrin repeat protein (DARPin), such as abicipar pegol.

In one example, this ligand is NCE (the so-called New Chemical Entity in the art), eg sunitinib.

In one example, this condition is selected from the group consisting of angiogenesis, neoangiogenesis, ocular angiogenesis and solid tumor angiogenesis.

In one example, this condition is subretinal neovascularization.

In some examples, the disease or condition is an angioplastic disease or condition.

Optionally, the disease or condition is an angiogenic eye condition or angiogenic cancer, such as solid tumors, gastrointestinal cancers, colorectal cancers, liver cancers, breast cancers, ovarian cancers, Non-small cell lung cancer, thyroid cancer, oral cancer, or blood cancer.

Optionally, this angiogenic disease or condition is selected from the group consisting of psoriasis, rheumatoid arthritis, hemangiomas, angiofibroma, diabetic retinopathy, corneal neovascularization and neovascular glaucoma.
Optionally, the disease or condition is or includes vascular permeability (eg, ocular vascular permeability), edema or inflammation in said human.

For example, the disease or condition is cerebral edema (eg, edema associated with brain injury, stroke or brain tumor); edema associated with inflammatory disorders (eg, associated with psoriasis or arthritis, rheumatoid arthritis or asthma); burns and Related edema; ascites or pleural effusion (eg, associated with tumor, inflammation or trauma); chronic airway inflammation; capillary leakage syndrome; septicemia; renal disease (eg, associated with increased protein leakage); eye damage (eg, associated with increased protein leakage) For example, it is selected from the group consisting of age-related edema degeneration or diabetic retinopathy).

In some examples, this method is to reduce or reduce the risk of VEGF-A-mediated endothelial cell maintenance or proliferation in said angioplastic disease or condition in humans.

In one example, this angioplastic disease or condition is an angioplastic disease or condition listed above, such as an angioplastic eye condition.

In one example, this method is to reverse neovascularization in the VEGF-A-mediated disease or condition in humans.

In one example, this method is to treat or reduce the risk of neoangiogenesis in humans with VEGF-A ligand-refractory neoangiogenesis.

In some cases, this human has been diagnosed with the disease or condition prior to said administration of the ligand.

For example, this human is partially or completely resistant to treatment with anti-VEGF-A ligands such as aflibercept, sunitinib, bevacizumab or ranizumab.
In some examples, this ligand is administered by intravenous or subcutaneous administration and / or is included in an injectable preparation.

2. The method of Concept 1, where the condition is an eye condition, eg diabetic retinopathy or retinopathy of prematurity.

In one example, this condition is diabetic retinopathy (DR).

In one example, this condition is age-related macular degeneration (AMD), eg, wet AMD.

In one example, this condition is polypoid choroidal angiopathy (PCV).

In one example, this condition is retinal hemangiomatous growth (RAP).

In one example, this condition is diabetic macular edema (DME).

In one example, this condition is retinal vein occlusion (RVO).

In one example, this condition is retinopathy of prematurity (ROP).

In one alternative, instead of "a method of treating or reducing the risk of a disease or condition mediated by VEGF-A in humans", the present invention relates to "a method of improving visual acuity in humans".

3. The method of concept 1 or 2 in which the SNP is selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039 and rs699946.

In one example, this SNP is rs699947, and optionally this state is DR.

In one example, this SNP is rs833061 and, optionally, this state is DR.

In one example, this SNP is rs2010963, and optionally this state is DR.

In one example, this SNP is rs3025039, and optionally this state is DR.

In one example, this SNP is rs699946, and optionally this state is DR.

4. Method 1 or 2 in which the SNP is selected from the group consisting of rs699947, rs2146323, rs1413711, rs699946, rs833068, rs833069 and rs3025000.

As shown in Table 19, these SNPs are associated with improved ocular response after eye treatment (eg, photodynamic therapy (PDT) or VEGF-A antagonism).

In one example, this SNP is homozygous with respect to rs699947, eg, said SNP.

In one example, this SNP is homozygous with respect to rs2146323, eg, said SNP.

In one example, this SNP is homozygous with respect to rs1413711, eg, said SNP.

In one example, this SNP is homozygous with respect to rs699946, eg, said SNP.

In one example, this SNP is homozygous with respect to rs833068, eg, said SNP.

In one example, this SNP is homozygous with respect to rs833069, eg, said SNP.

In one example, this SNP is homozygous with respect to rs3025000, eg, said SNP.

5. The method of concept 3 or 4, wherein the human is homozygous for said SNP.

In another example, this human is heterozygous with respect to said SNP.

6. A method of any one of concepts 2-5, further comprising treating humans with photodynamic therapy.

For example, the method further comprises treating the human with PDT at the same time as or after administration of the ligand to the human.

7. The disease or condition is cancer, the method of concept 1.

8. The method of Concept 7, where the SNP is rs699947 or rs1570360.

In one example, this SNP is homozygous with respect to rs699947, eg, said SNP.

In one example, this SNP is homozygous with respect to rs1570360, eg, said SNP.

9. Humans have (a) HTRA1 nucleotide sequences containing SNP rs11200638; (b) ARMS2 nucleotide sequences containing SNP rs10490924; (c) CFH nucleotide sequences containing T at position SNP rs800292, rs1061170 or A at position rs3766404; (d) Complementary component 2 (C2) nucleotide sequence containing SNP rs547154; (e) Prothrombin nucleotide sequence containing SNP rs1799963; (f) MTHFR nucleotide sequence containing SNP rs1801133; (g) Factor V nucleotide containing SNP rs6025 Sequence; or (h) any method of the aforementioned concept comprising a VEGFR2 nucleotide sequence comprising SNP rs4576072 or rs6828477.

In one example, this human contains an HTRA1 nucleotide sequence containing SNP rs11200638 (eg, in homozygous form).

In one example, this human comprises an ARMS2 nucleotide sequence containing SNP rs10490924 (eg, in homozygous form).

In one example, this human contains a CFH nucleotide sequence containing SNP rs800292 (eg, in homozygous form).

In one example, this human comprises a CFH nucleotide sequence containing SNP rs1061170 (eg, in homozygous form).

In one example, this human contains a CFH nucleotide sequence containing SNP rs3766404 (eg, in homozygous form).

In one example, this human contains a complement component 2 (C2) nucleotide sequence containing SNP rs547154 (eg, in homozygous form).

In one example, this human contains a prothrombin nucleotide sequence containing SNP rs1799963 (eg, homozygous).

In one example, this human contains an MTHFR nucleotide sequence containing SNP rs1801133 (eg, in homozygous form).

In one example, this human contains a factor V nucleotide sequence containing SNP rs6025 (eg, homozygously).

In one example, this human contains a VEGFR2 nucleotide sequence containing SNP rs4576072 (eg, in homozygous form).

In one example, this human contains a VEGFR2 nucleotide sequence containing SNP rs6828477 (eg, in homozygous form).

10. Humans have (a) HTRA1 nucleotide sequences containing SNP rs11200638; (b) ARMS2 nucleotide sequences containing SNP rs10490924; (c) CFH nucleotide sequences containing SNP rs800292; or (d) complement component 2 containing SNP rs547154. (C2) A method of any one of concepts 2-6, comprising a nucleotide sequence.

These SNPs are risk factors for eye condition.

In one example, the condition of the eye is DR or ROP, because this method treats or reduces the risk of DR or ROP.

11. Human has (i) ARMS2 nucleotide sequence containing G at position SNP rs10490924; (ii) CFH nucleotide sequence containing T at position SNP rs1061170 or rs3766404; (iii) Prothrombin nucleotide sequence containing SNP rs1799963; (iv ) MTHFR nucleotide sequence comprising SNP rs1801133; (v) Factor V nucleotide sequence comprising SNP rs6025; or (vi) VEGFR2 nucleotide sequence comprising SNP rs4576072 or rs6828477, any one of concepts 2-6.

These SNPs are associated with improved response after treatment of eye diseases or conditions (eg, PDT and / or VEGF-A antagonism).

In one example, the condition of the eye is DR, because this method treats or reduces the risk of DR.

SNPs associated with improved response after anti-VEGF-A treatment of eye diseases or conditions
12. A method of treating or reducing the risk of a disease or condition mediated by VEGF-A in humans, which is an anti-VEGF-A that specifically binds to human VEGF-A to said human. It comprises the step of administering a ligand (eg, an anti-VEGF-A trap, antibody or antibody fragment), wherein the human is (i) ARMS2 nucleotide sequence containing G at position SNP rs10490924; (ii) SNP rs1061170. A CFH nucleotide sequence containing T at position or T at position rs3766404; or (iii) a VEGFR2 nucleotide sequence containing SNP rs4576072 or rs6828477, wherein this ligand is the Asp and sequence corresponding to position 204 of SEQ ID NO: 42. Contains a human gamma-1 heavy chain constant region comprising an amino acid selected from the group consisting of Leu corresponding to position 206 of number 42, and where said human comprises an IGHG1 * 01 human heavy chain constant region gene segment. , Or this human expresses an antibody comprising a human gamma-1 heavy chain constant region containing the selected amino acid.

These SNPs are associated with improved response after anti-VEGF-A treatment of eye diseases or conditions.

In one example, this eye condition is DR, and this method is to treat or reduce the risk of DR.

In one example, this human contains an ARMS2 nucleotide sequence containing a G (eg, homozygous) at the position of SNP rs10490924.

In one example, this human contains a CFH nucleotide sequence containing a T (eg, homozygous) at the position of SNP rs1061170.

In one example, this human contains a CFH nucleotide sequence containing a T (eg, homozygous) at the position of SNP rs3766404.

In one example, this human contains a VEGFR2 nucleotide sequence containing SNP rs4576072 (eg, in homozygous form). In one example, this human contains a VEGFR2 nucleotide sequence containing SNP rs6828477 (eg, in homozygous form).

13. The method of Concept 12, wherein the condition is an eye condition, eg diabetic retinopathy or retinopathy of prematurity.

In one example, this condition is diabetic retinopathy (DR).

In one example, this condition is age-related macular degeneration (AMD), eg, leaching AMD.

In one example, this condition is polypoid choroidal angiopathy (PCV).

In one example, this condition is retinal hemangiomatous growth (RAP).

In one example, this condition is diabetic macular edema (DME).

In one example, this condition is retinal vein occlusion (RVO).

In one example, this condition is retinopathy of prematurity (ROP).

In one alternative, instead of "a method of treating or reducing the risk of a disease or condition mediated by VEGF-A in humans," the present invention relates to "a method of improving vision in humans."

14. The method of concept 12 or 13, wherein the human further comprises VEGF-A SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039 and rs699946.

In one example, this SNP is rs699947.

In one example, this SNP is rs833061.

In one example, this SNP is rs2010963.

In one example, this SNP is rs3025039.

In one example, this SNP is rs699946.

15. A method of any one of concepts 12-14, further comprising the step of treating a human with photodynamic therapy.

For example, the method further comprises treating the human with PDT at the same time as or after administration of the ligand to the human.

16. Concept 12 methods in which the disease or condition is cancer.

17. The method of concept 16 in which humans further include VEGF-A SNP rs699947 or rs1570360.

In one example, this SNP is homozygous with respect to rs699947, eg, said SNP.

In one example, this SNP is homozygous with respect to rs1570360, eg, said SNP.

Antagonism of VEGF-A and PDGF-B pathways
PDGF-B is also known as IBGC5, PDGF-2, PDGF2, SIS, SSV and c-sis. Platelet-derived growth factor receptor beta (PDGFRB) is also known as CD140B, IBGC4, IMF1, JTK12, PDGFR-1 and PDGFR1.

PDGF is a growth factor that can play a role in new angiogenesis of the eye, a mitogen of peripheral cells, and may also be a smooth muscle cell, fibroblast and other mesenchymal cell type. It has been proven. Peripheral cells are thought to be important for the maintenance of established blood vessels and may be important for the maturation process in eye conditions such as choroidal angiogenesis. It has been hypothesized that there is a period of endothelial cell VEGF dependence that overlaps with the period of responsiveness to PDGF withdrawal in newly formed new blood vessels. Probable mature blood vessels may be associated with resistance to anti-VEGF blockade, or rapid recurrence of choroidal angiogenesis.

PDGF has been demonstrated to stimulate angiogenesis and peripheral cell replacement. Loss of peripheral cells in retinal vessels is thought to be associated with vascular abnormalities and instability, including the formation of microaneurysms and vascular permeability. Peripheral cell loss due to PDGF blockade may be associated with regression of neoangiogenesis during maturation. Peripheral cells produce VEGF-A under selected conditions, thereby potentially protecting endothelial cells in the presence of systemic suppression of VEGF-A.

Am J Pathol. June 2006; 168 (6): pp. 2036-53; "Inhibition of platelet-derived growth factor B signaling enhances the efficacy of anti-vascular endothelial growth factor therapy in multiple models of ocular neovascularisation"; Jo N See. Jo et al. Demonstrated increased inhibition of neoangiogenesis in experimental models with specific blockade of both VEGF-A and PDGF-B compared to blockade with VEGF-A alone. In addition, new angioplasty became more resistant to VEGF-A blockade as it progressed. Inhibition of VEGF-A and PDGF-B together caused regression of neoangiogenesis that did not respond to anti-VEGF-A alone.

The following is also quoted:-
Angiogenesis. July 2014; 17 (3): pp. 553-62. doi: 10.1007 / sl0456-013-9402-5. Epub October 24, 2013; "Antagonism of PDGF-BB suppresses subretinal neovascularization and enhances the effects of blocking VEGF-A2 "; Dong A et al.
MAbs. January-February 2010; 2 (l): pp. 20-34, Epub January 2, 2010; "A dual-targeting PDGFRbeta / VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo "; Mabry R et al.

A treatment regimen that combines inhibition of peripheral and endothelial cell function through the use of simultaneous administration of anti-VEGF-A targeting and PDGF-BB pathway targeting is a paracrine and autoclinic VEGF signaling in endothelial cells. Useful for blocking both.

Therefore, this aspect of the invention provides:
18. The method of any of the aforementioned concepts, further comprising the step of antagonizing PDGF-B in said human.
In the example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc.) to humans. REGN2176-3 contains aflibercept.

19. The method of Concept 18, which comprises the step of administering the anti-PDGF-B ligand to humans simultaneously or sequentially with the anti-VEGF-A ligand.

In some examples, anti-VEGF-A and anti-PDGF-B ligands are included together by pharmaceutical formulations administered to humans. In the example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc.) to humans.
In one example, a bispecific ligand (eg, a trap or antibody or antibody fragment) is administered, where the ligand comprises a human VEGF-A binding site and a human PDGF-B binding site.

In one example, this anti-PDGF-B ligand antagonizes human PDGF-B encoded by the PDGF-B nucleotide sequence of (i).

In some examples, the anti-PDGF-B ligand is an anti-PDGF-B antibody or fragment, an anti-PDGF-B trap, an anti-PDGF-B aptamer [eg, E10030 or FOVISTA ™] or an anti-PDGF-B NCE (so-called tech). Includes or consists of New Chemical Entity) in the field.

Examples of suitable anti-PDGF-B pathway antibodies, and variable regions for incorporation into antibodies for use in the present invention, are disclosed in WO2014109999A1 and disclosed (and specifically such antibodies and variable). The sequence of regions (including the sequence of regions), as well as the methods of testing and the conditions of medical disease and use (as appropriate for the present invention) are incorporated herein as if expressly described. Disclosure of features that can be incorporated into the concepts herein is provided.
In one example herein, the anti-PDGF-B ligand specifically binds to a human PDGF-B dimer (ie, the so-called PDGF-BB).

20. The method of Concept 18, which comprises the step of administering the anti-PDGFR-B ligand to humans simultaneously or continuously with the anti-VEGF-A ligand.

In some examples, anti-VEGF-A and anti-PDGFR-B ligands are included together by pharmaceutical formulations administered to humans. In one example, a bispecific ligand (eg, a trap or antibody or antibody fragment) is administered, wherein the ligand comprises a human VEGF-A binding site and a human PDGFR-B binding site. See Dong et al., Which provides a suitable example.

In one example, the PDGFR-B ligand antagonizes human PDGFR-B encoded by the PDGF-B nucleotide sequence of (ii).

In one example herein, anti-PDGFRB antagonizes receptor binding to a human PDGF-B dimer (ie, so-called PDGF-BB) [eg, where this PDGF-B is in (i). Encoded by the nucleotide sequence].

Suitable PDGFRβ-specific inhibitors are disclosed, for example, in WO2008130704, the disclosures and sequences of which are incorporated herein by reference.

21. The method of any of the aforementioned concepts in which a human has been administered a human PDGF-B or PDGFR-B antagonist prior to administration of the anti-VEGF-A ligand.

Antagonism of VEGF-A and angiopoietin pathways Angiopoietin is a ligand for the endothelial cell receptor Tie2 and is essential for vascular development and angiogenesis. Unlike other family members, angiopoietin-2 (Ang2) is strongly upregulated by endothelial cells at sites of angioplasty and remodeling of blood vessels, including tumors. Enhanced antitumor effects have been observed in preclinical models with combined blockade of both VEGF and Ang2.

The Ang2 pathway functions to reconstruct blood vessels and is specifically active during both embryological and pathological angioplasty. However, it is rarely found in the mature quiescent vascular system. Therefore, blockade of combined Ang2 / VEGF results in decreased survival and maturation of new vessels and inhibition of pathological leakage. Ang2 inhibition also blocked inflammation-driven vascular remodeling in model systems in which VEGF inhibition was ineffective, suggesting that the combined blockade has the ability for greater therapeutic benefit. To.

Therefore, the present invention further provides:-
22. The method of any one of concepts 1-17, comprising the step of antagonizing angiopoietin (eg, Ang2) in the human.

In one example, the method comprises administering Nesbuckhamb (Regeneron Pharmaceuticals, Inc.) to humans.

23. The method of Concept 22, which comprises the step of administering an anti-Ang2 ligand (eg, Nesvacumab) to a human simultaneously or continuously with the anti-VEGF-A ligand.

In some examples, the anti-Ang2 ligand comprises or consists of an anti-Ang2 antibody or fragment, an anti-Ang2 trap, an anti-Ang2 aptamer or an anti-Ang2 NCE (the so-called New Chemical Entity).

24. The method of Concept 22, wherein the anti-Ang2 receptor (eg, anti-human Tie2) ligand is administered to humans simultaneously or sequentially with the anti-VEGF-A ligand.

25. The method of any one of concepts 1-24, wherein the human is administered a human Ang2 or Ang2 receptor (eg, Tie2) antagonist prior to administration of the anti-VEGF-A ligand.

26. A group of anti-VEGF-A ligands (eg, anti-VEGF-A antibody, antibody fragment or VEGF-A trap) consisting of Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42. Contains a human gamma-1 heavy chain constant region containing an amino acid selected from; and where said human contains the IGHG1 * 01 human heavy chain constant region gene segment, or this human contains the selected amino acid. A method of any of the aforementioned concepts for expressing an antibody comprising a human gamma-1 heavy chain constant region comprising.

Optionally, this constant region is contained by the Fc region of this ligand.

In one embodiment, the ligand of the invention is a VEGF-A trap comprising or consisting of the polypeptide of SEQ ID NO: 127, eg, the polypeptide of SEQ ID NO: 127.

27. The anti-VEGF-A ligand (eg, anti-VEGF-A antibody, antibody fragment or VEGF-A trap) is Pro corresponding to position 72 of SEQ ID NO: 44, Asn corresponding to position 75 of SEQ ID NO: 44, SEQ ID NO: Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Phe corresponding to position 76 of 44, Val corresponding to position 161 of SEQ ID NO: 44 and Ala corresponding to position 257 of SEQ ID NO: 44. And here, the human comprises an IGHG2 * 01 human heavy chain constant region gene segment, or the human expresses an antibody comprising a human gamma-2 heavy chain constant region containing the selected amino acid, Concepts 1-. Any one of the 25 methods.

Optionally, this constant region is contained by the Fc region of this ligand.

For example, this ligand contains VEGF- containing one or more human VEGF-A receptor domain sequences fused to a human gamma-1 or -2 heavy chain constant region, eg, a constant region as defined in Concept 26 or 27. A trap. In one example, such a trap comprises a first and second VEGF-A receptor domain sequence (eg, it contains only the two species) (eg, a first and second human Flt1 domain sequence; Or first and second human KDR domain sequences; or Flt domain sequences and KDR domain sequences). In one example, this trap is a dimer of the following polypeptides (from N-terminus to C-terminus): human Flt1-Ig-like C2-2 domain sequence and human KDR Ig-like C2-3 domain sequence (concept 26). Includes (fused into the human gamma-1 heavy chain constant region, or the human gamma-2 heavy chain constant region of concept 27). In one example, this constant region is part of the antibody Fc region.

A suitable human Flt1 Ig-like C2-2 type domain sequence for use in the present invention is shown in SEQ ID NO: 129. Suitable human KDR Ig-like C2-3 type domain sequences for use in the present invention are shown in SEQ ID NO: 130.

28. The method of any one of concepts 1-27, wherein the anti-VEGF-A ligand is a human anti-VEGF-A antibody, antibody fragment or VEGF-A trap.

29. Humans containing the VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP prior to the administration step (where humans are concept 1 or 12 humans). A method of any of the aforementioned concepts, including the step of selecting.

30. A method of any one of concepts 1-28, wherein the human has been determined to include the VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP.

31. Including the step of determining that a human contains the VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP, optionally, this determining step is for humans. Any one method of concepts 1-28 performed prior to administration of the ligand.

32. The method of concept 31, wherein the determining step comprises assaying a human-derived biological sample for said VEGF-A, HTRA1, ARMS2, CFH, C2, MTHFR, prothrombin, factor V or VEGFR2 SNP. ..

33. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of Concept 32, in which this assaying step is performed in a multiplex format.

34. Anti-VEGF-A ligand for use in any one of the methods of Concepts 1-33 (eg, anti-VEGF-A antibody, antibody fragment or VEGF-A trap).

35. Ligand of concept 34, wherein the anti-VEGF-A ligand is a human anti-VEGF-A antibody, antibody fragment or VEGF-A trap (eg, aflibercept).

36. A ligand of concept 34 or 35, wherein the ligand is administered intravenously or subcutaneously and / or is contained in an injectable preparation.

The 1000 Genomes Project shows that C2 SNP rs547154 has an average cumulative allelic frequency of 10% across all human populations; the frequency for the AFR population is 15%, and the frequency for the following human populations is also above average: Shown: PUR, IBS, GBR, ASW, LWK, YRI. Thus, if a human pedigree belongs to one of these populations, this human may have an increased risk of eye disease or condition or other VEGF-A-mediated disease or condition, and the present invention , Including the step of administering a ligand to such a human. Thus, in certain embodiments, the human comprises a C2 SNP rs547154 and is of AFR, PUR, IBS, GBR, ASW, LWK or YRI pedigree. For example, this human is of AFR pedigree.

VEGF-A SNP rs1413711 is shown in the 1000 Genomes Project to have a mean cumulative allelic frequency of 64% across all human populations; the frequencies for the AFR and ASN populations are 80% and 71%, respectively, and the following humans: Over-average frequencies are also shown for populations: ASW, LWK, YRI, PUR, CHB, CHS and JPT. Thus, if a human pedigree belongs to one of these populations, this human may have improved treatment of eye diseases or conditions, and the present invention administers a ligand to such a human. Includes the process of Thus, in certain embodiments, the human comprises VEGF-A SNP rs1413711 (eg, homozygously) and is of AFR, ASN, ASW, LWK, YRI, PUR, CHB, CHS or JPT lineage. .. For example, this human is of AFR pedigree. For example, this human is of ASN pedigree. In one example, this method is to improve visual acuity in said humans.

The 1000 Genomes Project shows that VEGF-A SNP rs699946 has an average cumulative allelic frequency of 26% across all human populations; the frequency for the ASN population is 42%, which is also above average for the following human populations: Frequency is indicated: PUR 34%, CHB 45%, CHS 41% and JPT 40%. Thus, if a human pedigree belongs to one of these populations, this human may have improved treatment of eye diseases or conditions, and the present invention administers a ligand to such a human. Includes the process of Thus, in certain embodiments, the human is a human with VEGF-A SNP rs699946 (eg, homozygous) and of ASN, ASW, PUR, CHB, CHS or JPT lineage. For example, this human is of ASN pedigree. In one example, this method is to improve visual acuity in said humans.

The VEGF-A SNP rs3025000 is shown in the 1000 Genomes Project to have an average cumulative allelic frequency of 26% across all human populations; the frequencies for the AMR, ASN and EUR populations are 30%, 42% and 27%, respectively. Yes, the following human populations also show above-average frequencies: MXL (29%), PUR (38%), CHB (45%), CHS (41%), JPT (40%), CEU (30%) ), GBR (27%), IBS (39%) and TSI (31%). Thus, if a human pedigree belongs to one of these populations, this human may have improved treatment of eye diseases or conditions, and the present invention administers a ligand to such a human. Includes the process of Thus, in certain embodiments, the human comprises VEGF-A SNP rs3025000 (eg, in homozygous form) and AMR, ASN, EUR, MXL, PUR, CHB, CHS, JPT, CEU, GBR, IBS or A human of TSI pedigree. For example, this human is of AMR pedigree. For example, this human is of ASN pedigree. For example, this human is of EUR pedigree.

The prothrombin SNP rs1799963 is almost exclusive to Caucasians; this SNP has been shown in the 1000 Genomes Database to have above-average cumulative allelic frequencies in the following human populations: AMR, EUR, TSI, GBR and PUR. Thus, if a human pedigree belongs to one of these populations, this human may have improved treatment of eye diseases or conditions, and the present invention administers a ligand to such a human. Includes the process of Thus, in certain embodiments, the human comprises prothrombin SNP rs1799963 (eg, homozygous) and is of Caucasian, AMR, EUR, TSI, GBR or PUR pedigree. For example, this human is of Caucasian descent. For example, this human is of EUR pedigree. In one example, this condition is CNV.

MTHFR SNP rs1801133 has been shown in the 1000 Genomes Database to have above-mean cumulative allelic frequencies in the following human populations: AMR, ASN, EUR, CHB, CLM, IBS, JPT, MXL, PUR and TSI. Thus, if a human pedigree belongs to one of these populations, this human may have improved treatment of eye diseases or conditions, and the present invention administers a ligand to such a human. Includes steps. Thus, in certain embodiments, the human comprises MTHFR SNP rs1801133 (eg, homozygously) and is of AMR, ASN, EUR, CHB, CLM, IBS, JPT, MXL, PUR or TSI pedigree. For example, this human is of Caucasian descent. For example, this human is of AMR pedigree. For example, this human is of ASN pedigree. For example, this human is of EUR pedigree. For example, this human is of EUR pedigree. In one example, this condition is CNV.

VEGFR2 SNP rs4576072 has been shown in the 1000 Genomes Project to have an average cumulative allelic frequency of 8% across all human populations; 10% and 11% frequencies for the AMR and EUR populations, respectively, and also for the following human populations: Shows above average: PUR (14%), CEU (9%), GBR (13%) and TSI (13%). Thus, if a human pedigree belongs to one of these populations, this human may have improved treatment of eye diseases or conditions, and the present invention administers a ligand to such a human. Includes the process of Thus, in certain embodiments, the human comprises VEGFR2 SNP rs4576072 (eg, in homozygous form) and is of AMR, EUR, PUR, CEU, GBR or TSI lineage. For example, this human is of AMR pedigree. For example, this human is of EUR pedigree. In certain embodiments, 3 or 4 VEGFR2 SNPs from rs4576072 and rs6828477 provide improved treatment. Thus, for example, this human contains 3 or 4 copies of VEGFR2 SNP selected from rs4576072 and rs6828477, eg, this human is GG (ie, this human contains VEGFR2 SNP rs4576072 homozygous). And AA or AG (ie, this human contains VEGFR2 SNP rs6828477, eg, in homozygous form); or GA (rs4576072) and AA (rs6828477).

Administration of human VEGF-A ligand to humans containing the PDFG-B pathway SNP risk factor Endothelial cells (EC) in blood vessels under formation are pathological, including cancer, in normal tissue and during angioplasty. In both situations, it is stabilized by the replacement of peripheral cells. Endothelial cells in small blood vessels and capillaries interact with peripheral cells, which are mesenchymal-derived cells that provide important support for the formation and function of blood vessels. Many studies have described the importance of members of the platelet-derived growth factor (PDGF) family for replacement of peripheral cells and vascular endothelium during embryogenesis, and their productive associations. Notably, mice lacking the components of the PDGF-B / PDGFR-β signaling axis are essentially deficient in peripheral cells and eventually die from microbleeding due to vascular malformations. Blood. September 8, 2011; 118 (10): pp. 2906-2917, pre-published online July 21, 2011, doi: 10.1182 / blood-2011-01-331694, "Pericytes promote endothelial cell survival through induction" Of autocrine VEGF-A signaling and Bcl-w expression ”, Franco M et al. The authors describe that the budding of newly formed blood vessels replenishes peripheral cells primarily through the secretion of platelet-derived growth factor beta (PDGF-BB) by endothelial end cells. Peripheral cells respond by migrating along the substrate retention gradient of PDGF-BB and by a proliferative burst if they reach the tip of budding. Contrary to this, peripheral cells upregulate the expression of cell-bound VEGF-A as they mature and are replenished into newly formed blood vessels. The authors found that peripheral cells stimulate VEGF-A autoclination expression by tumor endothelial cells in addition to producing VEGF-A, which acts in a paracrine manner. The authors found that treatment regimens that combine inhibition of peripheral and endothelial cells are VEGF-targeted antibodies and PDGF-BB / PDGFRβ-targeted antibodies to block both paracrine and autoclinic VEGF signaling in EC. It is suggested that it works through the use of concomitant doses of.

Therefore, the present invention provides the following aspects:-
1. A method of treating or reducing the risk of VEGF-A-mediated diseases or conditions in humans, which is an anti-human VEGF-A ligand; an anti-human PDGF-B ligand or an anti-PDGFR-B ligand in humans. Including the step of administering to, where this human is from (i) rs142404523 (ie, C corresponding to position -776) and C at that position of rs1800818 (ie, C corresponding to position -735). PDGF-B nucleotide sequence containing SNPs selected from the group; or (ii) SNPs selected from the group consisting of rs246395 (ie, G corresponding to position 2601) and rs74943037 (ie, T corresponding to position 1391). A method comprising a PDGFR-B nucleotide sequence comprising.

Examples of suitable anti-VEGF-A ligands that are antagonists of human VEGF-A are aflibercept, sunitinib, ESBA1008, bevacizumab or ranizumab. In one example, this ligand is an anti-VEGF-A antibody or fragment (eg, ESBA1008, bevacizumab or ranizumab), an anti-VEGF-A trap (eg, a VEGF-A receptor binding site fused to the antibody Fc region, eg, Aflibercept), aptamers or NCEs (so-called New Chemical Entity [New Chemical Entity], eg, snitinib) are included or consist of them.

In the example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc.) to humans. REGN2176-3 contains aflibercept and anti-PDGF-B ligand.

In certain embodiments, the selected SNP (eg, rs246395) is associated with VEGF-A secretion from the peripheral cells in the human and, for example, the peripheral cells in the human (eg, peripheral cells of the eye or solid tumor in the patient). It is associated with the maintenance or increase of VEGF-A secretion from peripheral cells) at the site of. Therefore, administration of anti-VEGF-A ligand is also beneficial for targeting such secreted VEGF-A. This is useful, for example, when the disease or condition is angioplasty or neoangiogenesis, or is associated with one or both of these.

For PDGF-B, C at that position in rs1800818 (ie, -735C) has been shown in the 1000 genomic database to have an average cumulative allele frequency of 37% across all human populations; 62% and 41%, respectively. Frequency for the AFR and EUR populations (ie above average), and also above average for the following human populations: ASW60%, LWK58%, YRI68%, CEU46%, FIN46%, IBS50% and TSI38 %. Thus, if a human pedigree belongs to one of these populations, this human may be at increased risk of angioplasty or other VEGF-A-mediated diseases or conditions, and the invention , Including the step of administering a ligand to such a human. Thus, in certain embodiments, the human comprises a PDGF-B nucleotide sequence containing -735C, i.e., a C at that position of PDGF-B SNP rs1800818, where the human is AFR, EUR, ASW, LWK. , YRI, CEU, FIN, IBS or TSI pedigree. For example, this human is of AFR pedigree. For example, this human is of EUR pedigree. For PDGF-B, SNP rs142404523 (-776T> C) has been shown in the 1000 Genomes Project to have an average cumulative allele frequency of 8% across all human populations; 17% and 10%, respectively (ie, mean). Frequency for AFR and EUR populations (above), and also above average for the following human populations: ASW 16%, LWK 18%, YRI 16%, PUR 11%, CEU 11%, FIN 9 %, GBR 12% and IBS 18%. Thus, if a human pedigree belongs to one of these populations, this human may be at increased risk of angioplasty or other VEGF-A-mediated diseases or conditions, and the invention , Including the step of administering a ligand to such a human. Thus, in certain embodiments, the human comprises PDGF-B SNP rs142404523 and is of AFR, EUR, ASW, LWK, YRI, PUR, CEU, FIN, GBR or IBS pedigree. For example, this human is of AFR pedigree. For example, this human is of EUR pedigree.

For PDGFR-B, SNP rs246395 (2601A> G) has been shown in the 1000 genomic database to have an average cumulative allele frequency of 23% for G across all human populations; 29% and 30% (ie, respectively). Frequency for the AMR and EUR populations (above the average), and also for the following human populations, is shown to be above average: CLM (36%), MXL (25%), PUR (26%), CEU (39) %), GBR (30%), IBS (29%) and TSI (31%). Thus, if a human belongs to one of these populations, this human may be at increased risk of angioplasty or other VEGF-A-mediated diseases or conditions, and the present invention is described in this invention. Including the step of administering a ligand to such a human. Thus, in certain embodiments, the human comprises PDGFR-B SNP rs246395 and is of AMR, EUR, CLM, MXL, PUR, CEU, GBR, IBS or TSI pedigree. For example, this human is of AMR pedigree. For example, this human is of EUR pedigree.

For PDGFR-B, SNP rs74943037 has been shown in the 1000 Genomes Project to have an average cumulative allelic frequency of 3% for G across all human populations; a frequency of 10% (ie, above average) for the AFR population, Also shown to be above average for the following human populations: ASW 8%, LWK 14% and YRI 8%. Thus, if a human pedigree belongs to one of these populations, this human may be at increased risk of angioplasty or other VEGF-A-mediated diseases or conditions, and the invention , Including the step of administering a ligand to such a human. Thus, in certain embodiments, this human comprises PDGFR-B SNP rs74943037 and is of AFR, ASW, LWK or YRI lineage. For example, this human is of AFR pedigree.

2. Humans are homozygous for the selected SNP (eg, homozygous for rs246395 or homozygous for C at that position in rs1800818 [ie, homozygous for C corresponding to position -735]). There is a method of aspect 1.

Such homozygotes can increase the severity or risk of said disease or condition.

3. The method of aspect 1 or 2, wherein the human comprises a PDGF-B nucleotide sequence comprising a SNP of (i) and a PDFGR-B nucleotide sequence comprising an SNP of (ii). For example, this human contains a C in its position at rs1800818 and rs246395. In another example, this human contains a C in its position at rs1800818 and rs74943037.

For example, this human includes rs142404523 and rs246395. In another example, this human comprises rs142404523 and rs74943037.

4. VEGF-A nucleotide sequence containing SNPs selected from the group consisting of (a) rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360; (b) SNP rs11200638 HTRA1 nucleotide sequence containing; (c) ARMS2 nucleotide sequence containing SNP rs10490924; (d) Complementary factor H (CFH) nucleotide sequence containing SNP rs800292, rs1061170 or rs3766404; (e) Complementary component 2 containing SNP rs547154 C2) Nucleotide sequence; (f) Prothrombin nucleotide sequence containing SNP rs1799963; (g) MTHFR nucleotide sequence containing SNP rs1801133; (h) Factor V nucleotide sequence containing SNP rs6025; or (i') SNP rs4576072 or rs6828477 The method of any one of aspects 1-3, comprising a VEGFR2 nucleotide sequence comprising.

In one example, this human comprises the SNP of (a).

For example, this human contains rs699947, and optionally this condition is DR or solid tumor. For example, this human contains rs833061, and optionally this condition is DR or solid tumor. For example, this human contains rs2010963, and optionally this condition is DR or solid tumor. For example, this human contains rs3025039, and optionally this condition is DR or solid tumor. For example, this human contains rs699946, and optionally this condition is DR or solid tumor. For example, this human contains rs2146323.

For example, this human comprises rs1413711 and optionally this human is homozygous with respect to said SNP. For example, this human comprises rs833068, and optionally this human is homozygous with respect to said SNP. For example, this human comprises rs833069, and optionally this human is homozygous with respect to said SNP. For example, this human contains rs3025000. For example, this human includes rs1570360.

In one example, this human comprises the SNPs of (a) and (b). In one example, this human comprises the SNPs of (a) and (c). In one example, this human comprises the SNPs of (a) and (d). In one example, this human comprises the SNPs of (a) and (e). In one example, this human comprises the SNPs of (a) and (f). In one example, this human comprises the SNPs of (a) and (g). In one example, this human comprises the SNPs of (a) and (h). In one example, this human comprises the SNPs of (a) and (i').

Optionally, in any aspect herein, the ligand specifically binds or antagonizes VEGF-A, eg, VEGF-A encoded by the VEGF-A nucleotide sequence containing the SNP of (a). To do. In one example, this is a specific binding or binding to VEGF-A in an in vitro sample (eg, a serum or blood sample) from a human (where this human comprises the VEGF-A nucleotide sequence). It can be determined by its antagonism. Specific binding may be determined, for example, by SPR or ELISA as known to those of skill in the art. Those of skill in the art are also familiar with standard assays for VEGF-A antagonism. Further, in one example, the human genome comprises the SNP of (a) above.

5. The human comprises a human VEGF-A nucleotide sequence containing an SNP selected from the group consisting of rs699947, rs833061, rs2010963, rs3025039, rs699946, rs2146323, rs1413711, rs833068, rs833069, rs3025000 and rs1570360, and the ligand is said above. The method of any one of aspects 1 or 4 that antagonizes VEGF-A encoded by a nucleotide sequence.

In an example of any aspect herein, VEGF-A is a VEGF-A- (165) isoform.
6. The method of any one of aspects 1-5, further comprising the step of antagonizing PDGF-B in the human.

In the example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc.) to humans. REGN2176-3 contains aflibercept.

7. The method of embodiment 6, comprising the step of administering the anti-PDGF-B ligand to humans simultaneously or sequentially with the anti-VEGF-A ligand.

In some examples, anti-VEGF-A and anti-PDGF-B ligands are included together by pharmaceutical formulations administered to humans. In the example of any aspect herein, the method comprises administering REGN2176-3 (Regeneron Pharmaceuticals, Inc.) to humans. In one example, this anti-PDGF-B ligand antagonizes human PDGF-B encoded by the PDGF-B nucleotide sequence of (i).

In some examples, this anti-PDGF-B ligand is an anti-PDGF-B antibody or fragment, an anti-PDGF-B trap, an anti-PDGF-B aptamer (eg, each of which is incorporated herein by reference in its entirety. , U.S. Pat. No. 6,207,816; No. 5,731,144; No. 5,731,424; and No. 6,124,449, eg, E10030 [FOVISTA ™]) or anti-PDGF-B NCE (so-called new in the art). Contains or consists of New Chemical Entity).

Examples of suitable anti-PDGF-B pathway antibodies and variable regions for incorporation into antibodies for use in the present invention are disclosed in WO2014109999A1, which disclosure and, specifically, such antibodies and Similarly, the method of study, as well as the medical disease and condition for use (which is appropriate for the present invention), includes the sequences within the variable region, as if expressly described herein. Provided are disclosures of features that are incorporated herein by reference and may be incorporated into aspects of this specification.

In one example herein, the anti-PDGF-B ligand specifically binds to a human PDGF-B dimer (ie, the so-called PDGF-BB).

8. The method of embodiment 6, comprising the step of administering the anti-PDGFR-B ligand to humans simultaneously or sequentially with the anti-VEGF-A ligand.

In some examples, anti-VEGF-A and anti-PDGFR-B ligands are included together by pharmaceutical formulations administered to humans.

In one example, the PDGFR-B ligand antagonizes human PDGFR-B encoded by the PDGF-B nucleotide sequence of (ii).

In one example herein, anti-PDGFRB antagonizes receptor binding to the human PDGF-B dimer (ie, so-called PDGF-BB) [eg, where PDGF-B is the nucleotide of (i)). Encoded by an array]. Suitable PDGFRβ-specific inhibitors are disclosed, for example, in WO2008130704, the disclosures and sequences of which are incorporated herein by reference.

9. The method of any one of aspects 1-8, wherein the human is administered a human PDGF-B or PDGFR-B antagonist prior to administration of the anti-VEGF-A ligand.

10. The method of any one of aspects 1-9, wherein the condition is selected from the group consisting of angiogenesis, neoangiogenesis, ocular angiogenesis and solid tumor angiogenesis.

In one example, this condition is subretinal neovascularization.

11. The method of any one of aspects 1-9, wherein the disease or condition is an angioplastic disease or condition.

Optionally, the disease or condition is angiogenic eye condition or angiogenic cancer, such as solid tumors, gastrointestinal cancers, colorectal cancers, liver cancers, breast cancers, ovarian cancers. , Non-small cell lung cancer, thyroid cancer, oral cancer, or blood cancer.

Optionally, the angiogenic disease or condition is selected from the group consisting of psoriasis, rheumatoid arthritis, hemangiomas, angiofibroma, diabetic retinopathy, corneal neovascularization and neovascular glaucoma.

12. The method of any one of aspects 1-11, wherein the disease or condition is or comprises vascular permeability (eg, vascular permeability of the eye), edema or inflammation in said human.

For example, the disease or condition is cerebral edema (eg, edema associated with brain damage, stroke or brain tumor); edema associated with inflammatory disorders (eg, associated with psoriasis or arthritis, rheumatoid arthritis or asthma); burns. Edema associated with; ascites or pleural effusion (eg, associated with tumor, inflammation or trauma); chronic airway inflammation; capillary leakage syndrome; septicemia; renal disease (eg, associated with increased protein leakage); It is selected from the group consisting of disorders (eg, age-related edema degeneration or diabetic retinopathy).

13. The method of any one of embodiments 1-12 for reducing or reducing the risk of VEGF-A-mediated endothelial cell maintenance or proliferation in said angioplastic disease or condition in humans.

In one example, this angioplastic disease or condition is the angioplastic disease or condition referred to above, eg, an angioplastic eye condition.

14. The method of any one of aspects 1-12 for regressing neoangiogenesis in the VEGF-A-mediated disease or condition in humans.

15. Any one method of embodiments 1-12 for treating or reducing the risk of neoangiogenesis in a human, who has VEGF-A ligand refractory neoangiogenesis. For example, this human is partially or completely resistant to treatment with anti-VEGF-A ligands such as aflibercept, sunitinib, bevacizumab or ranizumab.

16. The method of any one of aspects 1-15, comprising the step of antagonizing angiopoietin-2 (Ang2) in the human.

Angiopoietin is a ligand for the endothelial cell receptor Tie2 and is essential for angiogenesis and angiogenesis. Unlike members of other families, angiopoietin-2 (Ang2) is strongly upregulated by endothelial cells at sites of angioplasty and revascularization (including tumors). Enhanced antitumor efficacy has been observed in preclinical models with a combination of blocking both VEGF and Ang2.

In one example, the method comprises administering Nesvacumab (Regeneron Pharmaceuticals, Inc.) to humans.

17. The method of embodiment 16, comprising the step of administering an anti-Ang2 ligand (eg, Nesvacumab) to humans simultaneously or sequentially with the anti-VEGF-A ligand. In some examples, this anti-Ang2 ligand comprises or consists of an anti-Ang2 antibody or fragment, an anti-Ang2 trap, an anti-Ang2 aptamer or an anti-Ang2 NCE (so-called New Chemical Entity). ..

18. The method of embodiment 16, wherein the anti-Ang2 receptor (eg, anti-human Tie2) ligand is administered to humans simultaneously or sequentially with the anti-VEGF-A ligand.

19. The method of any one of aspects 1-18, wherein the human has been administered a human Ang2 or Ang2 receptor (eg, Tie2) antagonist prior to administration of the anti-VEGF-A ligand.

20. An anti-TOI (eg, VEGF-A) ligand (eg, antibody, antibody fragment or TOI trap) for use in any one of aspects 1-19.

21. Ligand of embodiment 20, which is a human anti-VEGF-A antibody, antibody fragment or VEGF-A trap (eg, aflibercept).

22. Ligand of embodiment 20 or 21 for intravenous or subcutaneous administration and / or contained in an injectable preparation.

General optional features applicable to all examples, embodiments, concepts and embodiments Anti-TOI (eg, VEGF-A) ligand (eg, antibody, antibody fragment or TOI trap) is located in 204 of SEQ ID NO: 42. It comprises a human gamma-1 heavy chain constant region containing Asp corresponding to the position or Leu corresponding to position 206 of SEQ ID NO: 42, wherein the human comprises (i) IGHG1 * 01 human heavy chain constant region gene segment. Any method or ligand that comprises or that this human expresses an antibody comprising the human gamma-1 heavy chain constant region containing Asp corresponding to position 204 of SEQ ID NO: 42 or Leu corresponding to position 206 of SEQ ID NO: 42. Provided by choice.

For example, this ligand is a VEGF-A trap containing one or more human VEGF-A receptor domain sequences fused to a human gamma-1 heavy chain constant region, eg, a constant region as defined in the previous paragraph. Is. In one example, such a trap might be a first and second VEGF-A receptor domain sequence (eg, containing only two), eg, a first and second human Flt1 domain sequence; or a first and second. Contains 2 human KDR domain sequences; or Flt domain sequences and KDR domain sequences. In one example, this trap comprises a human Flt1-Ig-like C2-2 type domain sequence and a human KDR Ig-like C2-3 type domain sequence (eg, in this order from the N-terminus to the C-terminus).

In one embodiment, the ligand of the invention is a VEGF-A trap comprising or consisting of SEQ ID NO: 127 or a dimer of SEQ ID NO: 127, eg, SEQ ID NO: 127.

In one example, this condition is an angioplastic disorder of the eye.

In one example, this condition is AMD, age-related macular degeneration; BRVO, retinal vein branch occlusion; CNV, choroidal angiogenesis; mCNV, choroidal angiogenesis caused by pathological myopia; CRVO, central retinal vein occlusion; DME , Diabetic macular edema; glaucoma; macular edema; PCV, polypochoroidal angiopathy; RAP, retinal hemangiomatous growth; retinal neoangiogenesis; ROP, retinopathy of prematurity; and RVO, selected from the group consisting of retinal vein occlusion Will be done.

In one example, this condition is wet AMD.

In one example, this method is for the treatment of Adult-Onset Vitelliform Detachments Associated With Pattern Dystrophy associated with pattern dystrophy.

In one example, this method is for the treatment of patterned dystrophy.

In one example, this method is used in neovascular AMD, for example, by administering an anti-VEGF ligand [eg, aflibercept, becavizumab or ranibizumab] in addition to Fovista® (anti-PDGF BB). For the treatment of subretinal fibrosis.

In some examples, this human is over 50 years old, for example, this human is over 55, 60, 65 or 70 years old.

In some examples, this condition is an ocular condition, and anti-VEGF-A ligands are administered in combination with photodynamic therapy (PDT), for example, to treat or prevent PCV.

In some examples, this condition is an ocular condition, and the anti-VEGF-A ligand is administered, for example, in combination with laser treatment and / or IVTA treatment to treat or prevent DME.

In one example, this condition is an ocular condition, and the anti-VEGF-A ligand is administered in combination with laser photocoagulation and / or intravitreal injection of glucocorticoids.

In one example, this condition is an ocular condition, and the anti-VEGF-A ligand is administered in combination with PDT and triamcinolone acetonide. Optionally, this ligand is administered intravitreal.

In one example, this human suffers from hypertension, hyperlipidemia, diabetes mellitus and / or atherosclerotic angiopathy. Arbitrarily, this condition is RVO, and more optionally, this human is over 60 years old.

In one example, when RVO is referred to herein, it is ischemic (non-perfused) or non-ischemic (perfused) RVO.

In one example, when ROP is referred to herein, it is type 1 ROP.

In one example, this condition is ROP, and the anti-VEGF-A ligand is administered in combination with laser treatment.

In certain embodiments, the human has a body mass index (BMI) of 30 or greater and / or the human is a cigarette smoker. These are risk factors, for example, the disease or condition is an eye disease or condition.

Optionally, there is less than 3 or 2 weeks, or 2 weeks between the onset of symptoms of an eye disease or condition and the initiation of treatment with a ligand according to the invention.

In some examples, AMD is a neovascular (nv) AMD, a "wet" AMD or an atrophic or "dry" AMD.

In one example, an eye condition or disease (eg, AMD), when the ligand is administered to a human, is the Age-Related Eye Disease Study classification (eg, Augood CA et al., "Prevalence". According to the agerelated maculopathy in older Europeans: the European Eye Study (EUREYE), Arch Ophthalmol. 2006; see 124: 529e35), it is a stage IV disease.

In certain embodiments, the disease or condition is here an eye disease or condition (eg, AMD, PCV, DR or CNV) and the ligand is administered to a human by intraocular injection, eg, intravitreal injection. Will be done.

Persistent angioplasty can result in or exacerbate certain diseases such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibroma, diabetic retinopathy and neovascular glaucoma. Inhibitors of VEGF activity are useful in treating such diseases and other VEGF-induced pathological angioplasty and vascular permeability conditions such as tumor angioplasty. Thus, in certain embodiments of the invention, Example 5 provides a method (or ligand for use in that method) for treating or reducing the risk of a condition associated with angiogenesis, wherein the condition. Is psoriasis, rheumatoid arthritis, hemangiomas, angiofibroma, diabetic retinopathy or neovascular glaucoma. In certain embodiments, the present invention provides, in Example 5, a method (or ligand for use in that method) for treating or reducing the risk of tumor angiogenesis.

By way of example, but not limited to, the methods of the invention relate to vascular permeability, edema or inflammation, such as injury, stroke or tumor-related cerebral edema; inflammatory disorders such as arthritis including psoriasis or rheumatoid arthritis. Edema; Asthma; Systemic edema associated with burns; Ascites and pleural effusion associated with tumors, inflammation or trauma; Chronic airway inflammation; Capillary leak syndrome; Sepulemia; Renal disorders associated with increased protein leakage; It may be useful in treating clinical conditions characterized by eye disorders such as edema degeneration and diabetic retinopathy. Thus, in certain embodiments, the present invention provides, in Example 5, a method (or ligand for use in that method) for treating or reducing the risk of any one of these conditions.

In one example, the method of the invention comprises the step of continuously administering to a patient multiple doses of a VEGF antagonist. The methods of the invention include administration of multiple doses of a VEGF antagonist to a patient at a frequency of once every 8 weeks or longer.

Various routes of administration are considered for use in the methods of the invention, including, for example, topical or intraocular administration (eg, intravitreal administration). The ligand (or pharmaceutical formulation containing the ligand) may be administered to the patient by any known delivery system and / or method of administration. In certain embodiments, the ligand is administered to the patient by ocular, intraocular, intravitreal or subconjunctival injection. In other embodiments, the ligand may be administered to the patient by topical administration, eg, via eye drops containing the ligand or other liquid, gel, ointment or fluid, and is applied directly to the eye. You may. Other possible routes of administration include, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral.

The amount of anti-VEGF ligand given to the patient at each dose is in most cases a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to the dose of ligand that produces a detectable improvement in one or more symptoms or signs of angioplastic eye damage, or angioplastic. It means the dose of ligand that inhibits, prevents, reduces or delays the progression of eye damage. For anti-VEGF antibody or VEGF receptor-based chimeric molecules (eg, VEGF traps, eg, aflibercept), eg, VEGFR1R2-FcΔC1 (a), therapeutically effective amounts are from about 0.05 mg to about 5 mg, For example, about 0.05 mg, about 0.1 mg, about 0.15 mg, about 0.2 mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45 mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, About 0.65 mg, about 0.7 mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 1.0 mg, about 1.05 mg, about 1.1 mg, about 1.15 mg, about 1.2 mg, about 1.25 mg, about 1.3 mg, about 1.35 mg, about 1.4 mg, about 1.45 mg, about 1.5 mg, about 1.55 mg, about 1.6 mg, about 1.65 mg, about 1.7 mg, about 1.75 mg, about 1.8 mg, about 1.85 mg, about 1.9 mg, About 2.0 mg, about 2.05 mg, about 2.1 mg, about 2.15 mg, about 2.2 mg, about 2.25 mg, about 2.3 mg, about 2.35 mg, about 2.4 mg, about 2.45 mg, about 2.5 mg, about 2.55 mg, about 2.6 mg, about 2.65 mg, about 2.7 mg, about 2.75 mg, about 2.8 mg, about 2.85 mg, about 2.9 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mg, or about 5.0 mg of antibody or receptor It may be a body-based chimeric molecule.

Dosage Regiments Specific non-limiting examples of dosing regimens within the scope of the invention are for anti-TOI ligands (eg, anti-VEGF-A antibodies or traps, eg, aflibercept):
Ligand 2 mg (0.05 mL) administered by intravitreal injection once every 4 weeks (monthly)
b. Ligand 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 8 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks ).
c. For the first 8 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection less frequently (as assessed by a qualified healthcare professional).
d. For the first 8 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection given at the discretion (PRN) (as evaluated by a qualified healthcare professional).
e. Ligand 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 12 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks ).
f. For the first 12 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection less frequently (as assessed by a qualified healthcare professional).
g. For the first 12 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection given at the discretion (PRN) (as evaluated by a qualified physician).
h. Ligand 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 16 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks ).
i. For the first 16 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection less frequently (as assessed by a qualified healthcare professional).
j. For the first 16 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection given at the discretion (PRN) (as evaluated by a qualified healthcare professional).
k. Ligand 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 20 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks ).
l. For the first 20 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection less frequently (as assessed by a qualified healthcare professional).
m. For the first 20 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection given at the discretion (PRN) (as evaluated by a qualified healthcare professional).
n. Ligand 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 24 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks ).
o. For the first 24 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection less frequently (as assessed by a qualified healthcare professional).
p. For the first 24 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection given at the discretion (PRN) (as evaluated by a qualified healthcare professional).
q. Ligand 2 mg (0.5 mL) administered by intravitreal injection once every 4 weeks for the first 28 weeks, followed by 2 mg (0.05 mL) via intravitreal injection once every 8 weeks ).
r. For the first 28 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection less frequently (as assessed by a qualified healthcare professional).
s. For the first 28 weeks, once every 4 weeks, 2 mg (0.5 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcome (physician or other) 2 mg (0.05 mL) via intravitreal injection given at the discretion (PRN) (as evaluated by a qualified healthcare professional).
t. As a single initial dose, 2 mg (0.05 mL) of ligand administered by intravitreal injection, followed by visual and / or anatomical outcomes (assessed by physician or other qualified healthcare professional) As you do) Additional doses administered at the option (PRN).

Variations on the above dosing regimen are understood by those skilled in the art and are also within the scope of the present invention. For example, the amount of ligand and / or formulation volume administered to a patient varies based on the patient's characteristics, disease severity, and other diagnostic assessments (by a physician or other qualified healthcare professional). May be done.

If this method or ligand is to treat or reduce the risk of eye condition or disease, some methods improve Best Correct Visual Acuity in humans.

Best Corrected Visual Acuity:
Visual function of the eye may be assessed using the ETDRS protocol (Early Treatment Diabetic Retinopathy Study Group) at 4 meters. Sight examiners are usually certified to ensure a consistent measurement of BCVA.

This method, in one example method, prevents visual loss of 15 characters or more against the ETDRS chart at 52 weeks compared to baseline. Additional or alternative, this method results in one or more of the following: (a) change of character score from baseline to 52 weeks for ETDRS charts; (b) 52 weeks from baseline Earn more than 15 characters against the ETDRS chart up to; (c) change in total NEI VFQ-25 score from baseline to week 52; and (d) change in CNV area from baseline to week 52 (eg) , If this state is CNV).

In one embodiment, the ligand is a VEGF antagonist and comprises one or more VEGF receptor-based chimeric molecules (also referred to herein as "VEGF-trap" or "VEGFT"). An exemplary VEGF antagonist that can be used in the context of the present invention is a large amount containing two or more VEGF receptor-based chimeric molecules referred to herein as "VEGFR1R2-FcAC1 (a)" or "aflibercept". The body's VEGF-binding protein.

Various ligand or antibody routes of administration have been considered for use in the methods of the invention, including topical or intraocular administration (eg, intravitreal administration).

The method of the present invention optionally comprises the step of continuously administering to the patient multiple doses of the ligand, eg, a VEGF antagonist. As used herein, "continuously administered step" means that each dose of ligand is different at different times, eg, at predetermined intervals (eg, hours, days, weeks, or months). It means that it is administered to the patient on a daily basis. The present invention comprises the step of sequentially administering to a patient a single initial dose of a ligand followed by one or more secondary doses of a ligand followed by one or more tertiary doses of a ligand. Including.

The terms "primary dose," "secondary dose," and "tertiary dose" refer to the temporal sequence of ligand administration. Thus, the "first dose" is the dose given at the beginning of the treatment regimen (also called the "baseline dose"); the "secondary dose" is the dose given after the first dose; and A "tertiary dose" is a dose given after a secondary dose. The primary, secondary and tertiary doses all contain the same amount of ligand, but generally differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of ligand contained in the primary, secondary and / or tertiary doses varies from each other during the course of treatment (eg, optionally adjusted up or down). ).

(Example 6)
PD-L1 and PD-1
Application of PD-L1 variations in the present invention Human PD-L1 is also CD274, B7-H, B7H1, B7-H1, B7 homolog 1, MGC142294, MGC142296, PDCD1L1, PDCD1LG1, PDCD1 ligand 1, PDL1, programmed cell death 1 Also known as ligand 1 and programmed death ligand 1; and has Uniprot number Q9NZQ7 and NCBI gene ID number 29126. PD-L1 is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on human chromosome 9. PD-L1 expression is, for example, by viruses (eg, including HIV, HBV, HCV and HTLV, among others), by bacteria (eg, including Helicobacter pylori, among others), and by parasites (eg, including Schistosoma mansoni). , Is involved in avoiding the immune response involved in chronic infections. CD274 / PD-L1 expression is also associated with suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but tumor immunological clearance is rare. Part of this deficiency results from immunosuppression due to the tumor's microenvironment. PD-L1 expression on many tumors is a component of this inhibitory environment and can function in response to other immunosuppressive signals. PD-L1 expression includes breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary adenocarcinoma, gastric cancer, glioma, thyroid, thoracic epithelium, head and neck cancer In-situ shown for a wide variety of solid tumors (Brown JA et al., 2003. J. Immunol. 170: 1257-66; Dong H et al. 2002. Nat. Med. 8: 793-800. Hamanishi J, et al. 2007.Proc.Natl.Acad.Sci.USA 104: 3360-65; Strome SE et al. 2003.Cancer Res.63: 65 01-5; Inman BA et al. 2007.Cancer 109: 1499 ~ 505; Konishi J et al. 2004. Clin.Cancer Res.10: 5094 ~ 100; Nakanishi J et al. 2007. Cancer Immunol.Immunother.56: 1173-82; Nomi T et al. 2007. Clin.Cancer Res .13: 2151-57; Thompson RH et al. 2004.Proc.Natl.Acad.Sci.USA 101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. 2006 .Acta Histochem. 108: 19-24). In addition, PD-1 expression is upregulated in tumor-invasive lymphocytes, which can also contribute to tumor immunosuppression (Blank C et al., 2003. J. Immunol. 171: 4574-81). In ovarian cancer, PD-L1 expression is inversely correlated with intraepithelial (but not with stromal and invasive CD8T cells), which causes PD-L1 to migrate CD8T cells intratumorally. It is suggested to inhibit (Hamanishi J et al. 2007. Proc. Natl. Acad. Sci. USA 104: 3360-65). Translation of PD-L1 mRNA is enhanced by loss of PTEN and subsequent activation of Akt (a common event of tumorigenesis) (Parsa AT et al. 2007. Nat. Med. 13: 84-88). Most importantly, PD-L1 expression is desirable for kidney, ovarian, bladder, breast, gastric and pancreatic cancers, according to studies related to PD-L1 expression on tumors for disease outcome. It is shown to be strongly correlated with no prognosis (Hamanishi J et al. 2007. Proc. Natl. Acad. Sci. USA 104: 3360-65; Inman BA et al. 2007. Cancer 109: 1499-505; Konishi J et al. 2004. Clin.Cancer Res.10: 5094-100; Nakanishi J et al. 2007.Cancer Immunol.Immunother.56: 1173-82; Nomi T et al. 2007.Clin.Cancer Res.13: 2151 ~ 57 pages; Thompson RH et al. 2004. Proc. Natl. Acad. Sci. USA 101: 17174 ~ 79 pages; Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. 2006. Acta Histochem. 108: 19-24). In addition, these studies suggest that higher levels of PD-L1 expression on tumors may promote the progression of tumor stages and entry into deeper tissue structures.

The PD-1 pathway can also play a role in blood malignancies. PD-L1 is expressed in multiple myeloma cells but not in normal plasma cells (Liu J et al. 2007. Blood 110: 296-304). PD-L1 is expressed on several primary T-cell lymphomas, especially anaplastic large-cell T-lymphomas (Brown JA et al., 2003. J. Immunol. 170: 1257-66). PD-1 is highly expressed on T cells of vascular immunoprecursor lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al. 2006. Am. J.Surg.Pathol.30: 802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, PD-1 is expressed by T cells associated with lymphocytes and / or histocytic (L & H) cells. Microarray analysis using PD-1 ligation-induced gene retrieval suggests that tumor-related T cells respond to PD-1 signals in in situ in Hodgkin lymphoma (Chemnitz JM et al. 2007). .Blood 110: 3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T-cell leukemia and lymphoma (Shimauchi T et al. 2007. Int.J. Cancer 121: pp. 2585-90). These tumor cells are poorly responsive to TCR signals.

Studies in animal models have shown that PD-L1 on tumors inhibits T cell activation and tumor cell lysis and, in some cases, results in increased tumor-specific T cell death ( Dong H et al. 2002. Nat. Med. 8: 793-800; Hirono F et al. 2005. Cancer Res. 65: 1089-96). Tumor-related APCs can also utilize the PD-1: PD-L pathway to regulate antitumor T cell responses. PD-L1 expression in a population of tumor-related bone marrow DCs is upregulated by tumor environmental factors (Curiel T J et al., 2003. Nat. Med. 9: 562-67). Plasmacytoid dendritic cells (DCs) in B16 melanoma tumor-inflow region lymph nodes express IDO, which strongly activates the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cell contact with IDO-expressing DCs (Sharma MD et al. 2007. J. Clin. Invest. 117: 2570-82).

US8507663 discusses specific inhibition of CD274 / PD-L1 gene expression. Also also referred to are WO2011066389, WO2010036959, WO2010077634, WO03099196, WO2013181634, WO2007005874, US20090317368 and US7635757.

The inventor has made natural human variations in PD-L1 and identified the following variations of interest for application to the present invention.

This table reads as follows: For example, for rs12551333, the following means Asp49His, that is, histidine at position 49 encoded by the SNP rs12551333.

Citations
1. Asia Pac J Clin Oncol. 2014 Jun; 10 (2): e1-6. Doi: 10.1111 / ajco.12037. Epub 2012 Nov 21; "Association between single nucleotide polymorphism of PD-L1 gene and non-small cell lung cancer susceptibility in a Chinese population "; Chen Y et al.
The authors published the following:-
Purpose:
To assess the correlation between polymorphisms in the PD-L1 gene and susceptibility to non-small cell lung cancer (NSCLC) in the Chinese population.
Method:
A total of 293 Chinese patients with NSCLC and 293 age- and gender-matched controls from the same ethnic group were enrolled in this study. The A / C gene polymorphism at position 8923 in intron 4 of the PD-L1 gene was typed using the polymerase chain reaction restriction fragment length gene polymorphism (PCR-RFLP). Interactions between A / C genotype, allele frequency and NSCLC susceptibility were analyzed.
result:
The frequency of A / C genotypes was significantly different between NSCLC patients and controls. The frequency of AC and CC was higher in NSCLC patients than in controls (16.4 vs 8.9% and 1.0 vs 0.3%, respectively). The frequency of C alleles was higher in NSCLC patients than in controls (9.2 vs 4.8%). Significant differences in the frequency of A and C alleles were noted between the two groups [x (2) = 8.864, P = 0.003]. A greater risk of NSCLC was found in individuals carrying the C allele than in individuals carrying the A allele (OR = 2.203; 95% CI 1.262-3.242). In both mild smokers (≤20 pack-years) and heavy smokers (heavy smokers) (> 20 pack-years), individuals carrying the C allele are more likely than individuals carrying the A allele. Also had a high risk of NSCLC (mild smokers OR = 1.847, 95% CI 1.001-3.409; severe smokers OR = 3.252, 95% CI 1.196-8.845, respectively).
Conclusion:
The A / C gene polymorphism at position 8923 of the PD-L1 gene is associated with NSCLC susceptibility. PD-L1 polymorphisms play a role in NSCLC, especially in patients with the C allele.
2.Eur J Endocrinol. June 2008; 158 (6): pp. 817-22. Doi: 10.1530 / EJE-07-0649. Epub March 5, 2008.
"Association of an A / C single nucleotide polymorphism in programmed cell death-ligand 1 gene with Graves' disease in Japanese patients"; Hayashi M et al.
The authors have published:-
Objective: Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) inhibit T cell proliferation and activation. This inhibition downregulates the immune response. We studied the association between PD-L1 polymorphisms and Graves' disease (GD).
Design: We investigated the association between A / C gene polymorphism at position 8923 and GD in PD-L1 intron 4.
Patients: This study included 327 GD patients and 192 controls, of which 252 GD patients were followed up for 5-10 years or longer.
Measurements: The A / C gene polymorphism at position 8923 of PD-L1 intron 4 was typed using the PCR-restricted fragment length gene polymorphism method.
RESULTS: A / C genotype frequency was significantly different between GD patients and controls. The frequency of A / C and C / C was higher in GD patients than in controls. A / A frequency was lower in GD patients than in controls. The frequency of C alleles was higher in GD patients than in controls. A total of 252 GD patients were followed for more than 5-10 years; 200 discontinued antithyroid agents (ATD), while 52 continued to take ATD. Of these 200 cases, 176 continued to be in remission and 24 had recurrent hyperthyroidism. Significant differences in the duration of positive TBII, positive thyroid-stimulating antibodies, and ATD treatment were noted between patients in remission and those who had relapsed. A significant difference in the frequency of the A and C alleles was noted between the two. The frequency of C alleles was higher in GD patients who did not reach remission than in patients who did.
CONCLUSIONS: The A / C gene polymorphism at position 8923 in PD-L1 is associated with GD. PD-L1 polymorphisms play a role in the development of GD. GD patients with the C allele at position 8923 within the PD-L1 gene had difficulty reaching remission.

3. Rheumatology (Oxford). October 2011; 50 (10): pp. 1809-13. Doi: 10.1093 / rheumatology / ker211. Epub July 26, 2011; "Effects of genetic polymorphisms of programmed cell death 1 and its ligands on the development of ankylosing spondylitis "; Huang C et al.
Human subjects with the PD-1 GG genotype were at significantly greater risk for rigid spondylitis (AS) than subjects with the AA genotype. Subjects with the PD-L2 CT genotype had a lower risk for AS than subjects with the CC genotype. The combined genotype of PD-1 G-536A, PD-L1 A8923C and PD-L2 C47103T also appears to be associated with the development of AS.
Conclusion. This result suggests that PD-1 G-536A, PD-L1 A8923C and PD-L2 C47103T gene polymorphisms are associated with the presence of AS.

4.J Clin Endocrinol Metab. December 2009; 94 (12): 5139-45. Doi: 10.1210 / jc.2009-1404. Epub October 22, 2009; "Programmed death ligand 1 (PD-L1)" ) Gene variants contribute to autoimmune Addison's disease and Graves' disease susceptibility "; Mitchell AL et al.
The authors have published:-
Status:
Despite considerable consideration, the substantial amount of gene susceptibility to autoimmune diseases remains unknown. In recent years, single nucleotide polymorphisms (SNPs) in the programmed death ligand 1 (PD-L1) gene have been associated with Graves' disease (GD) in a cohort of Japanese patients. Our aim is to determine if the variant in PD-L1 is also associated with autoimmune Addison's disease (AAD), and to reproduce the previous association in patients with UK-derived GD. there were.
Design and patient:
We analyzed 8 SNPs in PD-L1 in a UK cohort of 315 AAD subjects and 316 healthy controls. We then reproduced our experiment in a cohort of 342 Norwegian AAD cases and 379 controls, as well as 496 UK GD subjects.
result:
Three of the eight SNPs studied (part of the haplotype block in the PD-L1 gene) showed modest association with both AAD and GD in the UK cohort, with the greatest evidence at marker RS1411262 [UK]. AAD odds ratio 1.33 (5-95% confidence interval 1.02-1.73), P (genotype) = 0.028; GD odds ratio 1.36 (5-95% confidence interval 1.07-1.72), P (genotype) = 0.033]. The association of genotypes with the same three markers was confirmed in the Norwegian AAD cohort [P (genotype) = 0.011 to 0.020]. The recessive effect of the most relevant allele was observed in both the AAD and GD cohorts.
Conclusion:
We confirm the role of PD-L1 variants in GD sensitivity and extend these findings to demonstrate an association in a cohort of two Scandinavian patients with AAD. PD-L1 is involved in increasing the number of known susceptibility loci that exert a modest effect on these autoimmune disorders.

5. J Clin Immunol. November 2007; 27 (6): pp. 563-7. Epub June 28, 2007; "Polymorphisms of genes for programmed cell death 1 ligands in patients with rheumatoid arthritis"; Wang SC et al.
The authors have published:-
To investigate the role of ligands in programmed cell death 1 (PD-L) in the pathogenicity of rheumatoid arthritis (RA), this study examined 129 patients with RA and 125 unrelated healthy controls. Registered in. PD-L1 and PD-L2 gene polymorphisms were determined by the polymerase chain reaction (PCR) / direct sequencing or PCR / reaction fragment length gene polymorphisms. The genotype distribution of PD-L1 6777C / G was not significantly different between patients with RA and healthy controls. There was also no significant difference in the allelic frequency of PD-L1 6777 C / G gene polymorphisms between patients with RA and controls. Similar findings could be found in the phenotype and allelic frequency of PD-L2 47103 C / T and 47139 T / C gene polymorphisms between patients with RA and controls. Patients with PD-L1 6777G had a higher prevalence of rheumatoid nodules than patients without PD-L1 6777G (p = 0.005, OR = 4.0, 95% CI = 1.5-10.9). In contrast, PD-L2 47103 C / T and 47139 T / C gene polymorphisms were not associated with the appearance of rheumatoid nodules. This study demonstrated that PD-L1 and PD-L2 polymorphisms were not associated with RA susceptibility in Taiwan. PD-L1 6777G was associated with the prevalence of rheumatoid nodules.

6. Hum Genet. June 2013; 132 (6): 641-8 pages. Doi: 10.1007 / s00439-013-1275-6. Epub February 21, 2013; "A miR-570 binding site polymorphism in the B7-H1 gene is associated with the risk of gastric adenocarcinoma "; Wang W et al.
Since overexpression of the B7-H1 protein has been reported to be closely associated with the progression of gastric cancer disease, the authors found that the 3'untranslated region (3') of B7-H1 at risk of developing gastric cancer. -UTR) examined the potential role of miRSNP. In this associated study of 205 patients with gastric adenocarcinoma and 393 non-cancerous controls, they found that the common C> G genotypic polymorphism (rs4143815) genotype distribution was between cases and controls. We found that there was a significant difference in [P = 1.32 × 10 (-8)]. Compared to CC homozygotes, GG homozygotes and G allelic carriers are at 3.73 times [P = 2.98 × 10 (-8)] and 1.85 times (P = 0.002) risk of gastric adenocarcinoma, respectively. Showed an increase. Stratified analysis showed that variant genotype has a strong association with the clinicopathological features of gastric cancer, including grade of differentiation, depth of tumor infiltration and stage of tumor nodule metastasis (TNM) (P < 0.001). The luciferase reporter assay showed that this SNP may be responsible for aberrant B7-H1 protein expression in gastric cancer by disrupting the interaction between miR-570 and B7-H1 mRNA. These results are consistent with our hypothesis, indicating that genetic polymorphisms that affect B7-H1 expression alter cancer susceptibility.

7. Clinical cancer research: an official journal of the American Association for Cancer Research / 15 (3): pp. 971-9; Pub Date: February 1, 2009; "Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma "; Qiang G et al.
The authors have published:-
Objective Abnormal expression of programmed cell death 1 ligands 1 and 2 (PD-Ls) on tumor cells weakens anti-tumor immunity, which results in tumor immunity evasion. In this study, we examined the expression of PD-Ls in human hepatocellular carcinoma (HCC) to define their prognostic significance after curative surgery.
Experimental Design Using immunohistochemistry, PD-L expression, as well as granzyme B + cytotoxicity and FoxP3 + regulatory T cell infiltration, were performed on tissue microarrays containing 240 randomly selected HCC patients who underwent surgery. investigated. The results were further validated in an independent cohort of 125 HCC patients. PD-L expression on HCC cell lines was detected by Western blot assay.
Results Patients with higher PD-L1 expression had a significantly worse prognosis than patients with lower expression. Patients with higher PD-L2 expression also had poor survival, but the difference in recurrence was not statistically significant. Multivariate analysis identified tumor expression of PD-L1 as an independent predictor of postoperative recurrence. No correlation was found between PD-L expression and granzyme B + lymphocyte infiltration, but a significant positive correlation was detected between PD-L expression and FoxP3 + lymphocyte infiltration. In addition, tumor-invasive cytotoxic and regulatory T cells were also independent prognostic factors for both survival and recurrence. The prognostic value of PD-L1 expression was validated on an independent dataset.
CONCLUSIONS: Our data suggest that PD-L1 status may be a new predictor of recurrence in patients with HCC, developing new therapies that target the PD-L1 / PD-1 pathway for this deadly malignancies. For the first time, it is suggested to provide a rationale for doing so.

Using this analysis, the present inventor is useful for addressing PD-L1-mediated diseases and conditions such as cancer, autoimmune or inflammatory diseases and conditions (described in more detail below). The following aspects of the invention have been devised. Therefore, the present invention provides the following aspects.

In one example, the invention is a method of treating or reducing the risk of a PD-L1-mediated disease or condition in a human in need thereof and is specific to the human PD-L1 protein relative to said human. Provided are methods that include the step of administering a ligand that binds specifically (eg, an antibody or antibody fragment). The present invention also provides a corresponding ligand. In certain embodiments, PD-L1 comprises, for example, one or more of said SNPs (eg, RS1411262 and / or rs4143815; or 8923C and / or 395C), including variations shown in Table 21. It is encoded by the PD-L1 nucleotide sequence. In a preferred embodiment, this variation is 8923C.

The present invention provides anti-PD-L1 ligands; as well as PD-L1 binding or targeting ligands as described herein. This ligand has various usefulness. Some of these ligands, for example, in specific binding assays, in the affinity purification of PD-L1 (particularly human PD-L1 or its ligands) to genotype or phenotype humans, PD -Useful in screening assays to identify other antagonists with L1 activity. Some ligands of the present invention are useful for inhibiting PD-L1-mediated activity.

Anti-PD-L1 ligands (eg, antibodies and antisense RNA) have been developed based on the targeting and neutralization of so-called "wild" human PD-L1, which is a common type. Although such treatments are useful for human patients carrying this type of human PD-L1, the inventor, although a rare type of PD-L1 in the human population, is also naturally present. It was considered useful for investigating the possibility of type targeting. In this way, the inventor is a useful target for human treatment, prevention and diagnosis (protein or) that is mediated by PD-L1 activity or appropriate for diseases and conditions associated therewith. We have reached an insight into the natural appearance and distribution of rare human PD-L1 types that can function (at the nucleic acid level). This provides tailor-made treatment, prevention and diagnosis, especially in humans who lack the common PD-L1 gene or protein.

Those skilled in the art are aware that SNPs or other changes that translate into amino acid variations can result in variability in the activity and / or higher-order structure of the human target to be targeted. This has led to great interest in personalized medicine that uses knowledge of genotyping and protein and nucleotide variability to more effectively tailor-make patient care and diagnosis. Therefore, the present invention provides tailor-made medicines and tests specifically directed to the rare PD-L1 polymorphic variant. Such forms or "alleles" (at the nucleotide level) contain one or more changes at the nucleotide and amino acid levels from the corresponding common types of nucleotide and amino acid sequences, ie, protein targets in humans. There are one or more non-synonymous changes (also known as "missenses") at the nucleotide level that translate into one or more corresponding changes in.

Moreover, the inventor surprisingly found that rare natural forms are present in humans much less frequently than common forms, but are nevertheless shown in large and ethnically diverse human populations. We have noticed that there are usually many human examples per ethnic group shown. Thus, by targeting such rare types, the inventor provides effective treatment, prevention or diagnosis across many human ethnic groups, thereby expanding the usefulness of the invention. I noticed. Specifically, human PD-L1 variations are associated with increased frequency or risk of PD-L1-mediated diseases or conditions such as cancer. The inventor analyzed such variations and devised a collection of variants in Table 21. Therefore, in this regard, the present invention provides various aspects, as shown below in this example.

The present inventor relates to guiding the selection of anti-TOI (PD-L1) ligands for administration to human patients for the treatment and / or prevention of PD-L1-mediated or related diseases or conditions. Understood that there are significant industrial and medical applications. In this way, the patient is administered tailor-made drugs and ligands for their needs (determined by the patient's genetic or phenotyping). Together with this, the present invention provides patient genotyping and / or phenotyping in connection with such treatments, which allows for proper adaptation of the drug to the patient. This increases changes in medical efficacy, reduces the probability of subordinate treatments with drugs or ligands that are not suitable for the patient (eg, poor efficacy and / or side effects), and misprescribes and disposes of the drug. Is avoided.

To develop this idea, in a non-limiting embodiment, the inventor decides to determine the setting of the human PD-L1 variant based on the following criteria, which are the criteria realized by the inventor. And provides useful medicines and diagnostics for tailor-made needs in the human population. In certain embodiments, the inventor has selected a variant having at least three of the following four criteria:-
Naturally occurring human TOI variations with a cumulative human allele frequency of 49 or 35% or less;
Naturally occurring human TOI variations with a total human genotype frequency of about 50% or less;
Naturally occurring human TOI variations found in many different human ethnic populations [using the standard classification of the 1000 Genomes Project; see Table 2 below]; and many such different ethnic groups. Naturally occurring human TOI variations found in many individuals distributed across populations.

In one example, the ligand of the invention comprises an anti-human PD-L1 binding site, where the binding site is a human or humanized binding site, eg, this binding site is human or humanized. Contains or consists of an antibody variable domain or multiple variable domains (eg, human VH / VL binding sites). Additionally or optionally, this ligand comprises one or more human antibody constant regions [eg, human antibodies CH1, CH2, CH3 (or all of them) or Fc]. In one example, this ligand is an antibody containing a human or humanized variable region and human constant region (eg, carrying one or more mutations to enhance or weaken Fc function in a human patient). ..

In one example, the invention provides a method of targeting PD-L1 in humans, the method comprising a human PD that comprises a PD-L1 variation as described herein for said human. -Contains the step of administering a ligand (eg, an antibody or antibody fragment) that specifically binds to the human PD-L1 protein encoded by the L1 nucleotide. In some cases, this human has or is at risk for a PD-L1-mediated disease or condition. In some examples, this method treats or reduces the risk of PD-L1-mediated disease or condition in humans.

In certain embodiments, (i) the antibody or fragment comprises a VH domain derived from recombination of a human VH segment, a human D gene segment and a human JH segment, which human VH segment comprises Framework 1 of SEQ ID NO: 40. And here the human comprises a VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human comprises a VH domain comprising Framework 1 of SEQ ID NO: 40. Express.

Preferably, additionally or alternatively, in certain embodiments, the anti-PDL-1 ligand (eg, antibody or fragment) is a gamma-1 constant region (eg, IGHG1 * 01) as described herein. Includes stationary region).

Additional or alternative, in certain embodiments, (i) this antibody or fragment is a human gamma-4 fold containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Containing a chain constant region, wherein the human comprises an IGHG4 * 01 human heavy chain constant region gene segment, or this human is found in Leu at position 189 or SEQ ID NO: 73 found in SEQ ID NO: 73. It expresses an antibody containing a human gamma-4 heavy chain constant region containing Arg at position 289.

In certain embodiments, the anti-PD-L1 ligand, antibody or fragment of the invention comprises an Fc region, where the Fc region is 234D, 234E, 234N, 234Q as numbered by the EU index shown in Kabat. , 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q , 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A, 263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 3 From 31N, 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W Contains at least one unnatural amino acid residue selected from the group. Optionally, this Fc region may contain additional and / or alternative unnatural amino acid residues known to those of skill in the art (eg, US Pat. No. 5,624,821; 6,277,375; 6,737,056). See PCT Patent International Publication Nos. WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752 and WO05 / 040217).

Ligands, antibodies or fragments according to the invention (eg, anti-PD-L1 antibodies or fragments) can be any disease or condition disclosed in, for example, US8507663, WO2011066389, WO2010036959, WO2010077634, WO03099196, WO2013181634, WO2007005874, US20090317368 or US7635757. The diseases and conditions of the disclosure are to treat, prevent or reduce (or risk) the risk of which is incorporated herein by reference for potential inclusion in one or more aspects of the specification. To treat or prevent or reduce). Guidance for obtaining and testing antibodies can also be found in these publications. Anti-PD-L1 ligands, antibodies or fragments suitable for use in the present invention are disclosed in these publications, the disclosure of which (including its sequences) is one or more aspects of this specification. Incorporated herein by reference for potential inclusion in.

Further included by the present invention is the use of anti-PD-L1 ligands, antibodies or fragments in the manufacture of medicaments for use in reducing or inhibiting PD-L1-mediated diseases or disorders in humans. PD-L1-mediated or related disorders treated with the ligands, antibodies or fragments of the invention include, for example:

Therefore, the ligands, antibodies or fragments of the present invention are useful as therapeutic agents in the treatment of conditions involved in PD-L1 expression and / or activity. One embodiment is a method of treatment comprising administering, among other things, an effective amount of a ligand, antibody or fragment of the invention to a patient in need thereof, wherein PD-L1 activation is functional. The consequences are reduced. Another embodiment is, among other things, a method of treatment, (i) identifying a patient exhibiting PD-L1 expression or activity, and (ii) an effective amount of a ligand of the invention for this patient. A method comprising the step of administering an antibody or fragment, wherein the functional consequences of PD-L1 activation are alleviated. An effective amount according to the invention is the severity of at least one symptom of a particular disease or disorder being treated by modulating (eg, reducing) the functional consequences of PD-L1 activation. An amount that modulates (eg, reduces or reduces) but does not necessarily cure the disease or disorder. Accordingly, one embodiment of the invention is a method of treating or reducing the severity of at least one symptom of any of the disorders referred to herein, for a patient in need thereof. An effective amount of one or more ligands, antibodies or fragments of the invention is administered alone or in a therapeutic regimen in combination with another suitable medicament known in the art or described herein. A method that involves steps, resulting in a reduction in the severity of at least one symptom of any disorder. Another embodiment of the invention is, among other things, a method of antagonizing at least one effect of PD-L1 by contacting or contacting it with one or more effective amounts of one or more ligands, antibodies or fragments of the invention. A method comprising the step of administering, which results in antagonism of at least one effect of PD-L1, eg, the ability to promote or maintain a cancer, autoimmune state or inflammatory state.

Tailor Made of Anti-PD-L1 Ligand As outlined herein (eg, in the context of PCSK9 in Example 1), the present invention is an ethnic population in which the TOI type of variant is found to meet the selection criteria of the present invention. It further includes the possibility of tailor-making human treatment by selecting antibody-based ligands with variable and / or constant domains based on the gene segments found in many humans. This also applies mutatis mutandis when the TOI is human PD-L1, as in this example. Thus, all disclosures herein relating to tailor-making variable and / or constant domains apply to this embodiment with respect to PD-L1 and can be combined with use in one or more embodiments herein. Is.

As described in Example 1, in one example, a ligand comprising an antibody VH domain from a recombination of a human IGHV gene segment comprising a nucleotide selected at a position in HCDR1 or FW3 that is variable in human is provided ( That is, here SNPs are present in humans).

Further information is provided in Table 4, which also shows variations at these locations and variant distributions across the 1000 Genomes Project database associated with many human populations.

In other embodiments, as described in more detail above, the invention is based on an alternative human VH gene segment, or V _K , V _λ or constant region gene segment, and the genotype of the human recipient and /. Alternatively, provide a phenotypically tailored ligand (see further Table 9 for representative variants).

In a further example, therefore there is:-
(i) The ligand comprises a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a recombinantly derived VH domain of the human JH segment, and the human VH segment is the framework 1 of SEQ ID NO: 40. The human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or the human expresses a VH domain comprising Framework 1 of SEQ ID NO: 40.
(ii) The ligand comprises a human VH segment IGHV3-7 * 01, a human D gene segment and a recombination-derived VH domain of a human JH segment, and the human contains the IGHV3-7 * 01 VH gene segment or is human. Expresses the VH domain derived from the recombination of the human VH segment IGHV3-7 * 01, the human D gene segment and the human JH segment.
(iii) The ligand comprises the human Vκ segment IGKV1-12 * 01 and the recombinantly derived Vκ domain of the human Jκ segment, and the human comprises the IGKV1-12 * 01 Vκ gene segment, or the human contains the human Vκ segment. It expresses the Vκ domain derived from recombination of IGKV1-12 * 01 and human Jκ segment.
(iv) The ligand comprises the Vκ domain from the recombination of the human Vκ segment and the human Jκ segment, and the human Vκ segment encodes (i) CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, said human. Does it contain the Vκ gene segment encoding CDR3 containing Pro at position 7 shown in SEQ ID NO: 36, or does human express the Vκ domain containing CDR3 containing Pro at position 7 shown in SEQ ID NO: 36? Or (ii) the Vκ gene segment encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38 and the human encoding FW3 containing Ser at position 15 set forth in SEQ ID NO: 38, or Humans express the Vκ domain containing FW3, which contains Ser at position 15 shown in SEQ ID NO: 38.
(v) The ligand contains a human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human is (i) IGHG1 * 01 human. An antibody containing a heavy chain constant region gene segment or containing a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4 is expressed. ..
(vi) The ligands are Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 and Contains a human gamma-2 heavy chain constant region containing an amino acid selected from the group consisting of Ala at position 257 shown in SEQ ID NO: 6, and whether the human contains (i) IGHG2 * 01 human heavy chain constant region gene segment. , Or human, Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and position 161 shown in SEQ ID NO: 6. Val or the antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown in SEQ ID NO: 6. In one example, this constant region is Fc-enhanced, eg, ADCC or CDC-enhanced. Those skilled in the art are known for techniques for enhancing ADCC or CDC (eg, C region mutations are known).
(vii) Here, the ligand comprises a human kappa chain constant region comprising Val at position 84 set forth in SEQ ID NO: 16 or Cys at position 87 set forth in SEQ ID NO: 16, where the human is (vii). i) IGKC1 * 01 Human kappa chain constant region containing the gene segment, or this human has a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16. Express the antibody containing.
(viii) the ligand comprises a human IGLC1 * 01 lambda chain constant region, said human containing (i) a human IGLC1 * 01 lambda chain constant region gene segment, or a human containing a human IGLC1 * 01 lambda chain constant region. Expresses an antibody containing.
(ix) The ligand comprises a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human. An antibody comprising a heavy chain constant region gene segment or containing a human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73 is expressed. ..
The (x) ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3 * 01) constant region gene segment, wherein the human is (i) said the first constant region gene. An antibody comprising a segment (eg, IGHG3 * 01) or containing a human gamma-3 heavy chain constant region encoded by the first human IGHG3 (eg, IGHG3 * 01) constant region gene segment is expressed. ..
(xi) The ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human epsilon heavy chain constant region encoded by the first constant region gene segment.
(xii) The ligand comprises the human mu heavy chain constant region encoded by the first human mu heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expresses an antibody comprising the human mu heavy chain constant region encoded by the first constant region gene segment.
(xiii) The ligand comprises the human alpha heavy chain constant region encoded by the first human alpha heavy chain constant region gene segment, and the human comprises (i) said said first constant region gene segment. Humans express antibodies that include the human alpha heavy chain constant region encoded by the first constant region gene segment.
The (xiv) ligand comprises the human delta heavy chain constant region encoded by the first human delta heavy chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human , Expressing an antibody comprising the human delta heavy chain constant region encoded by the first constant region gene segment.
The (xv) ligand comprises the human kappa light chain constant region encoded by the first human kappa light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human kappa light chain constant region encoded by the first constant region gene segment.
The (xvi) ligand comprises the human lambda light chain constant region encoded by the first human lambda light chain constant region gene segment, and the human comprises (i) the first constant region gene segment, or the human is , Expressing an antibody comprising the human lambda light chain constant region encoded by the first constant region gene segment.

In general, in one example, this ligand comprises a heavy chain constant region that is Fc-enhanced, eg, ADCC or CDC-enhanced. Those skilled in the art are known for techniques for enhancing ADCC or CDC (eg, known are C region mutations; defucosylation to enhance ADCC). This applies to any of the preceding Examples 1 herein.

Therefore, PD-L1 ligands may be advantageous for ligands containing the gamma-1 or gamma-2 constant region [eg, as in embodiments (i)-(xvi) above]. For example, this ligand comprises said gamma-1 constant region, eg, ADCC or CDC enhanced constant region satisfying embodiment (v).

In one example, this ligand (eg, an antibody) is conjugated to a cytokine selected from the group consisting of IL-2, IL-12, IL-15 or IL-21. In one example, this cytokine is IL-2.

In one example, for example, if the condition is a viral infection, the ligand (eg, antibody) comprises the gamma-4 constant region as defined herein, and optionally, Fc is IgG4. For example, IgG4 (S228P) was inactivated.

Determination of specific binding of the ligand of the present invention to the PD-L1 variant
Specific binding of the ligand of the present invention to the PD-L1 variant may be carried out using the SPR method described in Example 1.

As an example, therefore, the present invention provides the following aspects:-
1. A method of treating or reducing the risk of PD-L1 -mediated disease or condition in humans, which method is selected from the variations listed in Table 21 for said humans. It comprises the step of administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by a PD-L1 nucleotide sequence comprising a variation.

For example, in humans, a method of treating or reducing the risk of PD-L1 mediated disease or condition is provided, the method of which is selected from the variations listed in Table 21 for said human. Includes the step of administering an anti-PD-L1 ligand that specifically binds to human PD-L1 (eg, an anti-PD-L1 trap, antibody or antibody fragment) expressed by a PD-L1 nucleotide sequence that comprises the variation. Here, the human comprises a PD-L1 nucleotide sequence comprising the selected variation.

For example, a method of treating or reducing the risk of cancer in humans is provided, the method comprising a variation selected from the variations listed in Table 21 for said PD-L1 nucleotide. It comprises the step of administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by the sequence, wherein said human. Contains the PD-L1 nucleotide sequence containing the variations.

For example, a method of treating or reducing the risk of an autoimmune disease or condition in humans is provided, the method comprising a variation selected from the variations listed in Table 21 for said human PD. It comprises administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by the -L1 nucleotide sequence, wherein said human , Includes PD-L1 nucleotide sequence containing the selected variation.

For example, a method of treating or reducing the risk of an inflammatory disease or condition in a human is provided, the method comprising a variation selected from the variations listed in Table 21 for said human. It comprises the step of administering an anti-PD-L1 ligand (eg, an anti-PD-L1 trap, antibody or antibody fragment) that specifically binds to human PD-L1 expressed by the PD-L1 nucleotide sequence, wherein said human. Contains the PD-L1 nucleotide sequence containing the selected variation.

Here, specific binding is tested using a conventional method known to those of skill in the art, such as ELISA or SPR (eg, using methods as described herein) and sample PD-L1. May be done. For example, PD-L1 is provided by said human or another human-derived sample, such as a serum or blood sample.

In one example, this ligand comprises an anti-PD-L1 binding site that specifically binds to human PD-L1. Optionally, this binding site comprises one or more antibody variable domains, or one or more human PD-L1 receptor domains. In one example, the specific bond is a bond with a Kd of 1 mm or less (ie, 1 mM or stronger), such as a Kd of 1 nM or less; or 100 pM or less; or 10 pM or less. Specific binding can be determined, for example, by assessing the binding of the ligand to PD-L1 in the human or another human-derived sample containing the PD-L1. In some examples, this sample comprises said human serum, blood, feces, tissue, cells, urine and / or saliva.

In one example, this ligand is wher MEDI4736 (Medimmune), Davarumab, RG-7446 (Genentech, Roche, Chugai, MPDL3280A), MPDL3280A (Genentech), STI-A1014 (Sorrento Therapeutics Inc), MSB- 0010718C (Merck Serono); Selected from the group consisting of MDX1105 (Bristol-Myers Squibb) and BMS-936559 (Bristol-Myers Squibb). Optionally, this ligand is administered in combination with an anti-CTLA-4 ligand (eg, antibody or fragment), eg, tremelimumab. For example, MEDI4736 or davarumab is administered in combination with an anti-CTLA-4 ligand (eg, antibody or fragment), such as tremelimumab.

In one example, this ligand is an anti-PD-L1 antibody or antibody fragment, such as davarumab.

In one example, this ligand is an NCE (the so-called New Chemical Entity).

In some cases, the human has been diagnosed with the disease or condition prior to the administration of the ligand.

For example, this human is partially or completely resistant to treatment with anti-PD-L1 ligands, such as anti-PD-L1 NCE.
In some examples, this ligand is administered by intravenous or subcutaneous administration and / or is included in an injectable preparation.

2. The method of embodiment 1, wherein the nucleotide sequence comprises a nucleotide corresponding to SNP rS1411262, SNP rs4143815, 8923C or 395C.

3. The method of aspect 1 or 2, wherein the human comprises a PD-L1 nucleotide sequence comprising the selected variation.
Optionally, this human is homozygous for the variation. Alternatively, this human is heterozygous for the variation.
In some examples, this ligand comprises an antibody constant region (eg, an antibody Fc region).

4. The ligand contains a human gamma-1 heavy chain constant region containing an amino acid selected from the group consisting of Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42, and optionally. , The human contains an IGHG1 * 01 human heavy chain constant region gene segment, or the human expresses an antibody comprising a human gamma-1 heavy chain constant region containing the selected amino acid, of embodiments 1-3. Either one way.

For example, this ligand comprises and comprises a human gamma-1 heavy chain constant region comprising an amino acid selected from the group consisting of Asp corresponding to position 204 of SEQ ID NO: 42 and Leu corresponding to position 206 of SEQ ID NO: 42. In, the human contains an IGHG1 * 01 human heavy chain constant region gene segment, or the human expresses an antibody comprising a human gamma-1 heavy chain constant region containing the selected amino acid. In certain embodiments, this is combined with any of the examples shown under aspect 1 above.

Optionally, this constant region of this ligand is contained by an Fc region, eg, an enhanced Fc region.

Optionally, this ligand comprises the IGHG1 * 01 human heavy chain constant region.

5. The method of any one of aspects 1-4, wherein the disease or condition is cancer (eg, solid tumor), autoimmune disease or condition; or inflammatory disease or condition.
In some cases, the cancer is breast cancer, lung cancer (eg, non-small cell lung cancer), colon cancer, ovarian cancer, melanoma (eg, stage IV melanoma), bladder cancer, liver cancer (eg, eg). Hepatic cell cancer), kidney cancer, salivary adenocarcinoma, gastric cancer (stomach cancer), gastric cancer (gastric cancer), glioma, pancreatic cancer, thyroid cancer, thoracic epithelial cancer, head cancer and / or Cervical cancer, multiple myeloma, T-cell lymphoma, Hodgkin lymphoma, renal cell cancer, cervical cancer, colorectal cancer, hematological tumor, metastatic colorectal cancer, uterine or cervical cancer , Leukemia (eg, acute myeloid leukemia), diffuse large cell type B cell lymphoma, follicular center lymphoma, prostate cancer and Merkel cell carcinoma.

Alternatively, the disease or condition is a viral infection, such as HIV, hepatitis C or Ebola infection. Alternatively, the disease or condition is myelodysplastic syndrome.

In some cases, the condition is cancer, eg, metastatic cancer.

6. The method of any one of aspects 1-5, comprising selecting a human comprising the PD-L1 nucleotide sequence prior to the administering step, wherein this human is a human of embodiment 1.

7. The method of any one of aspects 1-6, wherein the human was determined to contain the PD-L1 nucleotide sequence.

8. Any of aspects 1-6, comprising a step of determining, wherein the human comprises the PD-L1 nucleotide sequence and optionally this step of determining is performed prior to administration of the antibody to this human. One way.

9. The method of embodiment 8, wherein the step of determining comprises assaying a human-derived biological sample for a PD-L1 nucleotide sequence containing the selected variation.

10. The step of assaying is a biological sample and
A. At least one oligonucleotide probe that can be specifically hybridized and identified in a biological sample to a nucleotide sequence containing the selected variation, or against antisense of said sequence. It comprises a sequence of at least 10 contiguous nucleotides that specifically hybridize, wherein the nucleic acid hybridizes to the selected variation or hybridizes to an antisense sequence. Thus, if at least one nucleotide sequence containing the selected variation is present, with an oligonucleotide probe forming a complex; and / or
b. At least one oligonucleotide probe comprising the sequence of said contiguous nucleotides of a nucleotide sequence containing the selected variation or containing an antisense sequence of at least 10 contiguous nucleotides, where contiguous. The step of contacting; with the oligonucleotide probe forming the complex, if the sequence of the nucleotides in the nucleotide contains the selected variation and thereby the nucleotide sequence containing the selected variation is present, and the complex. Aspect 9 comprising the step of detecting the presence or absence of a body, wherein by detecting the presence of this complex, a human is determined to contain a PD-L1 nucleotide sequence containing the selected variation. the method of.

11. The assaying step involves nucleic acid amplification and, optionally, one or more methods selected from sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification and / or this. The method of embodiment 10, wherein the step of assaying is performed in a multiplex format.

12. The method of any one of aspects 1-11, wherein the human is substantially resistant to, or is further determined to be, resistant to PD-L1 or PD-1 treatment.

13. Aspect 1 in which the human has received or has received PD-L1 or PD-1 treatment, or has reduced responsiveness to PD-L1 or PD-1 treatment. Any one method from ~ 11.

14. The method of embodiment 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

15. The method of any one of aspects 1-14 in which the human has been diagnosed with cancer, an autoimmune disease or condition; or an inflammatory disease or condition.

16. The method of any one of aspects 1-15, wherein the ligand is administered by intravenous or subcutaneous injection and / or is included in the preparation for injection.

17. The method of any one of aspects 1-16, wherein the ligand is human.

18. Anti-human PD-L1 ligand for use in any one of aspects 1-17 (eg, antibody, antibody fragment or human PD-L1 trap).

19. Ligand of embodiment 18, wherein the ligand is a human anti-human PD-L1 antibody (eg, davarumab), antibody fragment or human PD-L1 trap.

20. The ligand of embodiment 18 or 19, wherein said ligand is for intravenous or subcutaneous administration and / or is included in an injectable preparation.

For rs148170925, the 1000 Genome Phase 1 database is 60% for SNP deletion (ie, presence on SNP) and 40% for SNP absence (ie, presence of TG at chromosome position 9: 545464-25454643). The cumulative frequency over the entire human population is shown. The following human populations exceed the average frequency for the absence of SNPs: AMR (45%), EUR (54%), ASW (44%), CLM (42%), MXL (44%), PUR (49%) %), CEU (60%), FIN (57%), GBR (56%), IBS (46%) and TSI (46%); in certain embodiments of the invention, this human is of these pedigrees. It is one of. The following human populations exceed the average frequency for the presence of SNPs: AFR (67%), ASN (76%), LWK (77%), YRI (65%), CHB (73%), CHS (79%) , JPT (75%); In certain embodiments of the invention, this human is one of these pedigrees.

(Example 7)
TOI for cancer, autoimmune and inflammatory applications
(Example 7A)
Tumor Targeting and Immunology-Application of Oncology Tumor microenvironment is an important aspect of cancer biology and contributes to tumor development, tumor progression and response to treatment. The cells and molecules of the immune system are the basic components of the tumor microenvironment. Importantly, therapeutic strategies can suppress the immune system and specifically target tumor cells, which can lead to long-term regression and prevent recurrence in cancer patients, tumor-specific. It is particularly aroused thanks to its ability to induce typical immunological memory.

The composition and characteristics of the tumor microenvironment vary widely and are important in determining the anti-tumor immune response. Certain cells of the immune system, including, for example, natural killer cells, dendritic cells (DCs) and effector T cells, can drive a strong antitumor response. However, tumor cells often induce an immunosuppressive microenvironment, which supports the development of immunosuppressive populations of immune cells such as bone marrow-derived inhibitory cells and regulatory T cells. Understanding the complexity of tumor immunoregulation is important in the development of immunotherapy. Various strategies have been developed to enhance the anti-tumor immune response, including DC-based vaccines and antagonists of inhibitory signaling pathways to overcome "immune checkpoints".

The present invention provides a method comprising the step of administering an anti-TOI ligand to a human, the TOI present in the human as multiple variants with different one or more amino acid gene polymorphisms, the method of which , Including the step of administering a ligand to a human, the ligand comprising a first and second protein domain, wherein the first domain is a TOI variant comprising a first amino acid gene polymorphism. Specific binding to, where this second domain comprises a second gene polymorphism, and where this human is (i) a TOI; and (i) comprising said first amino acid gene polymorphism. ii) Express a protein domain containing the second gene polymorphism.

In particular, the invention provides a method of targeting a tumor in humans, eg, in this case, the TOI is a tumor antigen (eg, a tumor-specific or tumor-related antigen as is apparent to a skilled recipient). Is. For example, the TOI is a group consisting of human PD-L1, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA) and melanoma-related antigen (MAGE). Is selected from.

Optionally, the ligand comprises a human cytokine variant amino acid sequence, and optionally, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15 and IL-21, eg, here. This sequence is fused to the N-terminal of one or more heavy and / or light chains of the ligand if the ligand is a trap containing an antibody or fragment or Fc region.

In one example, the cytokine is human IL-2, and the sequence comprises a genetic polymorphism selected from 142I, 129D, 76L, 38L and 27T. For example, this sequence contains 142I and 129D. These positions are shown to be polymorphic in Ensembl and dbSNP and associated with SNP. In certain embodiments, the ligand comprises an IL-2 amino acid sequence that comprises one, more, or all of the genetic polymorphisms in the group. In one example, this cytokine comprises SEQ ID NO: 134.

In one example, the cytokine is human IL-21, and the sequence comprises a genetic polymorphism selected from 32Q, 40R, 43I, 98V, 106K, 112A, 113G, 134K and 135E. For example, this sequence contains 32Q and 40R. These positions are shown to be polymorphic in Ensembl and dbSNP and associated with SNP. In certain embodiments, the ligand comprises an IL-21 amino acid sequence that comprises one, more, or all of the genetic polymorphisms in the group.

One alternative method provides a method of immunotherapy for cancer, eg, in this case, the TOI is a human immunocellular antigen (eg, where this antigen is a human natural killer cell, dendritic cell (DC)). ) Or effector T cell antigen). In another example, TOI is the target of immune checkpoints.

In certain embodiments, the TOI is human PD-1. In certain embodiments, this TOI is human PD-L1 or PD-L2. In another embodiment, the target is human CTLA-4. In another embodiment, the target is human LAG-3. In another embodiment, the target is the human TIM-3. In certain embodiments, the TOI is human OX40 (and, optionally, this ligand antagonizes the TOI). In certain embodiments, this TOI is a human KIR. In certain embodiments, the TOI is human CD137 (and, optionally, this ligand antagonizes the TOI). In certain embodiments, the TOI is human CD27 (and, optionally, this ligand antagonizes the TOI). In one embodiment, this TOI is human CD70. In certain embodiments, this TOI is human B7-H3. In certain embodiments, this TOI is a human KIR. In some embodiments, the TOI is a human GITR (and, optionally, this ligand antagonizes the TOI). In one example, the ligand comprises a human cytokine variant amino acid sequence, as described above, and the cytokine is useful for cancer immunotherapy. If no agonist is mentioned, this ligand can antagonize, for example, TOI.

In one example, this ligand is selected from the ligands in Table 22 (Table 24).

In any alternative example, this method treats or reduces the risk of cancer, eg, any cancer disclosed herein (eg, disclosed in Examples 5, 6 or 7). As it is done).

Therefore, according to alternatives: A method of human cancer immunotherapy by targeting immune cell TOIs in humans is provided, which TOIs are as multiple variants with different amino acid gene polymorphisms. Present in humans, the method comprises administering a ligand to the human, which comprises the first and second protein domains, wherein the first domain is present. It specifically binds to a TOI variant containing a first amino acid gene polymorphism, where this second domain comprises a second gene polymorphism, and where this human is (i) said the first. TOI containing an amino acid gene polymorphism; and (ii) express a protein domain containing the second gene polymorphism.

The TOI is for, for example, PD-1 (which is also known as PDCD1, Programmed Cell Death 1, PD1 and CD279), CTLA-4, LAG-3 or TIM-3. For example, the TOI is human PD-1, and the first gene polymorphism is the PD-1 gene polymorphism described herein [eg, variations listed in Table 23]. .. For example, this genetic polymorphism is encoded by the PD-1 SNPs described herein.

In this alternative, it may be desirable to block the TOI (not necessarily as clear as the IgG1 format), and human IgG4 may be suitable for doing this in an immunotherapeutic environment. Thus, in certain embodiments, the ligand comprises a human gamma-4 constant region, wherein the constant region comprises said second domain. Importantly, according to the present invention, this constant region gene polymorphism is compatible with the human genome, thereby interfering with the immune response, which should be minimized in the current immuno-oncology environment. Minimize. To "block" a TOI is, for example, to antagonize the binding of the TOI to a cognate receptor or human biological target, eg, PD-1 binding to PD-L1 and / or PD-L2; B7. It means blocking CTLA-4 binding to -1 or B7-2; or CD28 binding to B7-1 or B7-2.

For the immunotherapeutic environment of the present invention, it may be advantageous for gamma-4 to be a gamma-4 constant region with 228Pro or 235Glu. In one example, this constant region is IgG4PE (ie, a gamma-4 constant region with 228Pro and 235Glu). In one example, this ligand comprises the human IGHG4 * 01 hinge S10> P constant region.

Thus, in one example, this ligand comprises any human gamma-4 constant region disclosed herein (eg, see below, in any of Example 1).

Therefore, the present invention provides the following concepts:-
1. In humans, a method of cancer immunotherapy in humans by targeting immune cell TOIs (eg, PD-1), the TOIs of which one or more amino acid gene polymorphisms differ. Present in humans as a variant of, the method comprises administering to the human a ligand (eg, an antibody or antibody fragment), the ligand comprising a first and second protein domain. Here, this first domain specifically binds to a TOI variant containing the first amino acid gene polymorphism, where this second domain contains the second gene polymorphism, and here. The human expresses (i) a TOI containing the first amino acid gene polymorphism; and (ii) a protein domain containing the second gene polymorphism, wherein the ligand is found in SEQ ID NO: 73. It comprises a human gamma-4 heavy chain constant region containing Leu at position 189 or Arg at position 289 found in SEQ ID NO: 73, where the human comprises the IGHG4 * 01 human heavy chain constant region gene segment or This human expresses an antibody containing a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73; and here this second domain. Is included by said gamma-4 heavy chain constant region of this ligand.

Optionally, the TOI is a human immune cell antigen (eg, where the antigen is a human natural killer cell, dendritic cell (DC) or effector T cell antigen).

Programmed death-1 (PD-1) receptors act as immunological checkpoints, limiting bystander tissue damage and preventing the development of autoimmunity during inflammatory reactions. PD-1 is expressed by activated T cells and modulates T cell effector function downward upon binding on antigen-presenting cells to its ligands, PD-L1 and PD-L2. In patients with cancer, expression of PD-1 on tumor-infiltrating lymphocytes and its interaction with ligands on tumors and immune cells in the tumor microenvironment weakens and suppresses anti-tumor immunity. The rationality of PD-1 blockade in tumor immunotherapy is supported. In a particularly preferred embodiment, a method of cancer immunotherapy in humans is provided by targeting PD-1 in humans.
2. The method of Concept 1 in which the first and / or second gene polymorphisms are encoded by SNPs each having a cumulative human allele frequency of less than 50%.

Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

3. The method of any of the aforementioned concepts in which the first domain is the antibody variable domain or receptor domain.

4. The method of any of the aforementioned concepts in which the second domain is the antibody domain, optionally the variable domain or constant domain of the antibody.

5. The method of any of the aforementioned concepts, where the second domain is the domain of the antibody Fc region.

6. The first and second domains are antibody variable domains, and optionally, this variable domain specifically binds to the first and second human TOIs, respectively, where this TOI is different, the concept. Any one method from 1 to 4.

7. The concept that the first and second domains are receptor domains, and optionally, these receptor domains specifically bind to the first and second human TOIs, respectively, where this TOI is different. Any one method from 1 to 4.

8. This first domain contains a third amino acid gene polymorphism, wherein the human expresses (iii) the domain containing the third amino acid gene polymorphism, the method of concept 6 or 7. ..
Optionally, this human expresses a second TOI variant. In one example, the second TOI is a human immune cell surface antigen or tumor antigen.
Optionally, this third genetic polymorphism is encoded by an SNP with a cumulative human frequency of less than 50%. Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

In one example, this first domain is an antibody variable domain (eg, any V domain of the invention described herein, such as human VH3-23 * 04 described herein) and ( The domain of iii) is an antibody variable domain. For example, this first domain and the domain of (iii) are VH domains. For example, the domain of the first domain and (iii) is a V _K domain. For example, this first domain and the domain of (iii) are Vλ domains.
In one example, this first domain is the receptor binding site and the domain (iii) is the receptor binding site domain.

9. Combined concepts 1, 2 and 8 methods.

10. The second domain is the domain of the human cytokine variant, optionally selected from the group consisting of IL-2, IL-12, IL-15 and IL-21, and the domain (ii) is the cytokine. One method of concepts 1-3, which is the domain of the variant.

In one example, this second domain is the domain of human IL-2, and the second genetic polymorphism is selected from the group consisting of 142I, 129D, 76L, 38L and 27T. For example, this second domain contains 142I and 129D. These positions are shown to be polymorphic and associated with SNPs in Ensembl and dbSNP. In certain embodiments, the ligand comprises an IL-2 amino acid sequence that comprises one, more, or all of the genetic polymorphisms in the group.

In one example, this second domain is the domain of human IL-21, and the second genetic polymorphism consists of the group consisting of 32Q, 40R, 43I, 98V, 106K, 112A, 113G, 134K and 135E. Be selected. For example, this second domain contains 32Q and 40R. These positions are shown to be polymorphic and associated with SNPs in Ensembl and dbSNP. In certain embodiments, the ligand comprises an IL-21 amino acid sequence that comprises one, more, or all of the genetic polymorphisms in the group.

11. The method of any of the aforementioned concepts in which humans express the first and / or second domain.

12. Any method of the aforementioned concept in which the first, second and / or third genetic polymorphisms are found in at least 5 different human populations, respectively, as defined in Table 4 (Table 5).
For example, each of the first and second genetic polymorphisms is found in at least five different human populations, as defined in Table 4, respectively. For example, each of the first, second and genetic polymorphisms is found in at least five different human populations, as defined in Table 4 (Table 5), respectively.

Optionally, this first, second and / or third (see below) polymorphism is at least 9, 10, 11, as defined in Table 4, respectively. Found in 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human populations.

13. Concept 12 methods in which the first, second and / or third gene polymorphisms are found in at least 15 humans in the 1000 Genomes Phase I database, respectively.
In certain embodiments, the first, second and / or third genetic polymorphisms are distributed over many such different human populations at least 20, 25, 30, 35, 40, 45, 50, 55. , 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 cases of human individuals.

14. First, second and / or third gene polymorphisms are found in at least 5 different human populations, respectively, as defined in Table 4, and a cumulative human allele frequency of less than 50% A method of any of the aforementioned concepts, encoded by an SNP having.
Optional, frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1%.

15. The method of any of the aforementioned concepts, wherein the first, second and / or third gene polymorphisms are encoded by SNPs each having a total human genotype frequency of less than 50%.
Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

16. The method of any of the aforementioned concepts, wherein the first and second domains are human and, optionally, the ligand is fully human.

17. The method of any of the aforementioned concepts, wherein the ligand is an anti-TOI trap, antibody or antibody fragment.

18. The method of any of the aforementioned concepts in which the ligand specifically binds to two or more human TOIs, eg, the first and second TOIs.

19. The method of any of the aforementioned concepts, wherein the ligand is administered to a human by injection or the ligand is provided by an injectable preparation.

20. The human has been or has been genotyped positive for the TOI variant nucleotide sequence prior to administration of the ligand, or the method is prior to administration of the ligand. , A method of any of the aforementioned concepts, comprising the step of genotyping a human as positive for the variant nucleotide sequence.

21. Humans have been or have been genotyped positive for the TOI variant prior to administration of the ligand, or the method is said to be prior to administration of the ligand. A method of any of the aforementioned concepts, comprising the step of phenotyping a human as positive with respect to.

22. The method of any of the aforementioned concepts comprising the step of selecting a human containing the first TOI gene polymorphism prior to the administering step, wherein this human is a concept 1.

23. The method of any of the aforementioned concepts in which humans have been determined to contain a first and / or second TOI variant, or a nucleotide sequence encoding a TOI variant.

24. Includes the step of determining that a human contains a first TOI nucleotide sequence encoding a first gene polymorphism, optionally this step of determining is performed prior to administration of the ligand to humans. , Any of the aforementioned conceptual methods.

25. The method of Concept 24, wherein the determining step comprises assaying a human-derived biological sample for said nucleotide sequence.

26. The assay step is a biological sample and
aa At least one oligonucleotide probe that can be specifically hybridized and identified in a biological sample to a nucleotide sequence containing an SNP encoding a first gene polymorphism, or an anti-sequence of said sequence. It contains a sequence of at least 10 contiguous nucleotides that specifically hybridize to a sense, where the nucleic acid either hybridizes to the SNP or hybridizes to an antisense sequence. And if at least one nucleotide sequence containing said SNP is present, with an oligonucleotide probe forming a complex; and / or
bb At least one oligonucleotide probe comprising the sequence of said contiguous nucleotides of a nucleotide sequence comprising said SNP or containing an antisense sequence of at least 10 contiguous nucleotides, wherein said contiguous nucleotide With an oligonucleotide probe that forms a complex if the sequence comprises said SNP and thereby a nucleotide sequence containing said SNP is present;
The process of contacting, and
c. The method of Concept 25, comprising detecting the presence or absence of this complex, wherein by detecting the presence of this complex, humans are determined to contain a first TOI nucleotide sequence. ..

27. The assaying step comprises or optionally includes one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. Concept 25, in which the assaying step is performed in a multiplex format.

28. The method of any of the aforementioned concepts in which the human is substantially resistant to, or is further determined to be, substantially resistant to cancer treatment.

29. The method of any of the aforementioned concepts in which the human has, or has received, or is less responsive to cancer treatment.

30. The method of Concept 25, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

31. The method of any of the aforementioned concepts in which the human has been diagnosed with the disease or condition.

32. Anti-TOI ligands (eg, antibodies, antibody fragments or traps) for use in the methods of any of the aforementioned concepts. For example, this TOI is human PD-1 [eg, this ligand is pembrolizumab, lambbrolizumab or KEYTRUDA ™. In one example, this ligand is nivolumab or OPDIVO ™]. For example, the TOI is human CTLA-4, for example, the ligand is ipilimumab, YERVOY ™, tremelimumab, ticilimumab, veratacept or Nulojix ™.

In certain embodiments, the trap is a receptor Fc fusion (ie, a receptor domain fused to an antibody Fc region, such as a dimer), where the receptor is specific to the TOI. Join.

33. The ligand of concept 32, where the ligand is completely human.

34. The ligand is for administration by injection, or this ligand is provided by an injectable preparation; where this ligand is for topical or inhalation administration, or this ligand is for topical preparation. A ligand of concept 32 or 33, provided by a substance or inhalable preparation.

In one example, the first, second and / or third gene polymorphisms are:-
d. Has a cumulative human allele frequency of 15% or less;
e. Have a total human genotype frequency of 20% or less;
f. Found in at least 5 different human populations (eg, using the standard classification of the 1000 Genomes Project, as in the Phase I database);
g. Found in many individuals distributed across many such different populations.

In some examples, this criterion applies in connection with one or more human genome sequence databases as described herein. For example, this criterion is exactly what applies to the 1000 Genomes Database.

For example, in an example, embodiment or configuration of any aspect of the invention, the 1000 Genomes Database exposes 13 or Phase I. For example, the most recent version of the 1000 Genomes Database as of October 1, 2013 or October 1, 2014.

In one example, this human is homozygous for the first genetic polymorphism. Additional or alternative, this human is homozygous for the first genetic polymorphism. Additional or alternative, this human is homozygous for a third genetic polymorphism. For example, this human is homozygous for the first and second polymorphisms.

In one example, this method is by any one of the disclosures herein that comprises the steps of selecting, genotyping or phenotyping a human as positive for a TOI variant (ie, in the case of the present invention). Human PD-1 contains a genotype or a nucleotide sequence encoding it). In some examples, the method is by any one of the above concepts comprising the step of determining, or where the human is determined to include a variant TOI variant (eg, in the case of the present invention). , Human PD-1 contains a genetic polymorphism or a nucleotide sequence encoding it).

In some examples, this first genetic polymorphism is either the human PD-1 amino acid gene polymorphism described herein or encoded by the human PD-1 SNP described herein.
In one example, the TOI here is human PD-1, and this first genetic polymorphism is selected from the group consisting of rs7568402, rs28699177, rs28542728, rs118117097, rs28570544, rs118027315, rs191919871, rs6707556 and rs142909968. Coded by SNP.

The present invention further provides the following specific aspects.
1. A method of human cancer immunotherapy by targeting PD-1 in a human, the method comprising administering to the human an antibody or antibody fragment, wherein the antibody or The antibody fragment specifically binds to PD-1 containing the first amino acid gene polymorphism, where the antibody or antibody fragment is found in Leu at position 189 or SEQ ID NO: 73 found in SEQ ID NO: 73 289. Human gamma-4 heavy chain constant region containing Arg at position, where said human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is at position 189 found in SEQ ID NO: 73). (I express an antibody containing the human gamma-4 heavy chain constant region containing Arg at position 289 found in Leu or SEQ ID NO: 73); and (ii) encode PD-1 containing the first amino acid gene polymorphism. A method comprising a PD-1 nucleotide sequence (or this human expresses PD-1 containing the first amino acid gene polymorphism).

The first alternative aspect 1 provides:-
A method of cancer immunotherapy in humans by targeting PD-1 in humans, the method comprising administering an antibody or antibody fragment to a human, wherein the antibody or antibody. Fragments are SNPs (first gene polymorphisms) selected from the variation shown in Table 23 (selected from the group consisting of rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982) It specifically binds to PD-1 encoded by the containing PD-1 nucleotide sequence, where this antibody or antibody fragment is located at Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Containing a human gamma-4 heavy chain constant region comprising, wherein the human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is the Leu or sequence at position 189 found in SEQ ID NO: 73). It expresses an antibody containing a human gamma-4 heavy chain constant region containing Arg at position 289 found in number 73); and (ii) contains a PD-1 nucleotide sequence containing the selected SNP.

These SNPs are discussed further below.

The first alternative aspect 1 provides:-
A method of treating cancer in humans, the method comprising administering an antibody or antibody fragment to humans, wherein the antibody or antibody fragment comprises a PD containing a first amino acid gene polymorphism. Inhibits the binding of PD-L1 or PD-L2 to -1, where this antibody or antibody fragment contains human gamma at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73- It comprises a quadruple constant region, wherein the human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is at Leu at position 189 or SEQ ID NO: 73 found in SEQ ID NO: 73. (Expressing an antibody containing the human gamma-4 heavy chain constant region containing Arg at position 289); and (ii) the PD-1 nucleotide sequence (or) encoding PD-1 containing the first amino acid gene polymorphism. This human expresses PD-1 containing the first amino acid gene polymorphism). In one example, this human has PD-L1 SNPs disclosed in Example 6 or PD-1 nucleotide sequences containing SNPs selected from the group consisting of PD-1 SNPs rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982. Includes; Optionally, this human is determined to contain the SNP, or the method determines that the human contains the SNP prior to the step of administering the antibody or fragment. including.

In some examples, this antibody or fragment specifically binds to human PD-L1, PD-L2 or PD-1. In some examples, this antibody or fragment specifically binds to human PD-L1. In some examples, this antibody or fragment specifically binds to human PD-1.

For example, this PD-L1 is encoded by a PD-L1 nucleotide sequence that contains the PD-L1 gene polymorphism described in Example 6 or contains the PD-L1 SNP described in Example 6. For example, PD-1 comprises a PD-1 gene polymorphism described herein [eg, in Table 23] or is described herein [eg, in Table 23 (Table 25). In 25)] encoded by a PD-1 nucleotide sequence containing a PD-1 SNP (eg, an SNP selected from the group consisting of rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982). These SNPs are discussed further below.

2. The method of aspect 1, wherein the first genetic polymorphism or selected SNP is the nucleotide corresponding to the SNPs rs36084323, rs10204225, rs11568821, rs2227981 or rs2227982.

3. The method of aspect 1 or 2, wherein the human is homozygous for the first genetic polymorphism or selected SNP.

4. The method of any of the aforementioned embodiments, wherein the ligand comprises the IGHG4 * 01 human heavy chain constant region.

5. Cancer is renal cell carcinoma; hematological tumor; stage IV melanoma; non-small cell lung cancer; metastatic gastric cancer; breast cancer; solid tumor; bladder cancer; head and neck cancer, colonic rectal cancer, glia Any of the aforementioned aforementioned, selected from the group consisting of blastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, cervical cancer, myeloid leukemia (eg, acute myeloid leukemia or chronic myeloid leukemia). Aspect method. In certain embodiments, the cancer is not melanoma. In certain embodiments, the cancer is not lung cancer. In certain embodiments, the cancer is not non-small cell lung cancer.

6. The method of any of the aforementioned embodiments, comprising the step of selecting a human comprising the PD-1 nucleotide sequence of (ii) prior to the administering step, wherein this human is a human of embodiment 1.

7. The method of any of the aforementioned embodiments, wherein the human has been determined to contain the PD-L nucleotide sequence of (ii).

8. Including the step of determining that this human contains the PD-L nucleotide sequence of (ii), optionally, this determining step is performed prior to administration of this antibody to this human, optionally. The method of the above-described embodiment.

9. The method of embodiment 8, wherein the step of determining comprises assaying a human-derived biological sample for a PD-L nucleotide sequence containing the selected variation.

10. The assay step is the biological sample and
A. At least one oligonucleotide probe that can be specifically hybridized and identified in a biological sample to a nucleotide sequence containing the selected variation, or against antisense of said sequence. Containing a sequence of at least 10 contiguous nucleotides that specifically hybridize to the nucleic acid, either hybridize to the selected variation or hybridize to an antisense sequence. If at least one nucleotide sequence containing the selected variation is present, then with the oligonucleotide probe forming the complex; and / or
b. At least one oligonucleotide probe comprising, at least 10 consecutive nucleotide sequences of a nucleotide sequence containing the selected variation or comprising an antisense sequence of said contiguous nucleotides, where contiguous. The step of contacting; with the oligonucleotide probe forming the complex, if the sequence of the nucleotides contains the selected variation and thereby the nucleotide sequence containing the selected variation is present.
c. A step of detecting the presence or absence of this complex, wherein by detecting the presence of this complex, a step of determining that a human contains a PD-L1 nucleotide sequence containing the selected variation. The method of aspect 9, including.

11. The assaying step involves nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification and / or this. The method of embodiment 10, wherein the step of assaying is performed in a multiplex format.

12. The method of any of the aforementioned embodiments, wherein the human is substantially resistant to, or is further determined to be, resistant to cancer, PD-L1 or PD-1 treatment.
For example, this human has been further determined to be resistant or resistant to chemotherapeutic treatment.

13. The human has or has been treated with cancer (eg, chemotherapy), PD-L1 or PD-1, or has cancer (eg, chemotherapy), PD-L1. Alternatively, the method of any of the aforementioned embodiments, which is less responsive to PD-1 treatment.

14. The method of embodiment 9, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

15. The method of any of the aforementioned embodiments, wherein the ligand is administered by intravenous or subcutaneous injection and / or is included in the preparation for injection.

16. The method of any of the aforementioned embodiments, wherein the ligand is a human, eg, a human antibody or antibody fragment.

17. Anti-human PD-1 ligand (eg, antibody, antibody fragment or trap) for use in the method of any of the aforementioned embodiments.
For example, this ligand is pembrolizumab, lambrolizumab or KEYTRUDA ™. In one example, this ligand is nivolumab or OPDIVO ™.
In certain embodiments, the trap is a receptor Fc fusion (ie, for example, a receptor domain fused to the antibody Fc region as a dimer), where the receptor is on human PD-1. It binds specifically.

18. The ligand of embodiment 17, wherein the ligand is completely human.

19. The ligand of embodiment 17 or 18 where the ligand is for administration by injection or the ligand is provided by an injectable preparation; where this ligand is for topical or inhalation administration. Alternatively, this ligand is provided by a topical or inhalable preparation.

Optionally, in any of the configurations, concepts, aspects, embodiments or examples, one or more of the following optional features apply:

Optionally, this cancer includes renal cell carcinoma; hematological tumors; stage IV melanoma; non-small cell lung cancer; metastatic gastric cancer; breast cancer; solid tumors; bladder cancer; head and neck cancer, colonic rectum Tumor, gliablastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, cervical cancer, myeloid leukemia (eg, acute myeloid leukemia or chronic myeloid leukemia). In certain embodiments, the cancer is not melanoma. In certain embodiments, the cancer is not lung cancer. In certain embodiments, the cancer is not non-small cell lung cancer.

Optionally, this cancer is a renal cell carcinoma.

Optionally, the cancer is locally advanced or metastatic carcinoma, locally advanced or metastatic melanoma, or locally advanced or metastatic non-small cells. It is a lung carcinoma.

Optionally, this cancer is a urothelial cancer.

Optionally, the cancer is a recurrent or metastatic head and neck squamous cell carcinoma.

Optionally, the cancer is PD-L1-positive advanced or metastatic non-small cell lung cancer.

Optionally, the cancer is multiple myeloma.

Optionally, this cancer is an advanced melanoma.

Optionally, the cancer is an advanced Merkel cell cancer.

Optionally, the cancer is a solid tumor, eg, an advanced solid tumor.

In one example, this ligand comprises a human IgG4PE constant region.

In one example, this ligand is pembrolizumab, lambrolizumab or KEYTRUDA ™. In one example, this ligand is nivolumab or OPDIVO ™.

This TOI gene polymorphism is associated with cancer, such as any cancer disclosed herein.

The invention also presents anti-TOI ligands for use in any of the methods of cancer treatment or immunotherapy of the invention (eg, human PD-1, PD-L1 or PD-L2 traps, or anti-human PD-1, PD-L1 or PD-L2 antibody or fragment).

Optionally, this ligand (eg, antibody or antibody fragment) depends on one of the following options:-
(i) Here, the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a human JH segment, the human VH segment having SEQ ID NO: 40 Framework 1 is encoded, and where the human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human is the Framework 1 of SEQ ID NO: 40. Express the VH domain containing.
(ii) Here, this ligand contains a VH domain derived from recombination of the human VH segment, the human D gene segment and the human JH segment selected from the group consisting of IGHV3-33 * 01 and IGHV3-7 * 01. Moreover, the human contains the selected VH gene segment, or the human expresses the VH domain derived from the recombination of the selected human VH segment, the human D gene segment and the human JH segment.
Here (iii), the ligand comprises an IGKV3-11 * 01 and IGKV1-12 * 01 human V _K segments and V _K domains from recombinant human J _K segment is selected from the group consisting of, and, The human comprises the selected V _K gene segment, or the human expresses the V _K domain derived from the selected human V _K segment and human J _K segment recombination.
(iv) Here, this ligand contains a V _K domain derived from the recombination of the human V _K segment and the human J _K segment, and this human V _K segment is (i) Pro at position 7 shown in SEQ ID NO: 36. Containing CDR3 (and where said human contains the V _K gene segment encoding CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, or this human is located at position 7 set forth in SEQ ID NO: 36. (Expressing a V _K domain containing CDR3 containing Pro); or (ii) FW3 containing the Ser at position 15 shown in SEQ ID NO: 38 (and where the human is the Ser at position 15 shown in SEQ ID NO: 38). Contains a V _K gene segment encoding FW3 containing, or this human expresses a V _K domain containing FW3 containing Ser at position 15 set forth in SEQ ID NO: 38).

Optionally, instead of the gamma-4 constant region, this ligand (eg, trap, antibody or fragment) comprises a heavy chain constant region with one of the following options:-
(v) Here, the ligand comprises a human gamma-1 heavy chain constant region comprising Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4, and the human is here. , (I) IGHG1 * 01 Human gamma-1 weight containing the human heavy chain constant region gene segment, or this human containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4. Express an antibody that contains a chain constant region.
(vi) Here, this ligand is shown in Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and 161 shown in SEQ ID NO: 6. It comprises a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val at position Val and Ala at position 257 set forth in SEQ ID NO: 6, where the human is (i) IGHG2 * 01 human weight. This human contains the chain constant region gene segment, or this human is Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, and Phe at position 76 shown in SEQ ID NO: 6. , Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6 and expresses an antibody containing a human gamma-2 heavy chain constant region. In one example, this constant region is Fc-enhanced, eg ADCC or CDC-enhanced. Those skilled in the art are known for techniques for enhancing ADCC or CDC (eg, known are C region mutations).
(vii) Here, this ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3 * 01) constant region gene segment, wherein the human is (i). ) Includes the first constant region gene segment (eg, IGHG3 * 01), or this human is a human gamma-3 multiple encoded by the first human IGHG3 (eg, IGHG3 * 01) constant region gene segment. Express an antibody that contains a chain constant region.

Optionally, this ligand (eg, antibody or antibody fragment) depends on one of the following options:-
(viii) Here, this ligand comprises a human kappa chain constant region comprising Val at position 84 set forth in SEQ ID NO: 16 or Cys at position 87 set forth in SEQ ID NO: 16, where the human is (i). ) IGKC1 * 01 Human kappa chain constant region Contains a gene segment, or this human contains a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16. Express the antibody.
(ix) Here, this ligand comprises a human IGLC1 * 01 lambda chain constant region, wherein the human comprises (i) a human IGLC1 * 01 lambda chain constant region gene segment, or this human is , Human IGLC1 * 01 Expresses an antibody containing a lambda chain constant region.
(x) Here, the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment, wherein the human is (i) said the first constant. A region gene segment is included, or the human expresses an antibody comprising a human epsilon heavy chain constant region encoded by the first constant region gene segment.
(xii) Here, the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human mu heavy chain constant region encoded by the first constant region gene segment.
(xiii) Here, the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment, wherein the human is (i) said the first constant. A region gene segment is included, or the human expresses an antibody comprising a human alpha heavy chain constant region encoded by the first constant region gene segment.
(xiv) Here, the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human delta heavy chain constant region encoded by the first constant region gene segment.
(xv) Here, this ligand comprises the human kappa light chain constant region encoded by the first human kappa light chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human kappa light chain constant region encoded by the first constant region gene segment.
(xvi) Here, this ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human lambda light chain constant region encoded by the first constant region gene segment.

Citations
(a) Cancer Immunol Res. 2014 Sep; 2 (9): 846-56. Doi: 10.1158 / 2326-6066. CIR-14-0040. Epub 2014 May 28; "In vitro characterization of the anti-PD-1 antibody nivolumab, BMS-936558, and in vivo toxicology in non-human primates ", Wang C et al.
(b) Clin Biochem. 2014 May; 47 (7-8): 612-7. Doi: 10.1016 / j.clinbiochem.2013.12.023. Epub 2014 Jan 2; "Programmed death-1 (PD-1) polymorphisms in Chinese patients with esophageal cancer "; Qiu H et al.
(c) Gene. 2012 Oct 25; 508 (2): 229-32. doi: 10.1016 / j.gene.2012.07.059. Epub 2012 Aug 8; "Programmed death-1 gene polymorphism (PD-1.5 C / T) is associated with colon cancer "; Mojtahedi Z et al.
(d) Gastroenterol Hepatol Bed Bench. 2013 Fall; 6 (4): 178-82; "Programmed death-1 gene polymorphism (PD-1.5 C / T) is associated with gastric cancer"; Savabkar S et al.
(e) Breast Cancer Res Treat. 2011 Aug; 129 (1): 195-201. doi: 10.1007 / s10549-011-1440-3. Epub 2011 Apr 13; "PD-1 polymorphisms are associated with sporadic breast cancer in Chinese Han population of Northeast China "; Hua Z et al.

Human gene polymorphisms and SNPs
The inventor has assessed natural human variations in PD-1 and identified the following variations for application to the present invention.

Using this analysis, the inventor is useful in addressing diseases and conditions such as cancer, autoimmune or inflammatory diseases and conditions, the configurations, embodiments of the invention shown in Example 7. Concepts, examples and embodiments have been devised.

In certain embodiments, the human comprises an SNP selected from the SNPs in the first column of Table 23; optionally also this ligand is PD-1 encoded by a nucleotide sequence comprising said SNP. It specifically binds to.

In certain embodiments, the ligand (eg, trap, antibody or antibody fragment) specifically binds to PD-1 containing the corresponding variation as shown in Table 23 (Table 25) or Table 23 (Table 25). ) Is encoded by a nucleotide sequence containing or encoding an SNP or amino acid variation.

For example, this SNP is selected from rs36084323, rs10204225, rs11568821, rs2227981 and rs2227982. For example, this SNP is rs36084323. For example, this SNP is rs10204225. For example, this SNP is rs11568821. For example, this SNP is rs2227981. For example, this SNP is rs2227982.

In one example, this SNP and indication is an SNP / indication combination referred to in any of the references disclosed in this Example 7.

In one example, the indication is aplastic anemia, and this SNP is a G at that position in rs36084323.

(Example 7B)
Application of Autoimmunity, Inflammation and Other Diseases In some alternatives, instead of "methods of immunotherapy" or "methods of treating cancer," the invention is described as "autoimmunity," as shown in Example 7A. It may be read as "a method of treating or reducing the risk of a disease or condition" or "a method of treating or reducing the risk of an inflammatory disease or condition", and the TOI gene polymorphism is an autoimmune or inflammatory disease or condition. Related. Other non-cancer features of this example, which should be changed, are applicable to these alternatives. The TOI (eg, PD-1) contains the first gene polymorphism or is encoded by a nucleotide sequence containing the selected SNP.

For example, an autoimmune disease or condition is selected from the group consisting of rheumatoid arthritis, diabetes (eg, type I diabetes), Graves' disease, multiple sclerosis and systemic lupus erythematosus.

For example, this inflammatory disease or condition is selected from the group consisting of rheumatoid arthritis, rigid spondylitis, psoriasis, asthma, colitis and atopic dermatitis. For example, this method is to treat or reduce the risk of rigid spondylitis. In one example, this human is of Chinese (eg, CHS) pedigree.

Citations
(a) Vibanco-Perez N, Gutierrez-Franco J, Duran-Avelar M, Agraz-Cibrian J, Jimenez-Alvarez M, Pena-Virgen S and Zambrano-Zaragoza J (2013). Association of PD1.1 and PD1.6 polymorphisms with ankylosing spondylitis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). Doi: 10.3389 / conf.fimmu. 2013.02.0079.
Various SNPs in the PD1 gene have been identified, such as PD1.1 (rs36084323), PD1.3 (rs11568821), PD1.5 (rs2227981), and PD1.9 (rs2227982). Among these, PD1.3, PD1. 5, and PD1.9 have been associated with autoimmune disorders in different human populations. PD1.1 and PD1.6 are two single nucleotide polymorphisms, at -600 G / A (rs36084323), and +8669 G / A (rs10204225) respectively , that have been associated with others autoimmune diseases such as rheumatoid arthritis, type I diabetes mellitus, and systemic lupus erythematosus.
(b) Rheumatol Int. 2011 Feb; 31 (2): 209-13. doi: 10.1007 / s00296-009-1264-1. Epub 2009 Dec 12; "Programmed cell death 1 gene polymorphisms is associated with ankylosing spondylitis in Chinese Han population "; Liu X et al.
The frequencies of TC genotype and T allele of PD-1.9 were higher in the patients than those in the controls (P = 0.025). The frequencies of TC genotype and T allele of PD-1.9 were higher in the patients than those in the controls (P = 0.025). = 0.026 and 0.004). Moreover, the frequency of the CT haplotype (PD-1.5 C / T, PD-1.9 T / C) was significantly higher in AS patients than the controls (21.6 vs. 13.9%, P = 0.002). The CC haplotype was more common in the controls than in the patients.
(c) Tissue Antigens. 2003 Dec; 62 (6): 492-7; "Association of a putative regulatory polymorphism in the PD-1 gene with susceptibility to type 1 diabetes", Nielsen C et al.
Human PD-1 intronic 7146G / A SNP was found to be associated with the development of type 1 diabetes in humans.
(d) Inflammation. 2011 Dec; 34 (6): 707-12. doi: 10.1007 / s10753-010-9282-4; "Study of programmed cell death 1 (PDCD1) gene polymorphims in Iranian patients with ankylosing spondylitis", Soleimanifar N et al.
(e) Clin Exp Rheumatol. 2011 Jan-Feb; 29 (1): 13-8. Epub 2011 Feb 23; "Association of polymorphisms in the programmed cell death 1 (PD-1) and PD-1 ligand genes with ankylosing spondylitis in a Chinese population "; Yang Q et al.
(f) Arthritis Rheum. 2005 Jun; 52 (6): 1665-9; "Association of a PDCD1 polymorphism with renal manifestations in systemic lupus erythematosus"; Johansson M et al.
(g) Arthritis Rheum. 2005 Apr; 52 (4): 1058-62; "A new haplotype of PDCD1 is associated with rheumatoid arthritis in Hong Kong Chinese"; Kong EK et al.
(h) Ann Neurol. 2005 Jul; 58 (1): 50-7; "A PD-1 polymorphism is associated with disease progression in multiple sclerosis"; Kroner A et al.
(i) Leuk Lymphoma. 2013 Oct; 54 (10): 2251-4. Doi: 10.3109 / 10428194.2013.772605. Epub 2013 Mar 4; “Association between polymorphisms in PDCD1 gene and aplastic anemia in Chinese Han population; Wu Z et al ..

(Example 8)
Regulation of Iron The presence of multiple genetic disorders of iron metabolism suggests a genetic contribution to iron deficiency. To this end, the inventor has evaluated naturally occurring variations in human proteins of the iron homeostasis pathway, such as the hemoduverin-BMP6-hepcidin-ferroportin system.

BMP6 is also a bone morphogenetic protein 6, BMP-6, VGR-1, VG-1-R, VG-1-related protein, Vg1-related sequence, plant-related (TGFB-related) cytokine and plant-related growth factor. It is also known as (vegetal related growth factor) (TGFB related).

Matliptase-2 (also known as TMPRSS6) is an important regulator of the iron-regulating hormone hepcidin in the liver; matryptase-2 cleaves membrane-bound hemodaverin and results in bone morphogenetic protein (BMP ) Change signal transduction. Hemoduverin and hepcidin play important roles in retinal iron homeostasis; they are also expressed in the retina.

The present invention provides a method of iron homeostasis that comprises the step of administering an anti-TOI ligand to humans, the TOI being present in humans as multiple variants with different one or more amino acid gene polymorphisms. This method comprises the step of administering a ligand to a human, which comprises the first and second protein domains, wherein the first domain is the first amino acid gene. It specifically binds to a TOI variant containing the polymorphism, where this second domain comprises a second gene polymorphism, and where this human is (i) said the first amino acid gene polymorphism. TOI; and (ii) express a protein domain containing the second gene polymorphism. Particularly useful is the method in which the TOI is selected from the human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system.

A: Indications and humans optionally, this method increases or maintains increased blood iron, eg, to treat or reduce the risk of anemia; or to treat iron deficiency; or chronic To treat or reduce the risk of inflammatory anemia (ACI); or to treat or reduce the risk of anemia of chronic disease (ACD); or to treat or reduce the risk of cancer, kidney condition or GvHD-related anemia. To reduce; or to treat or reduce the risk of hemochromatosis.

In certain embodiments, the patient is a hemodialysis patient, and optionally the TOI is selected from the human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system (eg, the TOI is an A736V gene polymorphism). TMPRSS6 containing; or, for example, this human contains an A736V gene polymorphism).

In certain embodiments, the patient is a patient with coronary heart disease. In certain embodiments, the human suffers from an inflammatory disease or condition, such as acute acute inflammation.

In certain embodiments, this method is to treat or reduce the risk of hemochromatosis.

In certain embodiments, the method is to reduce or maintain reduced hepcidin levels in humans.

In certain embodiments, this method is to regulate (eg, increase; eg decrease) erythropoiesis in humans, optionally from the human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system. It is selected (eg, is the TOI TMPRSS6 containing the A736V gene polymorphism; or, for example, this human contains the A736V gene polymorphism).

In certain embodiments, this method is to treat or reduce the risk of iron overload in humans.

In certain embodiments, this method is to treat or reduce the risk of iron refractory iron deficiency anemia (IRIDA) in humans.

B: Human gene polymorphism and SNP
Any feature of this section B can be combined with any feature of section A above. For example, the human and / or TOI comprises an SNP as described below, and the method or ligand of the invention is according to one or more characteristics of A, comprising the step of administering an anti-TOI ligand.

The present inventor assessed the natural human variations of genes and proteins involved in iron homeostasis in humans and identified the following variations of interest for application to the present invention.

SNP rs111588693 encodes an amino acid in the region of the cleavage site of the precursor protein of BM6 and therefore it may be desired to target BMP6 encoded by the nucleotide sequence containing rs111588693 in humans, optionally. This human comprises a BMP6 nucleotide sequence containing said SNP.

In certain embodiments, the human comprises an SNP selected from the SNPs in the first column of Table 24; optionally also the ligand is a protein encoded by a nucleotide sequence comprising said SNP ( For example, it specifically binds to the human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system TOI).

In certain embodiments, the ligand (eg, trap, antibody or antibody fragment) is a TOI protein containing the corresponding variation as shown in Table 24 (eg, said human hemodaverin-BMP6-TMPRSS6-hepcidin). -It is encoded by a nucleotide sequence that specifically binds to a ferroptin-transferrin-based TOI) or contains or encodes an SNP or amino acid variation shown in Table 24.

For example, TOI is a human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin protein. In one example, this TOI is human hemodaverin. In one example, this TOI is human BMP6 (and optionally encoded by a BMP6 nucleotide sequence containing amino acid 28Q or SNP rs111588693; in certain embodiments, this human comprises said variation. BMP6 is expressed). In one example, this TOI is human hepcidin. In one example, this TOI is human ferroportin. In one example, the TOI is a human transferrin (and optionally encoded by a transferrin nucleotide sequence comprising SNP rs3811647; in certain embodiments, the human expresses transferrin comprising said variation). In one example, this TOI is human TMPRSS6 (and optionally contains one or more variations selected from the group consisting of A736V, Y141C, I212T, R271Q, S304L and C510S; or SNP rs855791 or rs1421312. Is encoded by a TMPRSS6 nucleotide sequence that comprises or encodes one or more variations selected from the group; in certain embodiments, the human expresses TMPRSS6 that includes the variation). For example, TMPRSS6 comprises variation A736V, and optionally this human expresses TMPRSS6 containing variation A736V. In one example, this TOI is human erythroferon. In one example, this TOI is the human hemochromatosis protein, HFE (and optionally encoded by an HFE nucleotide sequence containing amino acid 282Y or SNP rs1800562; in certain embodiments, this human is HFE containing the above variations is expressed). In some examples, the TOI is a product of a human CUBN nucleotide sequence, or the human comprises a CUBN nucleotide sequence, the nucleotide sequence comprising SNP rs10904850; in some embodiments, the human has said variation. Express the containing CUBN protein. CUBN is also known as a cubicilin and an endogenous factor cobalamin receptor. Ferroportin is also known as solute transporter family 40 member 1 (SLC40A1) or iron regulated transporter 1.
Hemoduverin is also known as hemoduverin, HFE2A, HJV, JH and RGMC.

In one example, this human comprises SNP rs2698530 on chr.2p14 and / or rs987710 on chr.22q11.

It may be desirable to block TOI in this alternative method (not necessarily as clear as the IgG1 format), and human IgG4 may be suitable for doing this in iron-regulated situations. Thus, in certain embodiments, the ligand comprises a human gamma-4 constant region. For iron regulation situations of the present invention, it may be advantageous for gamma-4 to be a gamma-4 steady region with 228Pro or 235Glu. In one example, the constant region is IgG4PE (ie, a gamma-4 constant region with 228Pro and 235Glu). In one example, this ligand comprises the human IGHG4 * 01 hinge S10> P constant region.

Thus, in one example, this ligand comprises any human gamma-4 constant region disclosed herein (see, eg, any of Example 1 below).

Citations
(a) Nutr Hosp. 2012 Nov-Dec; 27 (6): 2142-5. doi: 10.3305 / nh.2012.27.6.6154; "Intron SNP rs3811647 of the human transferrin gene modulates its expression in hepatoma cells"; Blanco-Rojo R et al.
Transferrin (Tf) exerts a crucial function in the maintenance of systemic iron homeostasis. The expression of the Tf gene is controlled by transcriptional mechanism. The authors conclude that A allele of SNP rs3811647 increases Tf expression in a manner that might underlie inter-individual variation in serum transferrin levels observed in different population groups.
(b) J Med Genet. 2013 Sep; 50 (9): 593-8. doi: 10.1136 / jmedgenet-2013-101673. Epub 2013 Jun 21; "Associations of common variants in HFE and TMPRSS6 with iron parameters are independent of serum hepcidin in a general population: a replication study "; Galesloot TE et al.
The authors found that HFE rs1800562 and TMPRSS6 rs855791 are the main determinants of HFE and TMPRSS6 related variation in serum iron, ferritin, transferrin saturation, and total iron binding capacity.
(c) BMC Nephrol. 2013 Feb 22; 14:48. Doi: 10.1186 / 1471-2369-14-48; "The A736V TMPRSS6 polymorphism influences hepcidin and iron degradation in chronic hemodialysis patients: TMPRSS6 and hepcidin in hemodialysis"; Pelusi S et al.
The authors reported the TMPRSS6 736 V variant was associated with higher hepcidin levels (p = 0.017). In patients without acute inflammation and overt iron deficiency (C reactive protein < 1 mg / dl and ferritin> 30 ng / ml; n = 86), hepcidin was associated with lower mean corpuscular volume (p = 0.002), suggesting that it contributed to iron-restricted erythropoiesis. In line with previous results, in patients without acute inflammation and severe iron deficiency the "high hepcidin" 736 V TMPRSS6 variant was associated with higher erythropoietin maintenance dose (p = 0.016), independently of subclinical inflammation (p = 0.02).
(d) PLoS One. 2012; 7 (6): e38339. doi: 10.1371 / journal.pone.0038339. Epub 2012 Jun 22; "Associations between single nucleotide polymorphisms in iron-related genes and iron status in multiethnic populations"; McLaren CE et al.
Three chromosomal regions showed association across multiple populations, including SNPs in the TF and TMPRSS6 genes, and on chromosome 18q21. A novel SNP rs1421312 in TMPRSS6 was associated with serum iron in whites (p = 3.7 × 10 (-6)) and replicated in African Americans (p = 0.0012). Twenty SNPs in the TF gene region were associated with total iron-binding capacity in whites (p <4.4 x 10 (-5)); six SNPs replicated in other ethnicities (p <0.01). SNP rs10904850 in the CUBN gene on 10p13 was associated with serum iron in African Americans (P = 1.0 × 10 (-5)).
(e) PLoS One. 2011 Mar 31; 6 (3): e17390. doi: 10.1371 / journal.pone.0017390; "Genome-wide association study identifies genetic loci associated with iron deficiency"; McLaren CE et al.
Five SNPs were identified in a Genome-Wide Association Study that met genome-wide statistical significance for association with at least one iron measure, rs2698530 on chr. 2p14; rs3811647 on chr. 3q22, a known SNP in the transferrin (TF) gene region rs1800562 on chr. 6p22, the C282Y mutation in the HFE gene; rs7787204 on chr. 7p21; and rs987710 on chr. 22q11 (GWAS observed P <1.51 × 10 (-7) for all). An association was observed between total iron binding capacity and SNP rs3811647 in the TF gene (GWAS observed P = 7.0 × 10 (-9). The analysis revealed strong associations between rs2698530 on chr. 2p14 and iron status outcomes.
(f) Hum Mutat. 2010 May; 31 (5): E1390-405. Doi: 10.1002 / hmu.21243; "Novel TMPRSS6 mutations associated with iron-refractory iron deficiency anemia (IRIDA)"; De Falco L et al.
Mutations leading to abrogation of matriptase-2 proteolytic activity in humans are associated with an iron-refractory iron deficiency anemia (IRIDA) due to elevated hepcidin levels. The authors describe 12 IRIDA patients belonging to 7 unrelated families and identify 10 (9 novel) TMPRSS6 mutations spread along the gene sequence: 5 missense, 1 non sense and 4 frameshift. The frameshift and non sense mutations are predict to result in truncated protein lacking the catalytic domain. The causal role of missense mutations (Y141C, I212T, R271Q, S304L and C510S) is demonstrated by in silico analysis, their absence in 100 control chromosomes and the high conservation of the involved residues. The C510S mutation in the LDLRA domain in silico model causes an intra-molecular structural imbalance that impairs matriptase-2 activation. We also assessed the in vitro effect on hepcidin promoter and the proteolytic activity of I212T and R271Q variants demonstrating a reduced inhibitory effect for the f ormer mutation, but surprisingly a normal function for R271Q which appears a silent mutation in vitro. Based on mRNA expression studies I212T could also decrease the total amount of protein produced, likely interfering with mRNA stability. Collectively, our results extend the pattern of TMPRSS6 mutations associated with IRIDA and propose a model of causality for some of the novel missense mutation.

The present invention provides the following concepts:-
1. A method of treating or reducing the risk of a disease or condition in humans, wherein the disease or condition is mediated by a TOI, wherein the TOI is a plurality of variants in which one or more amino acid gene polymorphisms differ. As present in humans, the method comprises the step of administering a ligand (eg, an antibody or antibody fragment) to the human, which contains the first and second protein domains. Here, this first domain specifically binds to a TOI variant containing the first amino acid gene polymorphism, where this second domain contains the second gene polymorphism, and where this human (I) expresses a TOI containing the first amino acid gene polymorphism; and (ii) expresses a protein domain containing the second gene polymorphism, and optionally this ligand is found in SEQ ID NO: 73189. A human gamma-4 heavy chain constant region containing Leu at position or Arg at position 289 found in SEQ ID NO: 73, wherein said human contains or comprises the IGHG4 * 01 human heavy chain constant region gene segment. Humans express an antibody containing a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73; and where this second domain is , Included by the gamma-4 heavy chain constant region of this ligand.

Optionally, this TOI is selected from the human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system. In one example, this TOI is human hemodaverin. In one example, this TOI is human BMP6. In one example, this TOI is human TMPRSS6. In one example, this TOI is human hepcidin. In one example, this TOI is human ferroportin. In one example, this TOI is human HFE. In one example, this TOI is human transferrin. The TOI of the "human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system" is a human protein (eg, expressed by the liver) that is part of an iron regulatory pathway that includes hemoduverin, BMP6, TMPRSS6, hepcidin, ferroportin and transferrin. Is done).

For example, this method may be for either the purpose or indication set forth in Section A. In one example, this method is to regulate iron in humans. In some examples, this iron is blood iron or circulating iron or free iron.

In one example, this method is a method of reducing anemia in humans.

2. The method of Concept 1 in which the first and / or second gene polymorphisms are encoded by SNPs, each with a cumulative human allelic frequency of less than 50%.
Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

3. The method of any of the aforementioned concepts in which the first domain is the antibody variable domain or receptor domain.

4. The method of any of the aforementioned concepts in which the second domain is the antibody domain, optionally the variable domain or constant domain of the antibody.

5. The method of any of the aforementioned concepts, where the second domain is the domain of the antibody Fc region.

6. The first and second domains are antibody variable domains, and optionally, this variable domain specifically binds to the first and second human TOIs, respectively, where this TOI is different, the concept. Any one method from 1 to 4.

Optionally, this second TOI is selected from the human hemoduverin-BMP6-TMPRSS6-hepcidin-ferroportin-transferrin system. In one example, this second TOI is human hemodaverin. In one example, this second TOI is human BMP6. In one example, this second TOI is human TMPRSS6. In one example, this second TOI is human hepcidin. In one example, this second TOI is human ferroportin. In one example, this second TOI is human HFE. In one example, this second TOI is human transferrin.

7. The concept that the first and second domains are receptor domains, and optionally, these receptor domains specifically bind to the first and second human TOIs, respectively, where this TOI is different. Any one method from 1 to 4.

8. This first domain contains a third amino acid gene polymorphism, wherein the human expresses (iii) the domain containing the third amino acid gene polymorphism, the method of concept 6 or 7. ..
Optionally, this human expresses a second TOI variant.

Optionally, this third genetic polymorphism is encoded by an SNP with a cumulative human frequency of less than 50%. Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

In one example, this first domain is an antibody variable domain (eg, any V domain of the invention described herein, such as human VH3-23 * 04 described herein). And the domain (iii) is an antibody variable domain. For example, this first domain and the domain of (iii) are VH domains. For example, the domain of the first domain and (iii) is a V _K domain. For example, this first domain and the domain of (iii) are Vλ domains.

In one example, this first domain is the receptor binding site and the domain (iii) is the receptor binding site domain.

9. Combined concepts 1, 2 and 8 methods.

10. The method of any of the aforementioned concepts in which humans express the first and / or second domain.

11. Any method of the aforementioned concept in which the first, second and / or third genetic polymorphisms are found in at least 5 different human populations, respectively, as defined in Table 4 (Table 5).
For example, each of the first and second genetic polymorphisms is found in at least five different human populations, as defined in Table 4, respectively. For example, each of the first, second and genetic polymorphisms is found in at least five different human populations, as defined in Table 4 (Table 5), respectively.

Optionally, this first, second and / or third (see below) polymorphism is at least 9, 10, 11, as defined in Table 4, respectively. Found in 12, 13, 14, 15, 16, 17, 18, 19 or 20 different human populations.

12. Concept 11 method in which the first, second and / or third gene polymorphisms are found in at least 15 humans in the 1000 Genomes Phase I database, respectively.
In certain embodiments, the first, second and / or third genetic polymorphisms are distributed over many such different human populations at least 20, 25, 30, 35, 40, 45, 50, 55. , 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140 or 150 cases of human individuals.

13. Cumulative humans with first, second and / or third genetic polymorphisms found in at least 5 different human populations, respectively, as defined in Table 4 and less than 50% A method of any aforementioned concept encoded by an SNP having an allelic frequency.
Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

14. The method of any of the aforementioned concepts, wherein the first, second and / or third gene polymorphisms are encoded by SNPs, each with a total human genotype frequency of less than 50%.
Optionally, this frequency is less than 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1%.

15. The method of any of the aforementioned concepts, wherein the first and second domains are human and, optionally, the ligand is fully human.

16. The method of any of the aforementioned concepts, wherein the ligand is an anti-TOI trap, antibody or antibody fragment.

17. The method of any of the aforementioned concepts, wherein the ligand specifically binds to two or more human TOIs, eg, the first and second TOIs.

18. The method of any of the aforementioned concepts, wherein the ligand is administered to a human by injection or the ligand is provided by an injectable preparation.

19. The human has been or has been genotyped positive for the TOI variant nucleotide sequence prior to administration of the ligand, or the method is prior to administration of the ligand. , A method of any of the aforementioned concepts, comprising the step of genotyping a human as positive for the variant nucleotide sequence.

20. Humans have been or have been genotyped positive for the TOI variant prior to administration of the ligand, or the method is said to be prior to administration of the ligand. A method of any of the aforementioned concepts, comprising the step of phenotyping a human as positive with respect to.

21. The method of any of the aforementioned concepts, comprising the step of selecting a human containing the first TOI gene polymorphism prior to the administration step, wherein this human is a human of Concept 1.

22. The method of any of the aforementioned concepts in which humans have been determined to contain a first and / or second TOI variant, or a nucleotide sequence encoding a TOI variant.

23. Including the step of determining that a human contains a first TOI nucleotide sequence encoding a first gene polymorphism, optionally, this determination step is performed prior to administration of the ligand to humans. , Any of the aforementioned conceptual methods.

24. The method of concept 23, wherein the determining step comprises assaying a human-derived biological sample for said nucleotide sequence.

25. The step of assaying is a biological sample and
At least one oligonucleotide probe that can be specifically hybridized and identified in a biological sample to a nucleotide sequence containing an SNP encoding a first gene polymorphism, or of said sequence. It contains a sequence of at least 10 contiguous nucleotides that specifically hybridize to the antisense, where the nucleic acid either hybridizes to the SNP or hybridizes to the antisense sequence. With an oligonucleotide probe that forms a complex when soybeaned and thereby at least one nucleotide sequence containing said SNP is present; and / or
b. At least one oligonucleotide probe comprising the sequence of said contiguous nucleotides of a nucleotide sequence comprising said SNP or containing an antisense sequence of at least 10 contiguous nucleotides, wherein the contiguous nucleotides. In the step of contacting the oligonucleotide probe forming the complex; and the step of detecting the presence or absence of the complex when the sequence contains the SNP and thereby the nucleotide sequence containing the SNP is present. A method of concept 24, which comprises the step of determining that a human contains a first TOI nucleotide sequence by detecting the presence of this complex here.

26. The assaying step comprises or optionally comprises one or more methods selected from nucleic acid amplification and optionally sequencing, next generation sequencing, nucleic acid hybridization and allele-specific amplification. Concept 24 method in which the step of assaying is performed in a multiplex format.

27. The method of any of the aforementioned concepts in which the human is substantially resistant to, or is further determined to be, substantially resistant to the treatment of anemia.

28. The method of any of the aforementioned concepts in which the human has, or has received, or is less responsive to anemia treatment.

29. The method of Concept 25, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

30. The method of any of the aforementioned concepts in which the human has been diagnosed with the disease or condition.

31. Anti-TOI ligands (eg, antibodies, antibody fragments or traps) for use in any of the aforementioned conceptual methods.
In certain embodiments, the trap is a receptor Fc fusion (ie, a receptor domain fused to an antibody Fc region, such as a dimer), where the receptor is specific to the TOI. Join.

32. Ligand of concept 31, which is completely human.

33. Ligand of concept 31 or 32 for administration by injection or provided by an injectable preparation.

In one example, the first, second and / or third gene polymorphisms are:-
h. Has a cumulative human allele frequency of 15% or less;
i. Have a total human genotype frequency of 20% or less;
j. Found in at least 5 different human populations (eg, using the standard classification of the 1000 Genomes Project, as in the Phase I database);
k. Found in many individuals distributed across many such different populations.

In some examples, this criterion applies in connection with one or more human genome sequence databases as described herein. For example, this criterion is exactly what applies to the 1000 Genomes Database.

For example, in an example, embodiment or configuration of any aspect of the invention, the 1000 Genomes Database exposes 13 or Phase I. For example, as of October 1, 2013, its most recent version of the 1000 Genomes Database.

In one example, this human is homozygous for the first genetic polymorphism. Additional or alternative, this human is homozygous for the first genetic polymorphism. Additional or alternative, this human is homozygous for a third genetic polymorphism. For example, this human is homozygous for the first and second polymorphisms.

In one example, this method is by any one of the disclosures herein comprising selecting, genotyping or phenotyping a human as positive for a TOI variant (ie, in the case of the present invention). Human PD-1 contains a genotype or a nucleotide sequence encoding it). In some examples, the method is by any one of the above concepts comprising the step of determining, or where the human is determined to include a variant TOI variant (eg, in the case of the present invention). , Human PD-1 contains a genetic polymorphism or a nucleotide sequence encoding it).

In some examples, this first genetic polymorphism is either the human BMP6 amino acid gene polymorphism described herein or encoded by the human BMP6 SNP described herein.

In one example, where this TOI is human BMP6, this first genetic polymorphism is encoded by SNP rs111588693.

The present invention further provides the following specific embodiments:-
1. A method of treating or reducing the risk of human anemia by targeting a human TOI selected from the group consisting of BMP6, ferroportin, hemoduverin, hepcidin and TMPRSS6 in humans, which is a method for treating or reducing the risk of human anemia. It comprises the step of administering an antibody or antibody fragment to the antibody or antibody fragment, wherein the antibody or antibody fragment specifically binds to the variant of the TOI containing the first amino acid gene polymorphism, wherein the antibody or antibody fragment Includes a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human weight. The chain constant region gene segment (or this human expresses an antibody containing a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73); and (ii) Includes a PD-1 nucleotide sequence encoding PD-1 containing the first amino acid gene polymorphism (or this human expresses PD-1 containing the first amino acid gene polymorphism).

Preferably, the trap, antibody or fragment of any configuration, concept, embodiment, example or embodiment comprises an IgG4PE constant region.

An anti-TOI (eg, anti-BMP6) antibody is used in any configuration, concept, embodiment, example or embodiment.

The first aspect provides:-
A method of treating or reducing the risk of anemia in humans, the method comprising administering to a human an anti-BMP6 trap, antibody or antibody fragment, wherein the trap, antibody or antibody fragment is: It specifically binds to human BMP6 encoded by a BMP6 nucleotide sequence containing SNP rs111588693, where this trap, antibody or antibody fragment is located at Leu at position 189 or position 289 found in SEQ ID NO: 73. Contains a human gamma-4 heavy chain constant region containing Arg, wherein the human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is at position 189 found in SEQ ID NO: 73). Human gamma containing Arg at position 289 found in Leu or SEQ ID NO: 73 expresses antibodies containing the 4-heavy chain constant region); and (ii) contains the BMP6 nucleotide sequence containing the SNP; optionally, this human , The method is determined to include the SNP, or the method comprises the step of determining that a human contains the SNP prior to administration of the antibody or fragment.

The second aspect provides:-
A method of treating or reducing the risk of anemia in humans, the method comprising administering to a human an anti-hemoduverin trap, antibody or antibody fragment, wherein the trap, antibody or antibody fragment. Specifically binds to human hemoduverin encoded by a hemoduverin nucleotide sequence containing SNP rs7540883, where this trap, antibody or antibody fragment is Leu at position 189 or SEQ ID NO: 73 found in SEQ ID NO: 73. Includes a human gamma-4 heavy chain constant region containing Arg at position 289 found in, where the human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is at SEQ ID NO: 73). It expresses an antibody containing a human gamma-4 heavy chain constant region containing Leu at position 189 or Arg at position 289 found in SEQ ID NO: 73); and (ii) contains a hemodoberin nucleotide sequence containing the SNP. Optionally, the human is determined to contain the SNP, or the method comprises the step of determining that the human contains the SNP prior to administration of the antibody or fragment.

The third aspect provides:-
A method of treating or reducing the risk of anemia in humans, the method comprising administering to a human an anti-ferroptin trap, antibody or antibody fragment, wherein the trap, antibody or antibody fragment is: It specifically binds to human ferroportin encoded by a ferroportin nucleotide sequence containing SNP rs11568350, where this trap, antibody or antibody fragment is at Leu at position 189 or SEQ ID NO: 73 found in SEQ ID NO: 73. A human gamma-4 heavy chain constant region containing Arg at position 289, which is found, wherein the human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is found in SEQ ID NO: 73 only. (Expressing an antibody containing a human gamma-4 heavy chain constant region containing Leu at position 189 or Arg at position 289 found in SEQ ID NO: 73); and (ii) containing a ferroportin nucleotide sequence containing the SNP; Optionally, the human is determined to contain the SNP, or the method comprises the step of determining that the human contains the SNP prior to administration of the antibody or fragment.

The fourth aspect provides:-
A method of treating or reducing the risk of anemia in humans, the method comprising administering to a human an anti-TMPRSS6 trap, antibody or antibody fragment, wherein the trap, antibody or antibody fragment is: It specifically binds to human TMPRSS6 encoded by a TMPRSS6 nucleotide sequence containing an SNP selected from the group consisting of rs855791, rs1421312, rs2543519, rs2235324, where this trap, antibody or antibody fragment is found in SEQ ID NO: 73. It contains a human gamma-4 heavy chain constant region containing Leu at position 189 or Arg at position 289 found in SEQ ID NO: 73, wherein the human is (i) IGHG4 * 01 human heavy chain constant region gene segment (or). This human expresses an antibody containing a human gamma-4 heavy chain constant region containing Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73); and (ii) selected above. Includes a TMPRSS6 nucleotide sequence containing an SNP; optionally, this human has been determined to contain the SNP, or the method indicates that the human contains the SNP prior to administration of this antibody or fragment. Includes the step of determining.

The fifth aspect provides:-
A method of treating or reducing the risk of anemia in humans, the method comprising administering to humans an anti-hepcidin trap, antibody or antibody fragment, wherein the trap, antibody or antibody fragment is SNP rs146776859. It specifically binds to human hepsidin encoded by a hepsidin nucleotide sequence containing, where this trap, antibody or antibody fragment is located at Leu at position 189 found in SEQ ID NO: 73 or Arg at position 289 found in SEQ ID NO: 73. Contains a human gamma-4 heavy chain constant region comprising (i) IGHG4 * 01 human heavy chain constant region gene segment (or this human is Leu at position 189 found in SEQ ID NO: 73 or Human gamma containing Arg at position 289 of SEQ ID NO: 73-expresses an antibody containing a heavy chain constant region); and (ii) contains a hepcidin nucleotide sequence containing said SNP; optionally, this human is said to be said. A method comprising the step of determining that an SNP is included, or that the method comprises the SNP prior to administration of the antibody or fragment.

2. The method of embodiment 1, wherein the human is homozygous for the selected SNP.

3. The method of any of the aforementioned embodiments, wherein the ligand comprises an IGHG4 * 01 human heavy chain constant region, eg, where this constant region is a gamma-4 constant region having 228 Pro and / or 235 Glu.

4. The method of any of the aforementioned embodiments in which the anemia is selected from the group consisting of chronic inflammatory anemia (ACI); anemia of chronic disease (ACD); or anemia associated with cancer, renal status or GvHD.

5. Prior to the step of administration, the step of selecting a human containing the BMP6, ferroportin, hemoduverin, TMPRSS6 or hepcidin nucleotide sequence of (ii), wherein the human is a human of embodiment 1, as described above. Aspect method.

6. The method of any of the aforementioned embodiments in which it has been determined that a human comprises the hepcidin nucleotide sequence of BMP6, ferroportin, hemoduverin, TMPRSS6 or (ii).

7. The step comprising determining that the human comprises the hepcidin nucleotide sequence of BMP6, ferroportin, hemoduverin, TMPRSS6 or (ii), optionally this determining step prior to administration of the antibody to this human. The method of any of the aforementioned embodiments made in.

8. The method of embodiment 7, wherein the step of determining comprises assaying a human-derived biological sample for a hepcidin nucleotide sequence containing BMP6, ferroportin, hemoduverin, TMPRSS6 or the selected variation, respectively.

9. The assay process is a biological sample and
aa At least one oligonucleotide probe that can be specifically hybridized and identified in a biological sample to a nucleotide sequence containing the selected variation, or specific to the antisense of said sequence. Contains a sequence of at least 10 contiguous nucleotides that hybridize with the nucleic acid, where the nucleic acid either hybridizes to the selected SNP or hybridizes to an antisense sequence. If at least one nucleotide sequence containing the selected SNP is present thereby, with an oligonucleotide probe forming a complex; and / or
bb At least one oligonucleotide probe comprising the sequence of said contiguous nucleotides of a nucleotide sequence containing the selected SNP or containing an antisense sequence of at least 10 contiguous nucleotides, contiguous here. The step of contacting; with an oligonucleotide probe forming a complex, if the sequence of nucleotides comprises the selected SNP and thereby a nucleotide sequence containing the selected SNP is present, and
c. A step of detecting the presence or absence of this complex, wherein by detecting the presence of this complex, humans can obtain a hepcidin nucleotide sequence containing BMP6, ferroportin, hemodoverin, TMPRSS6 or the selected SNP, respectively. The method of aspect 8 comprising the steps determined to include.

10. The assaying step comprises or / or includes nucleic acid amplification and, optionally, one or more methods selected from sequencing, next-generation sequencing, nucleic acid hybridization and allele-specific amplification. The method of embodiment 9 in which the step of assaying is performed in a multiplex format.

11. The method of any of the aforementioned embodiments, wherein the human is substantially resistant to anemia treatment (eg, EPO) or further determined to be substantially resistant.
For example, this human has been further determined to be resistant or resistant to anemia treatments (eg, EPO).

12. The method of any of the aforementioned embodiments, wherein the human has undergone or has undergone an anemic treatment (eg, EPO) or has had reduced responsiveness to the anemic treatment (eg, EPO).

13. The method of embodiment 8, wherein the biological sample comprises the human serum, blood, feces, tissue, cells, urine and / or saliva.

14. The method of any of the aforementioned embodiments, wherein the trap, antibody or fragment is administered by intravenous or subcutaneous injection and / or is included in the preparation for injection.

15. The method of any of the aforementioned embodiments, wherein the trap, antibody or fragment is human, eg, a human antibody or antibody fragment.

16. An anti-human TOI trap, antibody or antibody fragment for use in any of the aforementioned embodiments where the TOI is selected from BMP6, ferroportin, hemoduverin, TMPRSS6 and hepcidin.

In certain embodiments, the trap is a hepmodaverin-or receptor-Fc fusion (ie, a receptor domain fused to an antibody Fc region, such as a dimer), where the receptor is BMP6. , Ferroportin, hemoduverin, TMPRSS6 and hepcidin, specifically bind to or compete with the TOI.

17. Ligand of embodiment 17, wherein the trap, antibody or antibody fragment is completely human.

18. Ligand of embodiment 16 or 17, wherein the trap, antibody or antibody fragment is for administration by injection, or the trap, antibody or antibody fragment is provided by an injectable preparation.

Optionally, in any of the configurations, concepts, aspects, embodiments or examples, one or more of the following optional features apply:

Optionally, this ligand (eg, trap, antibody or antibody fragment) depends on one of the following options:-
(i) Here, the ligand comprises a VH domain derived from the recombination of a human VH segment (eg, human VH3-23 * 04), a human D gene segment and a human JH segment, the human VH segment having SEQ ID NO: 40 Framework 1 is encoded, and where the human comprises the VH gene segment encoding Framework 1 of SEQ ID NO: 40, or this human is the Framework 1 of SEQ ID NO: 40. Express the VH domain containing.
(ii) Here, this ligand contains a VH domain derived from recombination of the human VH segment, the human D gene segment and the human JH segment selected from the group consisting of IGHV3-33 * 01 and IGHV3-7 * 01. And here, the human comprises the selected VH gene segment, or the human expresses the VH domain derived from the recombination of the selected human VH segment, the human D gene segment and the human JH segment. ..
Here (iii), the ligand comprises an IGKV3-11 * 01 and IGKV1-12 * 01 human V _K segments and V _K domains from recombinant human J _K segment is selected from the group consisting of, and, Here, the human comprises the selected V _K gene segment, or the human expresses the V _K domain derived from the recombination of the selected human V _K segment and the human J _K segment.
(iv) Here, this ligand contains a V _K domain derived from the recombination of the human V _K segment and the human J _K segment, and this human V _K segment is (i) Pro at position 7 shown in SEQ ID NO: 36. Containing CDR3 (and where said human contains the V _K gene segment encoding CDR3 containing Pro at position 7 set forth in SEQ ID NO: 36, or this human is located at position 7 set forth in SEQ ID NO: 36. (Expressing a V _K domain containing CDR3 containing Pro); or (ii) FW3 containing the Ser at position 15 shown in SEQ ID NO: 38 (and where the human is the Ser at position 15 shown in SEQ ID NO: 38). Contains a V _K gene segment encoding FW3 containing, or this human expresses a V _K domain containing FW3 containing Ser at position 15 set forth in SEQ ID NO: 38).

Optionally, instead of the gamma-4 constant region, this ligand (eg, trap, antibody or fragment) comprises a heavy chain constant region with one of the following options:-
(v) Here, the ligand comprises a human gamma-1 heavy chain constant region comprising Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4, and the human is here. , (I) IGHG1 * 01 Human gamma-1 weight containing the human heavy chain constant region gene segment, or this human containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4. Express an antibody that contains a chain constant region.
(vi) Here, this ligand is shown in Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, and 161 shown in SEQ ID NO: 6. It comprises a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Val at position Val and Ala at position 257 set forth in SEQ ID NO: 6, where the human is (i) IGHG2 * 01 human weight. This human contains the chain constant region gene segment, or this human is Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, and Phe at position 76 shown in SEQ ID NO: 6. , Val at position 161 shown in SEQ ID NO: 6 or Ala at position 257 shown in SEQ ID NO: 6 and expresses an antibody containing a human gamma-2 heavy chain constant region. In one example, this constant region is Fc-enhanced, eg ADCC or CDC-enhanced. Those skilled in the art are known for techniques for enhancing ADCC or CDC (eg, known are C region mutations).
(vii) Here, this ligand comprises a human gamma-3 heavy chain constant region encoded by a first human IGHG3 (eg, IGHG3 * 01) constant region gene segment, wherein the human is (i). ) Includes the first constant region gene segment (eg, IGHG3 * 01), or this human is a human gamma-3 multiple encoded by the first human IGHG3 (eg, IGHG3 * 01) constant region gene segment. Express an antibody that contains a chain constant region.

Optionally, this ligand (eg, antibody or antibody fragment) depends on one of the following options:-
(viii) Here, this ligand comprises a human kappa chain constant region comprising Val at position 84 set forth in SEQ ID NO: 16 or Cys at position 87 set forth in SEQ ID NO: 16, where the human is (i). ) IGKC1 * 01 Human kappa chain constant region Contains a gene segment, or this human contains a human kappa chain constant region containing Val corresponding to position 84 shown in SEQ ID NO: 16 or Cys at position 87 shown in SEQ ID NO: 16. Express the antibody.
(ix) Here, this ligand comprises a human IGLC1 * 01 lambda chain constant region, wherein the human comprises (i) a human IGLC1 * 01 lambda chain constant region gene segment, or this human is , Human IGLC1 * 01 Expresses an antibody containing a lambda chain constant region.
(x) Here, the ligand comprises a human epsilon heavy chain constant region encoded by a first human epsilon heavy chain constant region gene segment, wherein the human is (i) said the first constant. A region gene segment is included, or the human expresses an antibody comprising a human epsilon heavy chain constant region encoded by the first constant region gene segment.
(xii) Here, the ligand comprises a human mu heavy chain constant region encoded by a first human mu heavy chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human mu heavy chain constant region encoded by the first constant region gene segment.
(xiii) Here, the ligand comprises a human alpha heavy chain constant region encoded by a first human alpha heavy chain constant region gene segment, wherein the human is (i) said the first constant. A region gene segment is included, or the human expresses an antibody comprising a human alpha heavy chain constant region encoded by the first constant region gene segment.
(xiv) Here, the ligand comprises a human delta heavy chain constant region encoded by a first human delta heavy chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human delta heavy chain constant region encoded by the first constant region gene segment.
(xv) Here, this ligand comprises the human kappa light chain constant region encoded by the first human kappa light chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human kappa light chain constant region encoded by the first constant region gene segment.
(xvi) Here, this ligand comprises a human lambda light chain constant region encoded by a first human lambda light chain constant region gene segment, wherein the human is (i) said the first constant region gene. Contains a segment, or the human expresses an antibody comprising the human lambda light chain constant region encoded by the first constant region gene segment.

Claims (22)

An anti-TOI ligand for use in humans in a manner that treats or prevents a target (TOI) -mediated disease or condition that exists as a different polymorphic variant in humans, said method to humans. Including the step of administering the ligand, the ligand was encoded by a nucleotide sequence containing an SNP (first SNP) having a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. A ligand that specifically binds to a TOI variant; the human comprises a nucleotide sequence encoding a TOI containing said SNP. A method of treating or preventing a target (TOI) -mediated disease or condition in humans that is present as a different polymorphic variant in humans, wherein less than 50% of the cumulative human allele frequency and / Alternatively, it comprises the step of administering an anti-TOI ligand that specifically binds to a TOI variant encoded by a nucleotide sequence containing an SNP (first SNP) having a total human genotype frequency of less than 50%; said human. A method comprising a nucleotide sequence encoding a TOI containing an SNP. Less than 50% cumulative human allele frequency and / or 50% in the manufacture of pharmaceuticals for use in methods of targeting targeted targets (TOIs) in humans and treating or preventing TOI-mediated diseases or conditions. The use of an anti-TOI ligand that specifically binds to a human TOI variant comprising an amino acid sequence encoded by a TOI nucleotide sequence comprising an SNP (first SNP) having a total human genotype frequency of less than, said TOI. , Present as different polymorphic variants in humans, said humans comprising a nucleotide sequence encoding a TOI containing said SNP, used. The ligand comprises a heavy chain gamma-1,2,3 or 4 constant region encoded by a nucleotide sequence containing the second SNP, and the human has a heavy chain gamma-1,2 containing the second SNP. , 3 or 4 The ligand, method or use according to any one of claims 1 to 3, which comprises 3 or 4 constant regions, respectively. The ligand comprises a human antibody variable domain derived from a recombination of a human V gene segment and a human J gene segment (and optionally, if the variable domain is a VH domain, a human D gene segment); the human genome , And / or the human expresses an antibody comprising an antibody variable domain derived from the recombination of the human V gene segment and the human J gene segment (and optionally the human D gene segment). , The ligand, method or use according to any one of claims 1 to 4. The ligand, method or use according to any one of claims 1 to 5, wherein each SNP encodes a non-synonymous amino acid variation and / or each SNP encodes an amino acid that is surface-exposed on a target. .. The ligand, method or use according to any one of claims 1 to 6, wherein the ligand is an antibody or an antibody fragment; or the ligand is a trap. The method comprises determining that the human is positive for the TOI variant, and optionally the determining step determines that the human is positive for the nucleotide variant encoding the TOI variant. The ligand, method or use according to any one of claims 1 to 7, which comprises a step. The ligand specifically binds to two or more different TOI variants, each encoded by a nucleotide sequence containing an SNP with a cumulative human allele frequency of less than 50% and / or a total human genotype frequency of less than 50%. The ligand, method or use according to any one of claims 1 to 8, wherein it is possible. The ligand, method or use according to any one of claims 1 to 9, wherein the TOI variant has been determined or is determined to be present in at least two different human ethnic populations. Any one of claims 1-10, wherein the cumulative human allele frequency is a frequency in a database of naturally occurring sequences containing at least 1000 sequences sampled from at least 15 different human populations. The ligand, method or use described in the section. A kit for treating or preventing a TOI-mediated condition or disease as defined in any one of claims 1 to 11, comprising said ligand and having a first SNP of at least two different ethnic populations. Found in; optionally combined with a label or instruction for use in the treatment and / or prevention of the disease or condition in humans; optionally, the label or instruction is issued by the regulatory agency. Includes license number; optionally, kit containing injection pen or IV container containing ligand. The ligand comprises a human gamma heavy chain CH1 domain encoded by a CH1 nucleotide sequence; the human genome comprises a gamma heavy chain constant region nucleotide sequence identical to the CH1 nucleotide sequence and / or said human. The ligand, method, use or kit according to any one of claims 1 to 12, which expresses an antibody comprising the human gamma CH1 domain. The ligand comprises a human gamma heavy chain CH2 domain encoded by a CH2 nucleotide sequence; the human genome comprises a gamma heavy chain constant region nucleotide sequence identical to the CH2 nucleotide sequence and / or said human. The ligand, method, use or kit according to any one of claims 1 to 13, which expresses an antibody containing the human gamma CH2 domain. The ligand comprises a human gamma heavy chain CH3 domain encoded by a CH3 nucleotide sequence; the human genome comprises a gamma heavy chain constant region nucleotide sequence identical to the CH3 nucleotide sequence and / or said human. The ligand, method, use or kit according to any one of claims 1 to 14, which expresses an antibody comprising the human gamma CH3 domain. The ligand comprises a human gamma heavy chain CH4 domain encoded by a CH4 nucleotide sequence; the human genome comprises a gamma heavy chain constant region nucleotide sequence identical to the CH4 nucleotide sequence and / or said human. The ligand, method, use or kit according to any one of claims 1 to 15, which expresses an antibody containing the human gamma CH4 domain. The ligand comprises a human gamma heavy chain Fc region encoded by an Fc nucleotide sequence; the human genome comprises a gamma heavy chain constant region nucleotide sequence identical to the Fc nucleotide sequence and / or said human. The ligand, method, use or kit according to any one of claims 1 to 16, which expresses an antibody containing a human gamma Fc region. The ligand, method, use or kit according to any one of claims 13 to 17, wherein the human is genotyped or genotyped positive for the heavy chain constant region nucleotide sequence. 13. One of claims 13-18, wherein the human is phenotypized or phenotypically positive for the gamma heavy chain constant domain, CH1, CH2, CH3, CH4 or Fc. Ligand, method, use or kit. The ligand, method, use or of any one of claims 1 to 19, wherein said ligand comprises a human IGHG1 * 01 gamma-1 heavy chain constant region; or a human IGHG2 * 01 gamma-1 heavy chain constant region. kit. The ligand is
a) Contains the human gamma-1 heavy chain constant region containing Asp at position 204 shown in SEQ ID NO: 4 or Leu at position 206 shown in SEQ ID NO: 4, and the human has the IGHG1 * 01 human heavy chain constant region gene segment. Or does the human express an antibody comprising a human gamma-1 heavy chain constant region containing Asp at position 204 set forth in SEQ ID NO: 4 or Leu at position 206 set forth in SEQ ID NO: 4;
b) Pro at position 72 shown in SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6, and SEQ ID NO: 6. Contains a human gamma-2 heavy chain constant region comprising an amino acid selected from the group consisting of Ala at position 257 shown, said human containing the IGHG2 * 01 human heavy chain constant region gene segment, or said human. Pro at position 72 shown in the selected SEQ ID NO: 6, Asn at position 75 shown in SEQ ID NO: 6, Phe at position 76 shown in SEQ ID NO: 6, Val at position 161 shown in SEQ ID NO: 6 or SEQ ID NO: 6 Does it express an antibody containing the human gamma-2 heavy chain constant region containing Ala at position 257 shown in?
c) Contains the human gamma-3 heavy chain constant region encoded by the IGHG3 * 01 constant region gene segment, and the human contains the IGHG3 * 01 constant region gene segment, or the human contains the IGHG3 * 01 constant region gene segment. Does it express an antibody containing the human gamma-3 heavy chain constant region encoded by?
(ii) A human gamma-4 heavy chain constant region containing Leu at position 189 shown in SEQ ID NO: 73 or Arg at position 289 shown in SEQ ID NO: 73, wherein the human is an IGHG4 * 01 human heavy chain constant region gene segment. , Or said human expresses an antibody comprising a human gamma-4 heavy chain constant region comprising Leu at position 189 set forth in SEQ ID NO: 73 or Arg at position 289 set forth in SEQ ID NO: 73; The ligand, method, use or kit according to any one of claims 1 to 20, which is contained in the gamma-4 heavy chain constant region of the ligand. The TOI is the following human TOI:
a) PCSK9 [Optionally, disease or condition is lipid disorder, hyperlipoproteinemia, hyperlipidemia, abnormal lipidemia, hypercholesterolemia, cardiac attack, stroke, coronary heart disease, atherosclerosis Select from sclerosis, peripheral vascular disease, lameness (eg, lameness associated with elevated cholesterol), type II diabetes, hypertension and cardiovascular disease or condition],
b) IL6R [Optionally, the disease or condition is inflammatory disease or condition, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis-like, psoriasis, bronchitis, gingitis, graft rejection, allogeneic transplantation One-sided rejection, graft-versus-host disease (GvHD), asthma, adult respiratory distress syndrome (ARDS), hemorrhagic shock, ulcerative colitis, Sjogren's syndrome, airway inflammation, Castleman's disease, periodontitis, atopic dermatitis, Selected from systemic erythematosus and coronary heart disease],
c) IL4Ra [Optionally select disease or condition from asthma, COPD, inflammatory bowel disease, fibrotic condition, allergy, graft rejection, delayed hypersensitivity, contact hypersensitivity, nasal polyps and sinusitis Ru],
d) VEGF-A [Optionally, disease or condition is eye condition, cancer; neovascularization, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD), polypoid choroid Selected from angiopathy (PCV), retinal hemangiomatous growth (RAP), diabetic macular edema (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
e) PDGF-B [Optionally, disease or condition is eye condition, cancer; neovascularization, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD), polypoid choroid Selected from angiopathy (PCV), retinal hemangiomatous growth (RAP), diabetic macular edema (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
f) PDGFR-B [Optionally, disease or condition is eye condition, cancer; neovascularization, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD), polypoid choroid Selected from angiopathy (PCV), retinal hemangiomatous growth (RAP), diabetic macular edema (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
g) Angiopoetin (eg Ang-2) [Optionally, disease or condition is eye condition, cancer; angiogenesis, neovascularization, diabetic retinopathy (DR), age-related macular degeneration (AMD) ), Polypoid choroidal angiogenesis (PCV), retinal hemangiomatous growth (RAP), diabetic macular degeneration (DME), retinal vein occlusion (RVO) and retinopathy of prematurity (ROP)],
h) Nav1.7 [Optionally, the disease or condition is pain, pruritus or channel disorder, such as chronic pain, neurogenic pain, painful diabetic neuropathy (PDN), post-herpes neuropathy (PHN). , Triangular nerve pain (TN), spinal cord injury pain, multiple sclerosis pain, phantom limb pain, post-stroke pain, chronic back pain, osteoarthritis pain, cancer-related pain, HIV-related pain, inflammatory pain, primary Select from extremity erythema (PE), paroxysmal extreme pain (PEPD) or channel disorder-related painlessness (CIP)],
i) PD-L1 [Optionally, the disease or condition is cancer, autoimmune disease or condition, viral infection and inflammatory disease or condition, eg, solid tumor, breast cancer, lung cancer (eg, non-small cell lung cancer), Colon cancer, ovarian cancer, melanoma (eg stage IV melanoma), bladder cancer, liver cancer (eg hepatocellular carcinoma), kidney cancer, salivary adenocarcinoma, gastric cancer (stomach cancer), gastric cancer (eg gastric cancer), glioma, pancreatic cancer, thyroid cancer, thoracic epithelial cancer, head cancer and / or cervical cancer, multiple myeloma, T-cell lymphoma, hodgkin lymphoma, renal cell cancer, offspring Miya cervical cancer, colorectal cancer, bloodline neoplasm, metastatic colorectal cancer, uterine or cervical cancer, leukemia (eg, acute myeloid leukemia), diffuse large B-cell lymphoma, follicular center lymphoma , Prostate cancer, Merkel cell carcinoma, HIV infection, hepatitis C infection or Ebola infection].
j) PD-1 [optionally, the disease or condition is, for example, renal cell carcinoma; bloodline neoplasm; stage IV melanoma; non-small cell lung cancer; metastatic gastric cancer; breast cancer; solid tumor; bladder cancer; head and neck Selected from partial cancer, colorectal cancer, glioblastoma, gastric cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, cervical cancer, myeloid leukemia (eg, acute myeloid leukemia and chronic myeloid leukemia) I have cancer],
k) BMP6 [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
l) Hemoduverin [optionally, the disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); anemia associated with cancer, kidney condition or GvHD; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
m) Hepcidin [optionally, the disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron refractory iron deficiency anemia (IRIDA)],
n) Ferropatin [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or anemia associated with GvHD; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
o) TMPRSS6 [Optional disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron-refractory iron deficiency anemia (IRIDA)],
p) Transferrin [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic diseased anemia (ACD); cancer, kidney condition or GvHD-related anemia; hemochromatosis And selected from iron refractory iron deficiency anemia (IRIDA)]; and
q) Human hemochromatosis protein (HFE) [Optionally, disease or condition is anemia; iron deficiency; chronic inflammatory anemia (ACI); chronic disease anemia (ACD); cancer, kidney condition or GvHD Related anemia; selected from hemochromatosis and iron refractory iron deficiency anemia (IRIDA)]
The ligand, method, use or kit according to any one of claims 1 to 21, selected from the group consisting of.