KR20140018299A - Methods of treating immune disorders with single domain antibodies against tnf-alpha - Google Patents

Abstract

The present invention relates to a TNFX binding molecule ozorarizumab (ATN-103), a method of using the molecule to treat immune disorders, including rheumatoid arthritis, and specific dosage regimens for treating rheumatoid arthritis.

Description

METHODS OF TREATING IMMUNE DISORDERS WITH SINGLE DOMAIN ANTIBODIES AGAINST TNF-ALPHA}

The present invention uses polypeptides comprising a single domain antigen binding (SDAB) molecule to Tumor Necrosis Factor alpha (TNFα), and a SDAB molecule to treat an immune disorder such as rheumatoid arthritis. It is about how to.

SDAB for TNFα is described, for example, in PCT publications WO 04/041862 and WO 06/122786. The anti-TNFα SDAB is associated with and / or mediated by TNFα, such as inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison disease, autoimmune hepatitis, autoimmunity Parotitis, type 1 diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, male infertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, sarcoidosis , Scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroid lobe, and vasculitis.

While implementing anti-TNF therapy in treatment regimens has improved the disease burden and quality of life in patients with inflammatory conditions, many of these therapies require regular injections of drugs, for example, weekly. Frequent hospital visits and injections pose a significant burden to the medical system, patients, and caregivers. Reducing such visits and providing self-administration options to patients and caregivers can be of significant personal and economic benefit. Not all anti-TNF therapies may be candidates for reduced frequency of treatment or candidates for self-injection, and therefore are effective when administered at longer intervals (eg, once every month or two months), and / or as self-injection. There is a need for new anti-TNF therapies.

Patients receiving tumor necrosis factor (TNF) drugs are at risk of serious infection (SI) caused by bacteria, mycobacteria, fungi, viruses, parasites, and other opportunistic infectious agents. Such infections may be related to the agent or medication taken with the treatment. Severe infections (SI) may result in hospitalization, death, or the need for medications such as antibiotics. Thus, it is desired to minimize severe infections while maintaining drug efficacy.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the above mentioned requirements. Treatment of human subjects with rheumatoid arthritis with certain dosage regimens using polypeptides comprising anti-TNFα SDAB significantly improved the disease state of the subject. Thus, in some embodiments, the present invention provides a multi-dose of 30-200 mg of a polypeptide comprising two anti-TNFα SDABs and anti-human serum albumin (HSA) SDAB to a human in need thereof. Including administering, wherein the dose is administered separately at a time interval of about 4 weeks or more, the method comprising treating an immune disorder in a human in need thereof. Modeling revealed that polypeptides containing anti-TNFα SDAB were effective at prolonged time intervals at increased doses without increasing clinically significant adverse effects. In addition, comparative modeling with contemporary anti-TNF inhibitors revealed that polypeptides comprising anti-TNFα SDAB combined the desired efficacy without any impact on the risk profile of the SI effect. Thus, in some embodiments, the invention provides a multi-dose of 30-400 mg of a polypeptide comprising two anti-TNFα SDABs and one anti-human serum albumin (HSA) SDAB to a human in need thereof. Wherein the dose is administered separately at a time interval of about 4 weeks or more, such as, for example, about 8 weeks or 2 months, for treating an immune disorder in a human in need thereof. It includes a method. In some embodiments, the present invention provides 30-400 mg of multiple polypeptides comprising two antitumor necrosis factor alpha (TNFα) SDAB and one antihuman serum albumin (HSA) SDAB to a human in need thereof. The polypeptide for use in treating an immune disorder in a human being in need thereof by administering a dose, wherein the dose is administered at least about four weeks apart, wherein the two antitumor necrosis Polypeptides comprising factor alpha (TNFα) SDAB and one antihuman serum albumin (HSA) SDAB.

In some embodiments, the anti-TNFα SDAB comprises three complementary determining regions (CDRs), wherein CDR1, CDR2, and CDR3,

(a) CDR1 is

(i) amino acid sequence DYWMY (SEQ ID NO: 22);

(ii) an amino acid sequence having at least 80% sequence identity with DYWMY (SEQ ID NO: 22); or

(iii) comprises an amino acid sequence that differs by only one amino acid from DYWMY (SEQ ID NO: 22);

(b) CDR2 is

(i) amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 23);

(ii) an amino acid sequence having at least 80%, 90%, or 95% sequence identity with EINTNGLITKYPDSVKG (SEQ ID NO: 23); or

(iii) comprises an amino acid sequence that differs two or one amino acid from EINTNGLITKYPDSVKG (SEQ ID NO: 23);

(c) CDR3 is

(i) amino acid sequence SPSGFN (SEQ ID NO: 24);

(ii) an amino acid sequence having at least 80% sequence identity with SPSGFN (SEQ ID NO: 24); or

(iii) includes an amino acid sequence that differs by one amino acid from SPSGFN (SEQ ID NO: 24).

In some embodiments, the anti-HSA SDAB comprises three CDRs (CDR1, CDR2, and CDR3), wherein

(a) CDR1 is

(i) amino acid sequence SFGMS (SEQ ID NO: 25);

(ii) an amino acid sequence having at least 80% sequence identity with SFGMS (SEQ ID NO: 25); or

(iii) contains an amino acid sequence that differs by only one amino acid from SFGMS (SEQ ID NO: 25);

(b) CDR2 is

(i) amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 26);

(ii) an amino acid sequence having at least 80%, 90%, or 95% sequence identity with SISGSGSDTLYADSVKG (SEQ ID NO: 26); or

(iii) comprises an amino acid sequence that differs two or one amino acid from SISGSGSDTLYADSVKG (SEQ ID NO: 26);

(c) CDR3 is

(i) amino acid sequence GGSLSR (SEQ ID NO: 27);

(ii) an amino acid sequence having at least 80% sequence identity with GGSLSR (SEQ ID NO: 27); or

(iii) includes an amino acid sequence that differs by one amino acid from GGSLSR (SEQ ID NO: 27).

In certain embodiments, at least one of the anti-TNFα SDABs comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2 (TNF30). In certain embodiments, the anti-HSA SDAB comprises an amino acid sequence having at least 80%, 90%, 95%, or 95% sequence identity with SEQ ID NO: 5 (ALB8). In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 (ozorarizumab). In certain embodiments, at least one of the SDABs is humanized.

In some embodiments, each of the two anti-TNFα SDABs and the anti-HSA SDABs are linked via a linker. The linker may be an amino acid sequence, which may be selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7.

Doses of the invention may be administered separately at time intervals of about 2 weeks, 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or 2 months or more.

Dosages of the present invention may comprise 10, 30, 80, 100, 120, 160, 180, 200, 225, 250, 275, 300, 320, 350, 375 or 400 mg of SDAB molecules, intravenously, subcutaneously Orally, orally, intraperitoneally, nasal, or sublingually.

The SDAB molecules of the invention can be formulated into pharmaceutically acceptable formulations. In some embodiments, the formulation is

(a) SDAB molecules at a concentration of about 1 mg / ml to 250 mg / ml, such as about 10 mg / ml to 250 mg / ml;

(b) a lyophilized protectant selected from sucrose, sorbitol, or trehalose at a concentration of about 5% to about 10%;

(c) a surfactant selected from polysorbate-80 or poloxamer-188 at a concentration of about 0.01% to 0.6%; And

(d) a buffer selected from histidine buffer at a concentration of about 10-20 mM or Tris buffer at a concentration of about 20-20 mM such that the pH of the formulation is about 5.0-7.5.

In some embodiments, the formulation is 1 mg / ml, 10 mg / ml, 30 mg / ml, 80 mg / ml, 100 mg / ml, 160 mg / ml 180 mg / ml, 200 mg / ml, or even 250 mg / ml of polypeptide, 20 mM histidine, 10% (w / v) sucrose, and 0.02% polysorbate-80, pH 6.0.

In some embodiments, the formulation is 1 mg / ml, 10 mg / ml, 30 mg / ml, 80 mg / ml, 100 mg / ml, 160 mg / ml 180 mg / ml, 200 mg / ml, or even 250 mg / ml of polypeptide, 20 mM histidine, 7.5% (w / v) sucrose, and 0.01% polysorbate-80, pH 6.0.

SDAB molecules of the invention may be administered for the treatment of immune disorders, and / or polypeptides of the invention may be used for inflammation, arthritis (rheumatic arthritis, psoriatic arthritis, ankylosing spondylitis, pediatric idiopathic arthritis and osteoarthritis, including COPD), asthma, inflammatory Intestinal diseases (including Crohn's disease and ulcerative colitis), multiple sclerosis, Addison's disease, autoimmune hepatitis, autoimmune mumps, type I diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, Immunity selected from the group consisting of systemic lupus erythematosus, male infertility, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, purulent cholangitis, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroid lobe, and vasculitis It can be used to treat disorders.

Humans treated with the SDAB molecules of the invention can be treated with methotrexate simultaneously.

In certain embodiments, the invention comprises administering 80 mg of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 once every four weeks to a human being in need thereof for treating rheumatoid arthritis, wherein the human is methotrexate. Consider a method of treating rheumatoid arthritis in humans in need of treatment for rheumatoid arthritis, being treated at the same time.

In a further particular embodiment, the present invention comprises administering once every four weeks a 160, 200, 320 or 400 mg polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to a human being in need of treatment for rheumatoid arthritis Wherein, optionally, a human is treated concurrently with methotrexate, contemplating a method of treating rheumatoid arthritis in a human in need of rheumatoid arthritis treatment.

In another specific embodiment, the present invention comprises administering 320 or 400 mg of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 about once every eight weeks or once a month to a human being in need of treatment for rheumatoid arthritis Wherein, optionally, the human is treated concurrently with methotrexate, contemplating a method of treating rheumatoid arthritis in a human in need thereof.

In a further aspect, the present invention provides rheumatoid arthritis in humans in need of rheumatoid arthritis treatment by administering 80 mg of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 about every four weeks to a human in need of rheumatoid arthritis treatment The polypeptide for use in treating A, wherein the human is directed to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the human is being treated simultaneously with methotrexate.

In a further aspect, the present invention requires the treatment of rheumatoid arthritis by administering a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 of 160, 200, 320 or 400 mg about every four weeks to a human being in need thereof. The polypeptide for use in treating rheumatoid arthritis in a human, wherein the human optionally relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the human is being treated simultaneously with methotrexate.

In another specific embodiment, the invention provides rheumatoid by administering 320 or 400 mg of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 about once every 8 weeks or once a month to a human being in need of treatment for rheumatoid arthritis As said polypeptide for use in treating rheumatoid arthritis in a human being in need of arthritis treatment, a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, optionally wherein the human being is treated simultaneously with methotrexate, is contemplated.

In a further aspect, the invention provides a polypeptide for use in treating an immune disorder in a human in need thereof, wherein the polypeptide comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (ozorarizumab) To a polypeptide.

1 shows the proportion of patients who achieved ACR20, 50, and 70 criteria for rheumatoid arthritis treatment at 8 and 16 weeks of each of the six treatment groups receiving ozorarizumab. In the dosing regimen administered every four weeks, subjects received either ozorarizumab or placebo on days 1, 4, 8, and 12 weeks. In the regimen administered every 8 weeks, subjects received ozorarizumab at Days 1 and 8 and placebo at 4 and 12 weeks.
2 shows ACR20 response rate over time by treatment group (LOCF).
3 shows the rate of DAS28 response over time by treatment group (LOCF).
4 shows median dose-ACR20 response (95% PI) of placebo corrected ATN-103. The dashed line shows the median simulated response of the specified comparator treatment.
5 shows the median simulated DAS response (95% PI) of ATN-103. The dashed line shows the median response of the specified comparator treatment.
6 expresses the utility of the drug in ACR20 (%) and SI (%). Each bubble label includes a simulated response 95% PI of each drug in its specified dosage regimen.

Justice

In order that the present invention may be more readily understood, certain terms are first defined. Further definitions are described throughout the detailed description.

As used herein, the articles "a" and "an" refer to one or more than one (eg, one or more) grammatical objects.

As used herein, the term "or" means the term "and / or" and is used interchangeably herein unless the context clearly dictates otherwise.

The terms "protein" and "polypeptide" are used interchangeably herein and include the SDAB molecules of the present invention.

“About” and “approximately” should generally mean the degree of error that is acceptable for the measured quantity, taking into account the nature or precision of the measurement method. Exemplary error degrees are within 20 percent (%) of a given value, or within a range of values, typically within 10%, and more typically within 5%.

For example, when the modifier "about," "approximately," "at least (or more than)," or "maximum" precedes a list of items, the term is intended to modify each item in the list. For example, the phrase "at least about 10% or at least 20%" should be interpreted as "at least about 10% or at least about 20%."

As used herein, the term "substantially identical" as used herein contains sufficient or minimal number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence, such that the first and second nucleotide sequences About 85%, 90%, 91%, 92%, 93 with a first nucleic acid sequence, such as a reference sequence, that encodes a polypeptide having a common functional activity, or encodes a common structural polypeptide domain, or a common functional polypeptide activity By nucleotide sequence having at least%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Fragments, derivatives, analogs, or variants, and any combination thereof, of the polypeptides of the invention are also included in the invention. When referring to a protein of the invention, the terms “fragment,” “variant,” “derivative,” and analog ”include any polypeptide possessing at least some of the functional properties of the corresponding native SDAB molecule. Fragments of polypeptides of the invention include proteolytic and deletion fragments, in addition to specific antigen binding fragments discussed elsewhere herein. Variants of the polypeptides of the invention may be due to fragments as described above, and due to amino acid substitutions, deletions, or insertions. Include polypeptides with altered amino acid sequences Variants may be naturally occurring or may be non-naturally occurring Non-naturally occurring variants may be prepared using mutagenesis techniques known in the art Variant polypeptides may include conservative or non-conservative amino acid substitutions, deletions, or additions. Derivatives of fragments of the name are polypeptides that have been modified to show additional features not found in natural polypeptides, including fusion proteins, variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein, “derivative” of a polypeptide refers to a polypeptide of interest having one or more residues chemically derivatized by reaction of flanking functional groups Polypeptides comprising one or more naturally occurring amino acid derivatives of 20 standard amino acids Also included as “derivatives.” For example, it may be substituted with 4-hydroxyproline instead of proline, with 5-hydroxylysine instead of lysine, and with 3-methylhistidine instead of histidine; Homoserine instead of serine; ornithine instead of lysine Can be substituted.

The term "functional variant" means a polypeptide that has an amino acid sequence that is substantially identical to a naturally occurring sequence, or that is encoded by a substantially identical nucleotide sequence, and that can have one or more activities of a naturally occurring sequence. do.

The homology or sequence identity between the sequences (the terms are used interchangeably herein) is calculated as follows.

In order to determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for the purpose of being optimally compared (e.g., among the first and second amino acid or nucleic acid sequences Gaps can be introduced in one or both, and sequences that are nonhomologous can be ignored for comparison purposes). In typical embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence.

The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position of the first sequence is occupied with the same amino acid residue or nucleotide as the corresponding position of the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" means amino acid or nucleic acid) Equivalent to "homology").

The percent identity between two sequences is a function obtained by dividing the number of identical positions by the sequence, taking into account the number of gaps, and the length of each gap, which need to be introduced to optimally align the two sequences.

Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between the two amino acid sequences is included in the GAP program in the GCG software package (available on the internet gcg.com), see Needleman & Wunsch, J. Mol . Biol . 48: 444-453 (1970), using either the Blosum 62 matrix or the PAM250 matrix, and gap weight = 16, 14, 12, 10, 8, 6, or 4, and length weight = Measured using 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between the two nucleotide sequences uses the GAP program in the GCG software package (available on the internet gcg.com) and uses the NWSgapdna.CMP matrix and gap weight = 40, 50, 60 , 70, or 80, and length weight = 1, 2, 3, 4, 5, or 6. One typical set of parameters (and what should be used unless otherwise noted) is the Blosum 62 scoring matrix with gap penalty = 1, gap expansion penalty = 4, and frameshift gap penalty = 5.

% Identity between two amino acid or nucleotide sequences is included in the ALIGN program (version 2.0), see E. Meyers and W. Miller CABIO, 4: 11-17 (1989), and can be measured using the PAM120 weight residue table, gap length penalty = 12 and gap penalty = 4.

For example, the nucleic acid and protein sequences described herein can be used as "query sequences" to perform searches against published databases to identify other family members or related sequences. Altschul, et al. J. Mol . Biol . 215: 403-10 (1990), by using the NBLAST and XBLAST programs (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules that characterize the invention. A BLAST protein search can be performed using the XBLAST program, score = 50, wordlength = 3 to obtain an amino acid sequence homologous to the protein (SEQ ID NO: 1) molecule characterizing the invention. To obtain a gapped alignment for comparison purposes, see Altschul et al., Nucleic Acids Res . 25: 3389-25 3402 (1997), Gapped BLAST can be used. When using the BLAST and Gap BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Family of amino acid residues with similar side chains are defined in the art. Such families include amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg glycine, asparagine, glutamine, Serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g. threonine, valine, isoleucine ) And amino acids having aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

The term "SDAB" refers to a single domain antigen binding molecule as described in WO 04/041862 and WO 06/122786. The term "SDAB molecule" refers to a polypeptide or other molecule comprising one or more SDABs. The terms “SDAB” and “SDAB molecule” are interchanged to mean a single single domain antigen binding unit (eg, TNF30 or ALB8) or a multivalent single domain antigen binding domain (eg, TNF55, TNF56, or ozorarizumab). May be used.

The "CDR" of the variable domain determines the cumulative definition of Kabat, Chothia, Kabat and Chothia, AbM, contact, and / or conformational definitions, or CDRs well known in the art. Amino acid residues in the hypervariable regions identified according to any method. Antibody CDRs can be identified as hypervariable regions originally defined by Kabat et al. See, eg, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The location of the CDRs can also be identified as the structural loop structures originally described by Chothia et al. See, eg, Chothia et al., 1989, Nature 342: 877-883. Another CDR identification approach is a compromise between Kabat and Chothia and includes the "AbM Definitions," derived from Oxford Molecular's AbM antibody modeling software (now Accelerys ^® ), or literature [ MacCallum et al., 1996, J. Mol. Biol., 262: 732-745, which includes "contact definitions" of CDRs based on observed antigenic contacts. In another approach, as referred to herein as the "stereotype definition" of a CDR, the location of the CDR can be identified as a residue that enthalpy contributes to antigen binding. See, eg, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166. Further other CDR boundary definitions may not strictly follow one of the above definitions and, nevertheless, the predictive or experimental fact that a particular residue or group of residues, or even the entire CDR, has no significant effect on antigen binding. It may be shortened or extended in light of the discovery, but will overlap with at least some of the Kabat CDRs. As used herein, CDR may refer to a CDR defined by any approach known in the art, including combinations of approaches. The methods used herein can use CDRs defined according to any of the above approaches. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact, and / or conformation definitions.

DETAILED DESCRIPTION OF THE INVENTION

Single domain antigen binding ( SDAB ) Molecule

Single domain antigen binding (SDAB) molecules include molecules whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that are naturally free of light chains, single domains derived from antibodies of conventional four chains, engineered domains, and single domain scaffolds other than those derived from antibodies Do not. The SDAB molecule can be any of those known in the art, or any single domain molecule to be known later. SDAB molecules can be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and cow.

According to one aspect of the invention, the SDAB molecule is a naturally occurring single domain antigen binding molecule, known as a heavy chain free of light chains. Such single domain molecules are disclosed, for example, in WO 94/04678 and Hamers-Casterman et al., Nature 363: 446-448 (1993). For clarity, the variable domain derived from naturally occurring light chain free heavy chain molecules may be described as VHH to distinguish from conventional VH consisting of four immunoglobulins. Such VHH molecules can be derived from, for example, species of camels such as camels, llamas, dromedary camels, alpacas and guanaco. Species other than camelaceae can produce heavy chain molecules that are naturally free of light chains; Such VHHs are included within the scope of the present invention.

SDAB molecules can be recombinant, CDR grafted, humanized, camelized, de-immunized and / or generated in vitro (eg, selected by phage display), as described in more detail below.

The term "antigen binding" is intended to include a portion of a polypeptide comprising a determinant that forms an interface that binds to a target antigen, or epitope thereof, such as a single domain molecule described herein. With respect to a protein (or protein mimetic), the antigen binding site typically comprises one or more loops (of four or more amino acids or amino acid mimetics) that form an interface that binds to the target antigen. Typically, the antigen binding site of a polypeptide, such as a single domain antibody molecule, comprises one or two or more CDRs, or more typically three, four, five or six or more CDRs.

The term "immunoglobulin variable domain" is often understood in the art as being identical or substantially identical to a VL or VH domain of human or animal origin. It should be appreciated that immunoglobulin variable domains may evolve in certain species, such as sharks and llamas, and differ from amino acid sequences from human or mammalian VL or VH.

However, the domain is still primarily involved in antigen binding. The term “immunoglobulin variable domain” typically includes one or two or more CDRs, or more typically three or more CDRs.

"Constant immunoglobulin domain" or "constant region" is intended to include immunoglobulin domains that are identical or substantially similar to CL, CH1, CH2, CH3, or CH4 domains of human or animal origin. See, eg, Hasemann & Capra, Immunoglobulins: Structure and Function, in William E. Paul, ed., Fundamental Immunology, Second Edition, 209, 210-218 (1989). The term "Fc region" refers to the Fc region of the constant immunoglobulin domain, or immunoglobulin domains substantially similar thereto, including immunoglobulin domains CH2 and CH3.

In certain embodiments, the SDAB molecule is a monovalent or multispecific molecule (eg, bivalent, trivalent, or tetravalent molecules). In other embodiments, the SDAB molecule is a monospecific, bispecific, trispecific, or quadspecific molecule. Whether a molecule is “monospecific” or “multispecific,” such as “bispecific”, refers to the number of different epitopes that react with a binding polypeptide. Multispecific molecules may be specific for different epitopes of the target polypeptides described herein, or may be specific for the target polypeptide as well as for heterologous epitopes such as heterologous polypeptides or solid support materials.

As used herein, the term "binding value" refers to the number of potential binding domains, such as antigen binding domains, present in the SDAB molecule. Each binding domain specifically binds to one epitope. If the SDAB molecule contains more than one binding domain, each binding domain can specifically bind to the same epitope, in the case of an antibody with two binding domains, it is termed "bivalent monospecific", or It can specifically bind to different epitopes, in the case of SDAB molecules with two binding domains, it is termed "bivalent bispecific". SDAB molecules can also be bispecific and bivalent for each specificity (which is termed a "bispecific tetravalent molecule"). Bispecific bivalent molecules, and methods for their preparation, are described, for example, in US Pat. No. 5,731,168; 5,807,706; 5,807,706; 5,821,333; 5,821,333; And US Publication Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated herein by reference. Bispecific trivalent molecules, and methods for their preparation, are described, for example, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated herein by reference. Further examples of multispecific and multivalent molecules are described in WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; US Patent No. 4,474,893; No. 4,714,681; 4,925,648; 4,925,648; 5,573,920; 5,573,920; And 5,601,819; Tutt et al., J. Immunol . 147: 60-69 (1991); And Kostelny et al., J. Immunol . 148: 1547-1553 (1992).

In certain embodiments, the SDAB molecule is a single chain fusion polypeptide comprising one or more single domain molecules without complementary variable domains or immunoglobulin constant regions, such as Fc regions, that bind to one or more target antigens. Examples of exemplary target antigens recognized by antigen binding polypeptides include tumor necrosis factor alpha (TNFα). In certain embodiments, the antigen binding single domain molecule binds to a serum protein, such as, for example, human serum protein, selected from one or more of serum albumin (eg, human serum albumin (HSA) or transferrin).

TNF alpha

Tumor necrosis factor alpha (TNFα) is known in the art to be associated with inflammatory disorders such as rheumatoid arthritis, Crohn's disease, ulcerative colitis, and multiple sclerosis. Both TNFα and its receptors (CD120a and CD120b) have been studied in detail. Its bioactive form of TNFα is a trimer. There are several jeonryakbeop the development of a TNFα antagonist action with an anti-TNFα antibodies, which are commercially available as in the present example, Remicade (Remicade), and Humira ^® (Humira) ^®. Numerous examples of TNFα binding single domain antigen binding molecules are disclosed in WO 04/041862, WO 04/041865, and WO 06/122786, all of which are incorporated herein by reference in their entirety.

Further examples of single domain antigen binding molecules are disclosed in US2006 / 286066, US2008 / 0260757, WO 06/003388, US2005 / 0271663, and US2006 / 0106203, all of which are incorporated herein by reference in their entirety. In certain embodiments, an SDAB molecule of the invention comprises an amino acid sequence of Table 1 below, or an amino acid sequence having about 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Table 1 Include SDAB. In alternative embodiments, the SDAB molecules of the invention may comprise amino acid sequences that differ from the sequences in Table 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. have.

In other embodiments, single, dual, triple and other multispecific single domain antibodies against TNFα and serum proteins such as HSA are contemplated. Many examples of multispecific TNFα and HSA binding polypeptides are disclosed in WO 06/122786, the contents of which are incorporated herein by reference. Multispecific polypeptides of the invention may comprise an amino acid sequence of Table 1 or an amino acid sequence having about 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Table 1. In alternative embodiments, multispecific SDAB molecules of the invention comprise amino acid sequences that differ in the sequence of Table 1 from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. can do.

In specific embodiments, the TNFα binding SDAB molecule comprises one or more of the SDABs disclosed in WO 06/122786. For example, the TNFα binding SDAB molecule may be a monovalent, divalent, or trivalent TNFα binding polypeptide disclosed in WO 06/122786.

Exemplary TNFα binding SDABs disclosed in WO 06/122786 include, but are not limited to, TNF1, TNF2, TNF3, and humanized forms thereof (eg, TNF29, TNF30, TNF31, TNF32, TNF33). Further examples of monovalent TNFα binding SDABs are disclosed in Table 8 of WO 2006/122786. Exemplary bivalent TNFα binding SDAB molecules include, but are not limited to, TNF55 and TNF56 (disclosed in WO 06/122786) comprising two TNF30 SDABs that are linked through a peptide linker to form a single fusion polypeptide. Further examples of bivalent TNFα binding SDAB molecules are disclosed in Table 19 of WO 06/122786 as TNF4, TNF5, TNF6, TNF7, TNF8.

In certain embodiments, anti-TNFα SDAB comprises three complementarity determining regions (CDR1, CDR2, and CDR3), wherein CDR1 comprises amino acid sequence DYWMY (SEQ ID NO: 22); An amino acid sequence having at least 80% sequence identity with DYWMY (SEQ ID NO: 22); Or an amino acid sequence that differs by only one amino acid from DYWMY (SEQ ID NO: 22); CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 23); Amino acid sequences having at least 80%, 90%, or 95% sequence identity with EINTNGLITKYPDSVKG (SEQ ID NO: 23); Or comprises an amino acid sequence that differs two or one amino acid from EINTNGLITKYPDSVKG (SEQ ID NO: 23); CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 24); Amino acid sequences having at least 80% sequence identity with SPSGFN (SEQ ID NO: 24); Or one amino acid difference from SPSGFN (SEQ ID NO: 24).

In some embodiments, the HSA binding SDAB comprises an amino acid sequence of Table 1, or an amino acid sequence having about 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of Table 1. In alternative embodiments, an anti-HSA SDAB of the invention may comprise an amino acid sequence that differs from the sequence of Table 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids. Can be.

In other embodiments, the HSA binding SDAB molecule comprises one or more of the SDABs disclosed in WO 06/122786. For example, the HSA binding SDAB molecule can be a monovalent, divalent, trivalent HSA binding SDAB molecule disclosed in WO 06/122786. In other embodiments, the HSA binding SDAB molecule can be a monospecific or multispecific molecule having one or more binding specificities for HSA. Exemplary HSA binding SDABs include, but are not limited to, ALB1 and its humanized forms (eg, ALB6, ALB7, ALB8, ALB9, ALB10) disclosed in WO 06/122786.

In certain embodiments, the anti-HSA SDAB comprises three CDRs (CDR1, CDR2, and CDR3), wherein CDR1 comprises the amino acid sequence SFGMS (SEQ ID NO: 25); Amino acid sequences having at least 80% sequence identity with SFGMS (SEQ ID NO: 25); Or an amino acid sequence that differs by only one amino acid from SFGMS (SEQ ID NO: 25); CDR2 has the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 26); Amino acid sequences having at least 80%, 90%, or 95% sequence identity with SISGSGSDTLYADSVKG (SEQ ID NO: 26); Or comprises an amino acid sequence that differs two or one amino acid from SISGSGSDTLYADSVKG (SEQ ID NO: 26); CDR3 comprises the amino acid sequence GGSLSR (SEQ ID NO: 27); Amino acid sequences having at least 80% sequence identity with GGSLSR (SEQ ID NO: 27); Or an amino acid sequence that differs by one amino acid from GGSLSR (SEQ ID NO: 27).

In other embodiments, two or more single domain molecules of the SDAB molecule are fused as genes or polypeptide fusions with or without a linking group. The linker may be any linker that will be apparent to those skilled in the art. For example, the linking group can be a biocompatible polymer that is 1 to 100 atoms in length. In one embodiment, the linking group comprises or consists of a polyglycine, polyserine, polylysine, polyglutamate, polyisoleucine, or polyarginine residue, or a combination thereof. For example, the polyglycine or polyserine linker may comprise 5, 7, 8, 9, 10, 12, 15, 20, 30, 35, and 40 or more glycine and serine residues. Exemplary linkers that may be used include Gly-Ser repeats, eg, (Gly) 4-Ser (SEQ ID NO: 19) repeats consisting of one, two, three, four, five, six, seven or more repeats. do. In some embodiments, the linker has the following sequence: (Gly) 4-Ser- (Gly) 3-Ser (SEQ ID NO: 20) or ((Gly) 4-Ser) n (SEQ ID NO: 21), wherein n is 4, 5 or 6). In some embodiments, the linker comprises SEQ ID NO: 6 (GS9) or SEQ ID NO: 7 (GS30).

In one exemplary embodiment, the SDAB molecules of the invention comprise two SDAB domains (eg, two camelid variable regions) that bind to a target antigen, such as TNFα, and one single that binds to serum proteins, such as HSA. It may consist of a single chain polypeptide fusion consisting of domain antibody molecules (eg, camelid variable regions).

A polypeptide referred to herein as “ozorarizumab” is a humanized, trivalent, bispecific TNFα inhibitory fusion protein. The fusion protein is derived from camelids and has a high degree of sequence and structural homology with human immunoglobulin VH domains. Its single polypeptide chain consists of two binding domains for TNFα and one for HSA and two to nine amino acid G-S linkers linking these two domains. A detailed description of ozorarizumab is provided in WO 06/122786 (herein referred to as TNF60), the contents of which are incorporated herein by reference.

SDAB  Molecular manufacturing

The SDAB molecules of the invention consist of one or more single domain molecules (e.g., SDAB) generated (e.g., selected by phage display) that are recombinant, CDR grafted, humanized, camelized, de-immunized and / or in vitro. Can be. Techniques for generating antibodies and SDAB molecules and recombinantly modifying them are known in the art and are described in detail below.

Many methods known to those skilled in the art can be used to obtain antibodies. For example, monoclonal antibodies can be prepared by generating hybridomas according to known methods. The hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE ™) assays to One or more hybridomas can be identified that produce specifically binding SDAB. Certain forms of specific antigens such as recombinant antigens, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptides thereof can be used as immunogens.

One exemplary method for preparing antibodies and SDAB molecules includes screening protein expression libraries, such as phage or ribosomal display libraries. Phage display is described, for example, in US Pat. No. 5,223,409; Smith Science 228: 1315-1317 (1985); WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; And WO 90/02809.

In addition to using display libraries, certain antigens can be used to immunize non-human animals such as rodents such as mice, hamsters, or rats. In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, large fragments of the human Ig locus can be used to engineer mouse strains that are defective in mouse antibody production. Hybridoma technology can be used to prepare and select antigen specific monoclonal antibodies derived from genes with the desired specificity. See, eg, XENOMOUSE ™, Green et al., Nature Genetics 7: 13-21 (1994), US20030070185, WO 96/34096, and PCT Application No. PCT / US96 / 05928.

In another embodiment, SDAB molecules can be obtained from non-human animals and then modified, such as humanized, deimmunized, and / or chimerized. Such SDAB molecules can be prepared using recombinant DNA techniques known in the art. Various approaches to preparing chimeric antibodies and SDAB molecules have been described. See, eg, Morrison et al., Proc. Natl . Acad . Sci USA 81: 6851 (1985); Takeda et al., Nature 314: 452 (1985); US Patent Nos. 4,816,567 and 4,816,397; European Patent Publications EP171496 and 0173494; And British Patent GB 2177096B. For example, humanized SDAB molecules can also be prepared using transgenic mice that express human immunoglobulin genes but not endogenous human immunoglobulin genes.

Winter has described an exemplary CDR transplantation method that can be used to prepare the humanized SDAB molecules described herein (US Pat. No. 5,225,539). All of the CDRs of a particular SDAB molecule may be substituted with at least a portion of a non-human CDR, or only some of the CDRs may be substituted with a non-human CDR. Only substitution of the required number of CDRs for binding the SDAB molecule to the predetermined antigen is necessary.

Humanized SDAB molecules can be generated by substituting an equivalent sequence from a human variable domain for a sequence of variable domains not directly involved in antigen binding. Exemplary methods for generating humanized antibodies or fragments thereof include Morrison Science 229: 1202-1207 (1985); Oi et al., BioTechniques 4: 214 (1986); And US Patent No. 5,585,089; 5,693,761; 5,693,761; 5,693,762; 5,693,762; 5,859,205; 5,859,205; And 6,407,213.

The method comprises isolating, manipulating, and expressing a nucleic acid sequence encoding all or part of an immunoglobulin variable domain from at least one of a heavy or light chain. The nucleic acid can be obtained from hybridomas as well as from other sources, which produce SDAB molecules for a predetermined target, as described above. The recombinant DNA encoding the humanized SDAB molecule can then be cloned into an appropriate expression vector.

In certain embodiments, humanized SDAB molecules can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions, and / or reverse mutations. The modified immunoglobulin molecule can then be prepared by any of several techniques known in the art (see, eg, Teng et al., Proc . Natl . Acad . Sci USA . 80: 7308-7312. (1983); Kozbor et al., Immunology Today 4: 7279 (1983); Olsson et al., Meth . Enzymol , 92: 3-16 (1982)], WO 92/06193 or EP 0239400. Techniques for humanizing SDAB molecules are also provided in WO 06/122786.

SDAB molecules can also be modified by “de-immunization” or specific deletion of human T cell epitopes by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy chain variable domain of the SDAB molecule can be analyzed for peptides that bind type II MHC; The peptides represent potential T cell epitopes (as defined in WO 98/52976 and WO 00/34317). For the detection of potential T cell epitopes, a computer modeling approach called “peptide threading” can be applied, further comprising the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. A database of human type II MHC binding peptides can be searched for motifs present in. This motif binds to any of the 18 major type II MHC allotypes and thus constitutes a potential T cell epitope. The potential T cell epitope detected can be removed by substituting a few amino acid residues in the variable domain, or preferably by a single amino acid substitution. Typically, conservative substitutions are made.

Although not exclusively, amino acids that are common at positions in human germline antibody sequences are usually available. Human germline sequences are described, eg, in Tomlinson et al., J. Mol . Biol . 227: 776-798 (1992); Cook et al., Immunol. Today 16 (5): 237-242 (1995); Chothia et al., J. Mol . Biol . 227: 799-817 (1992); And Tomlinson et al., EMBO J. 14: 4628-4638 (1995). The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled in Tomlinson, LA. Et al. RC Center for Protein Engineering, Cambridge, UK). Such sequences can be used, for example, as a source of human sequence sequences for framework regions and CDRs. Consensus human skeletal regions can also be used, as described, for example, in US Pat. No. 6,300,064. SDAB molecules can be made by living host cells genetically engineered to make such proteins. Methods of genetically manipulating cells to make proteins are well known in the art. See, eg, Ausubel et al., Eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing a nucleic acid that encodes a protein and allows it to be expressed into a living host cell. Such host cells may be bacterial cells, fungal cells, or preferably animal cells grown in culture. A bacterial host cell is Escherichia coli (Escherichia coli ) cells, including but not limited to. Examples of suitable E. coli strains include HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that does not cleave foreign DNA. Fungal host cells that can be used include Saccharomyces cerevisiae , Pichia pastoris ) and Aspergillus cells. Some examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38. New animal cell lines can be established by methods well known to those skilled in the art (eg, by transformation, viral infection, and / or selection). Optionally, the protein can be secreted into the medium by the host cell.

Deformed SDAB  molecule

The formulations of the invention may contain one or more SDAB molecules having an amino acid sequence that differs from the amino acid sequence of a naturally occurring domain such as the VH domain in one or more amino acid positions in one of the framework regions. For example, the amino acid sequence of some of the SDAB molecules of the present invention, such as a humanized SDAB molecule, may be combined with an amino acid sequence of a domain in which one or more amino acid positions occur naturally in one or more backbone regions, such as a naturally occurring VHH domain. Can be different.

The present invention also encompasses formulations consisting of derivatives of SDAB molecules. Such derivatives may generally be obtained by modification of one or more residues of the amino acid residues forming the SDAB molecule, and / or the SDAB molecule disclosed herein, and in particular by chemical and / or biological (eg, enzymatic) modification. have. As well as examples of such modifications, examples of amino acid residues in the SDAB molecular sequence that can be modified in this manner (ie on any protein backbone, but preferably on the side chain), methods that can be used to introduce such modifications and Techniques, and potential uses and advantages of the modifications, will be apparent to those skilled in the art.

For example, such modifications may include the introduction of one or more functional groups, functional residues or functional moieties into or onto the SDAB molecule (eg, by covalent bonds or in any other suitable manner), and in particular And the introduction of one or more functional groups, functional moieties or functional moieties that impart one or more desired properties or functionality to the SDAB molecule. Examples of such functional groups will be apparent to those skilled in the art.

For example, such modifications may increase the half-life, solubility, and / or absorption of the SDAB molecule (eg, by covalent bonds or in any other suitable manner), and / or the immunogenicity and / or the SDAB molecule. Or reduce toxicity and / or eliminate or attenuate any undesirable side effects of the SDAB molecule, and / or impart other beneficial properties to the SDAB molecule, and / or reduce the undesirable property of the SDAB molecule. ; Or the introduction of one or more functional groups to perform any combination of two or more of the following. Examples of such functional groups, and examples of techniques for introducing them, will be apparent to those of ordinary skill in the art, which generally includes pharmaceutical protein modifications as well as all functional groups and techniques mentioned in the general background of the art cited herein above. And functional groups and techniques that are known per se as being intended for, and in particular for modification of antibodies or antibody fragments (including ScFv and single domain antibodies), see Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing. Co., Easton, PA (1980). Such functional groups may be linked to, for example, directly (eg, covalently), or optionally via a suitable linker or spacer to the SDAB molecule of the invention, which will also be apparent to those skilled in the art. Techniques widely used to ameliorate the half-life of a pharmaceutical protein and / or to reduce its immunogenicity include suitable pharmacologically acceptable polymers such as poly (ethyleneglycol) (PEG) or derivatives thereof such as methoxypoly (Ethylene glycol) or mPEG). Generally, PEGylation used in the art is used for any suitable form of PEGylation, such as, but not limited to, antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv); See, eg, Chapman, Nat . Biotechnol . , 54: 531-545 (2002); Veronese and Harris, Adv . Drug Deliv . Rev. 54: 453-456 (2003); Harris and Chess, Nat . Rev. Drug . Discov ., 2, (2003); And WO 04/060965. Various reagents for PEGylating proteins are also commercially available, for example, from Nektar Therapeutics (USA).

Preferably, site directed PEGylation is used, in particular via cysteine residues (see, eg, Yang et al., Protein Engineering 16 (10): 761-770 (2003). For example, for this purpose, PEG may be attached to cysteine residues naturally present in the SDAB molecule, and the SDAB molecule may be modified to suitably introduce one or more cysteine residues for the attachment of PEG Or an amino acid sequence comprising one or more cysteine residues for the attachment of PEG to the N and C termini of the SDAB of the invention, all of which are performed by using protein manipulation techniques known per se to those skilled in the art. Can be.

Preferably, for SDAB molecules, the molecular weight is greater than 5,000, such as greater than 10,000 to less than 200,000, such as 100,000; For example, PEG in the range of 20,000-80,000 is used.

With regard to PEGylation, in general, the present invention also preferably discloses that the PEGylation can (1) increase half-life in vivo; (2) reduce immunogenicity; (3) provide one or more additional beneficial properties per se known for PEGylation; (4) no more than essentially 90%, preferably, more than 50%, as determined by a suitable assay, such as described in the Examples below, and / or without essentially affecting the affinity of the SDAB molecule, and Not reduce the affinity by more than 10%); (5) It should be noted that it includes any SDAB molecule PEGylated at one or more amino acid positions in such a way as not to affect any of the other desirable properties of the SDAB molecule. Suitable PEG groups and methods for attaching them specifically or nonspecifically will be apparent to those skilled in the art.

PEGylated SDAB is disclosed, for example, in US Provisional Application No. 61 / 265,307, filed Jul. 16, 2011, which is incorporated herein by reference.

In some embodiments, the PEGylated SDAB comprises a modified SDAB molecule linked to a PEG polymer, wherein the PEG polymer molecule is a branched PEG polymer molecule selected from the group consisting of Formulas (a)-(h):

<Formula (a)-(h)>

In some embodiments, PEGylated SDAB is

(i) one or more single antigen binding domains that bind to one or more targets;

(ii) nonpeptidic linkers; And

(iii) comprises one or more polymer molecules,

Wherein the non-peptidic linker is a moiety of formula (I):

&Lt; Formula (I) >

Where

W1 and W2 are each independently selected from a bond or NR1;

Y is a bond, C1-4 alkylene substituted with 0-2 Ra present, or pyrrolidine-2,5-dione;

X is O, a bond, or absent;

Z is O, NR 3, S or a bond;

R 1 and R 3 are each independently hydrogen or C 1-6 alkyl;

R2 is absent or is one or more polymer moieties;

Ra is selected from hydroxyl, C 1-4 alkyl or C 1-4 alkoxy;

m is 0 or 1;

n is 0, 1, 2 or 3;

p is 0, 1, 2, 3 or 4;

In a further embodiment, PEGylated SDAB is linked to PEG via a linker represented by the formula:

.

.

In certain embodiments, the modified SDAB molecule comprises the following structure:

.

.

Another generally less preferred variant includes N-linked or O-linked glycosylation, generally as part of co-translational and / or post-translational modifications, depending on the host cell used to express the SDAB molecule.

SDAB How to manufacture

SDAB molecules can be made by living host cells genetically engineered to make such proteins. Methods of genetically manipulating cells to make proteins are well known in the art. See, eg, Ausubel et al., Eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). The method includes introducing a nucleic acid into a living host cell that encodes a protein and allows it to be expressed. Such host cells can be bacterial cells, fungal cells, or animal cells grown in culture. Bacterial host cells include, but are not limited to Escherichia coli cells. Examples of suitable E. coli strains include HB101, DH5a, GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that does not cleave foreign DNA. Fungal host cells that can be used include, but are not limited to, Saccharomyces cerevisiae, Pchia pastoris and Aspergillus cells. Some examples of animal cell lines that can be used are CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and WI38. New animal cell lines can be established by methods well known to those skilled in the art (eg, by transformation, viral infection, and / or selection). Optionally, the protein can be secreted into the medium by the host cell.

In some embodiments, SDAB molecules can be produced by bacterial cells, such as E. coli cells. For example, when SDAB is encoded by a sequence in a phage display vector comprising an inhibitory stop codon between a display entity and a bacteriophage protein (or fragment thereof), the vector nucleic acid is transferred into a bacterial cell capable of inhibiting the stop codon. Can be. In this case, SDAB is not fused to gene III protein and is secreted into the periplasm and / or medium.

SDAB molecules can also be produced by eukaryotic cells. In one embodiment, the SDAB molecule is a yeast cell, such as pichia (see, eg, Powers et al. J Immunol Methods 251: 123-35 (2001)), Hansenula , or Saccharomyces Expressed in.

In one embodiment, the SDAB molecule is produced in mammalian cells. Typical mammalian host cells expressing clone antibodies, or antigen binding fragments thereof, include Chinese Hamster Ovary cells (CHO cells) (see, eg, Kaufman and Sharp, Mol. Biol. 159: 601-621). 1982), dhfr-CHO, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980) as used with DHFR selectable markers. Cells, lymphocytic cell lines such as NS0 myeloma cells and SP2 cells, COS cells, and cells derived from transgenic animals such as transgenic mammals, for example, cells are breast epithelial cells.

In addition to nucleic acid sequences encoding SDAB molecules, recombinant expression vectors can carry additional sequences, such as sequences that control the replication of the vector in host cells, and additional sequences such as selectable marker genes. Selectable marker genes facilitate the selection of host cells into which vectors are introduced (eg, US Pat. Nos. 4,399,216; 4,634,665; and 5,179,017). For example, linear selectable marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to the host cell into which the vector is introduced.

In an exemplary system for recombinant expression of SDAB molecules, recombinant expression vectors encoding single domain antibody chains are introduced into dhfr-CHO cells by calcium phosphate mediated transfection. Antibody genes in recombinant expression vectors may be derived from enhancer / promoter regulatory elements (eg, SV40, CMV, adenovirus, etc., eg, CMV enhancer / AdMLP promoter regulatory element or SV40 enhancer / AdMLP promoter regulation, to drive high levels of gene transcription). Element is operatively connected. Recombinant expression vectors also carry a DHFR gene that allows CHO cells transfected with the vector to be selected using methotrexate selection / amplification. Selected transformant host cells can be cultured to express the antibody chains and an intact single domain is recovered from the culture medium. Recombinant expression vectors can be prepared using standard molecular biology techniques, transfecting host cells, screening for transformants, culturing host cells, and recovering antibody molecules from the culture medium. For example, some SDAB molecules can be isolated by affinity chromatography.

In one embodiment, the SDAB molecules are purified as described in WO 10/056550. In an exemplary embodiment, SDAB comprises contacting a mixture of SDAB and contaminant (s) with a Protein A based support and / or an ion exchange support under conditions such that the SDAB can support or attach to the support; SDAB removes one or more contaminants by washing the bound support under conditions such that it remains on the support, and selectively elutes SDAB from the support by eluting the adsorbed SDAB molecules with elution buffer to remove SDAB from the one or more contaminants. Purify.

SDAB molecules can also be produced by transgenic animals. For example, US Pat. No. 5,849,992 describes a method for expressing antibodies in the mammary gland of a transgenic mammal. A transgene comprising a milk-specific promoter and a nucleic acid encoding an antibody molecule, a signal sequence for selection, is constructed. Milk produced by the female of the transgenic mammal contains a single domain of interest secreted therein. Antibody molecules can be purified from milk or can be used directly for some applications.

Formulation

The SDAB of the present invention may be formulated in any pharmaceutically acceptable formulation. The formulation may be liquid or dried. The formulations may be produced by mixing, drying, lyophilization, vacuum drying, or any known method of formulating pharmaceutical compositions.

Pharmaceutical formulations may be formulated as solutions, microemulsions, dispersants, liposomes, or other structured structures suitable for high concentrations of protein. Sterile injectable solutions can be prepared by incorporating the agents described herein in the appropriate solvent in the appropriate amount with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization, if desired. Generally, dispersants can be prepared by incorporating the agents described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated below. Proper fluidity of the liquid can be maintained, for example, by the use of a coating such as lecithin, in the case of a dispersant, by maintaining the required particle size and by the use of surfactants.

Agents that delay absorption, such as monostearate salts and gelatin, may be included in the composition to enable the injectable composition to be absorbed over an extended period of time.

Formulations of SDAB molecules include SDAB molecules, compounds that act as cryoprotectants, and buffers. The pH of the formulation is generally pH 5.5-7.0, preferably about pH 6. In some embodiments, the formulation is stored as a liquid. In other embodiments, the formulation is prepared as a liquid and then dried, eg, by lyophilization, spray drying, before storage. The dried formulation may be used as a dry compound, eg as an aerosol or powder, or may be reconstituted at its original concentration or at another concentration, such as water, buffers, or other suitable liquids.

After long term storage as a frozen liquid, the SDAB molecular purification process is designed to deliver SDAB molecules into a formulation suitable for freeze drying (eg, using histidine / sucrose). The formulation is lyophilized using the protein at a specific concentration. The lyophilized formulation is then reconstituted with a suitable diluent (eg, water) as needed to reconstitute the original formulation component at a concentration equal to or higher than that of the desired concentration, typically pre-lyophilization. Can be dissolved.

Depending on the volume of water or diluent added to the lyophilisate, relative to the volume of the original lyophilized liquid, the reconstitution of the lyophilized formulation may produce a formulation whose concentration differs from the original concentration (ie, the concentration prior to lyophilization). have. Suitable agents can be identified by assaying one or more parameters for antibody integrity.

The SDABs of the invention can be formulated as described in WO 10/077422. SDABs of the invention can be formulated by the following exemplary process: after lyophilization of a 10 mixture consisting of SDAB, lyophilizer, surfactant, and buffer; The lyophilized mixture is reconstituted in diluent to prepare a formulation. In certain embodiments, the SDABs of the invention are formulated by the following process: expressing SDAB in cell culture; Purification of the SDAB via a chromatographic purification step and / or an ultrafiltration / diafiltration step; Sucrose at a concentration of about 5-10%, Polysorbate 80 at a concentration of about 0.01-0.02%, and histidine buffer at a concentration of about 10-20 mM or Tris at a concentration of about 20 mM so that the pH of the formulation is about 5 to 7.5. The concentration of SDAB is adjusted to about 10-250 mg / ml in the formulation containing the buffer.

Treatment method

The SDAB molecule is administered to a subject (eg a human subject) alone or in combination with a second agent, such as a second, therapeutic or pharmacologically active agent, to treat a TNFα related disorder such as inflammatory or autologous. Immune disorders can be treated or prevented (eg, alleviation or amelioration of one or more symptoms associated with the disorder).

A polypeptide comprising an SDAB molecule of the invention, such as two anti-tumor necrosis factor alpha (TNFα) SDABs and one anti-human serum albumin (HSA) SDAB, is used alone or herein in humans in need of treating or preventing an immune disorder. It can be used in combination with a second agent as described in the treatment or prevention of an immune disorder.

The term “treating” refers to the therapy in an amount, manner, and / or mode effective to ameliorate symptoms, symptoms, or parameters associated with the disorder, or to be detectable to a person skilled in the art to a statistically significant degree or to prevent progression of the disorder. It means to run. When used for therapeutic purposes, treatment can improve, cure, maintain, or shorten the duration or duration of the disorder or condition in a subject. When used therapeutically, a subject may exhibit partial or complete symptom findings. In a typical case, treatment will improve the subject's disorder or condition to be detectable by the physician, or worsen the worsening of the disorder or condition. The effective amount, mode, or mode may vary from subject to subject, and can be adapted to the subject.

As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the terms "subject" and "patient" refer to animals such as mammals, including non-primates (eg, cattle, pigs, horses, donkeys, goats, camels, cats, dogs, guinea pigs, rats). , Mouse, sheep) and primates (eg, monkeys such as cynomolgus monkeys, gorilla chimpanzees and humans).

Non-limiting examples of immune disorders that can be treated include autoimmune disorders such as arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, lupus-associated arthritis or ankylosing spondylitis), scleroderma, systemic lupus erythematosus, Sjogren Syndromes, vasculitis, multiple sclerosis, autoimmune thyroid lobes, dermatitis (atopic dermatitis and eczema dermatitis), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, colitis, diabetes (type I); Inflammatory conditions of the skin (eg, dry); Acute inflammatory conditions (eg, endotoxins, sepsis and septicemia, toxic shock syndrome and infectious diseases); Transplant rejection and allergies. In one embodiment, the TNFα associated disorder is an arthritis disorder, such as rheumatoid arthritis, rheumatoid arthritis (RA) (eg, moderate to severe rheumatoid arthritis), osteoarthritis, psoriatic arthritis, or ankylosing spondylitis, polyarticular pediatric Juvenile idiopathic arthritis (JIA); Or a disorder selected from one or more of psoriasis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and / or multiple sclerosis.

In certain embodiments, the SDAB molecule (or agent) is administered or used in combination with a second therapeutic agent. For example, for TNF-SDAB, the second agent can be an anti-TNF antibody or a TNF binding fragment thereof, wherein the second TNF antibody has an epitope specificity different from the TNF binding SDAB molecule of the agent. Other non-limiting examples of agents that can be co-formulated with TNF binding SDAB include, for example, cytokine inhibitors, growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, cytotoxic agents, and cells Proliferation inhibitors. In one embodiment, nonsteroidal antiinflammatory agents (NSAIDs); Corticosteroids (including prednisolone, prednisone, cortisone, and triamcinolone); And disease modifying anti-rheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine (Plaquenil) and sulfasalazine, leflunomide (Arava ^® ), tumors Necrosis factor inhibitors (etanercept (Enbrel ^® ), infliximab (Remide ^® ) (with or without methotrexate), and adalimumab (with Humira ^® ), anti-CD20 antibodies (eg Rituxan) ^® ), soluble interleukin 1 receptors such as anakinra (Kineret), gold, minocycline (Minocin ^® ), penicillamine, and cytotoxic agents (azathioprine, cyclophosphamide, and Additional agents, including but not limited to cyclosporin, are standard therapies for arthritis. Such combination therapies may be advantageous in that lesser doses may be used for the therapeutic agent administered, thereby avoiding possible toxicity or complications associated with various monotherapy.

When the additional agent is methotrexate, the dose of methotrexate may range from about 7.5 to about 25 mg per week.

SDAB molecules can be administered or used in the form of liquid solutions (eg, injections and infusions). The composition can be administered by parenteral mode (eg, subcutaneous, intraperitoneal, or intramuscular injection), or by inhalation. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to a mode of administration other than enteral and topical, generally, by injection, as well as subcutaneous or intramuscular administration. But also intravenous, intracapsular, orbital, intracardiac, intradermal, intraperitoneal, coronal, subcutaneous, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions. In one embodiment, the formulations described herein are administered subcutaneously.

We hypothesized that the bispecific interaction site of ozorarizumab with TNFα would be particularly useful for treating immune disorders. Accordingly, the present invention comprises administering to a human being in need thereof, a polypeptide competing with a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (ozorarizumab). A method for treating an immune disorder.

Dosing regimen

Ozararizumab has been shown to be effective in treating rheumatoid arthritis when administered subcutaneously at time intervals of four weeks or more between doses. In contrast, the recommended dosing for Humira is performed every two weeks and the remicade should be administered intravenously. Thus, the dosage regimens of the present invention provide an advantage over the state of the art in the art.

The SDAB molecules of the present invention have been shown to be effective in treating a disease when administered at a dose of 30 mg to 80 mg every four weeks. Modeled based on this efficacy, the higher doses (e.g. 120-200 mg or 200-400 mg) of the SDAB molecules of the present invention, when administered every six or eight weeks or two months, It has been shown to be effective. This beneficial profile reduces the burden on injection.

In addition, modeling also showed that severe infection (SI) of the ozorarizumab treatment was lower than for etanercept and infliximab. In particular, compared to prior art anti-TNFα inhibitors, the SDAB molecules of the present invention have been demonstrated to provide a very desirable ratio of benefits (efficiency) to risk (SI effects).

Thus, the SDAB molecules of the invention can be administered every 4, 6, or 8 weeks at doses ranging from 30-200 mg. Specific effective amount is 30-400 mg. In certain embodiments, the dosage is about 5, 10, 15, 20, 25, 30, 80, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 320, 350, 375 or 400 mg SDAB molecule.

A single dose of about 30-200 mg or even 30-400 mg is approximately daily, every 2 days, twice a week, every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks Every other month or every 1 or 2 months.

In certain embodiments, a polypeptide comprising an amino acid sequence of about 30, 80, 120, 160, 200, 240, 280, 320, 360, or 400 mg dose of SEQ ID NO: 1 (ozorarizumab) is approximately every single human subject. It is administered every 4, 6, or 8 weeks or 2 months. In some embodiments, the subject is a subject suffering from rheumatoid arthritis.

In further embodiments, the dose of SDAB molecule is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or 400 mg.

In some embodiments, the dose is about 3.6, 10.8, 36, 72, 144, or 288 mg.

In certain embodiments, the SDAB molecule is PEGylated and the dose is about 3.6, 10.8, 36, 72, 144, or 288 mg.

When administered to pediatric patients, the dose should be adjusted to the weight of the patient. In certain embodiments the dose is about 0.1, 0.38, 1, 2, 3, 3.5, 4, 4.5 or 5 mg / kg.

Subjects can be treated simultaneously with methotrexate. In some embodiments, a subject can receive methotrexate for at least about 6 or 12 weeks prior to first receiving ozorarizumab. Methotrexate can be administered by any suitable route and the dose can range from about 7.5 to 25 mg weekly.

How to measure efficacy

The efficacy of any particular SDAB molecule or dosage regimen can be measured by methods available to those skilled in the art. In brief, during clinical trials, medical personnel can observe patients and assess disease status by any combination of criteria. Whether a patient's disease state improves is determined based on the criteria at various time points, and the efficacy of the therapy is assessed by plotting the combination of the decisions in the patient population.

In an exemplary embodiment, disease progression for rheumatoid arthritis can be measured by any or all of the criteria described in Table 2 below.

Example

Example  One

Ozararizumab was used to conduct a seamless, half-phase, randomized, stratified, double-blind, placebo-controlled study in rheumatoid arthritis (RA) patients. A total of 254 subjects were randomized in this study; One of the subjects was not treated and the remaining 253 subjects were included in the safety population as well as the modified intent-to-treat (mITT) population. Each subject was randomly assigned to only one treatment group and stratified based on never receiving a TNF inhibitor (TNFi) or prior TNFi use. As described in Table 3 below, a total of six treatment groups existed, with each group containing 40-45 randomized patients.

group process One 10 mg every 4 weeks (4 doses total) 2 10 mg every 8 weeks (placebo given at mid-week 4 visits) (2 doses in total) 3 30 mg every 4 weeks (4 doses total) 4 80 mg every 4 weeks (4 doses total) 5 80 mg every 8 weeks (placebo given at mid-week 4 visits) (2 doses in total) 6 Placebo every 4 weeks (total 4 doses)

The baseline characteristics of the six groups were generally similar; The extremely small but statistically significant difference (p = 0.048) observed between the groups was in DAS28, with the 80 mg Q4 group having the highest mean DAS28 (6.59). The age range of the subjects was 18 to 79 years (mean age 52.1 years), and the majority of the subjects (80.2%) were women (as expected in clinical trials in RA patients). The overall mean disease duration was 8.3 years, and 28.9% of the subjects had previously received TNFi treatment.

The primary population for efficacy analysis was the mITT population defined as all randomized subjects who received at least one dose of ozorarizumab. All patients were treated simultaneously with 7.5 to 25 mg dose of methotrexate weekly.

Each subject was injected subcutaneously (SC) with a single dose level of ozorarizumab or placebo at the indicated time points.

The primary comparison of interest was the comparison of each ATN-103 treatment group versus placebo.

Table 4 below shows the American College of Rheumatology 20 (ACR20) response rate at 16 weeks.

P values were obtained according to the Cochran-Mantel-Haenszel Test stratified without TNFi or based on previous TNFi use. Minimum risk (MR) weighting proposed by Mehrotra and Railkar to calculate layered confidence intervals based on no TNFi for adjusted differences for placebo or previous use of TNFi Fire method was used. The last observation carried forward (LOCF) approach was used to replace any missing data in the ACR element before deriving the ACR20 response.

1 shows the proportion of subjects who met the ACR20, ACR50, and ACR70 criteria at weeks 8 and 16. Some of the dosing regimens were more beneficial than placebo at the following endpoints: ACR20 (FIGS. 1 & 2), ACR50 (FIG. 1), DAS28 (FIG. 3), ACR-N, TJC, SJC, Pain VAS, HAQ-DI, CRP, Overall assessment of physicians, overall assessment of patients, general health VA5, and EULAR responses (data not shown).

Briefly, as shown in FIG. 1, at week 8, for 10 mg Q8, 36.6% of subjects achieved ACR20, 9.8% of subjects achieved ACR50, and 0% of subjects achieved ACR70; For 10 mg Q4, 45.2% of the subjects achieved ACR20, 11.9% of the subjects achieved ACR50, and 7.1% of the subjects achieved ACR70; For 30 mg Q4, 50% of subjects achieved ACR20, 32.5% of subjects achieved ACR50, and 15% of subjects achieved ACR70; For 80 mg Q8, 54.8% of subjects achieved ACR20, 23.8% of subjects achieved ACR50, and 4.8% of subjects achieved ACR70; For 80 mg Q4, 65.1% of subjects achieved ACR20, 25.6% of subjects achieved ACR50, and 2.3% of subjects achieved ACR70. At week 16, for 10 mg Q8, 48.8% of subjects achieved ACR20, 24.4% of subjects achieved ACR50, and 8.9% of subjects achieved ACR70; For 10 mg Q4, 52.4% of subjects achieved ACR20, 21.4% of subjects achieved ACR50, and 4.8% of subjects achieved ACR70; For 30 mg Q4, 60% of subjects achieved ACR20, 32.5% of subjects achieved ACR50, and 20% of subjects achieved ACR70; 80 mg Q8 was achieved, 59.5% of subjects achieved ACR20, 31% of subjects achieved ACR50, and 19% of subjects achieved ACR70; For 80 mg Q4, 72.1% of subjects achieved ACR20, 37.2% of subjects achieved ACR50, and 11.6% of subjects achieved ACR70.

As shown in FIG. 3, the mean change rates from baseline (placebo) for DAS28 baseline at week 4 were 1.39% for 10 mg Q4, 1.05% for 10 mg Q8, 1.62% for 30 mg Q4, 1.80% for 80 mg Q4 and 1.67% for 80 mg Q8. At week 8, the average rate of change from baseline for the DAS28 criterion was 1.59% for 10 mg Q4, 1.01% for 10 mg Q8, 1.78% for 30 mg Q4, 2.00% for 80 mg Q4, And 1.85% for 80 mg Q8. At week 12, the mean rate of change from baseline for the DAS28 criterion was 1.61% for 10 mg Q4, 1.72% for 10 mg Q8, 2.31% for 30 mg Q4, 2.30% for 80 mg Q4, And 2.20% for 80 mg Q8. At week 16, the mean rate of change from baseline for the DAS28 criterion was 1.60% for 10 mg Q4, 1.70% for 10 mg Q8, 2.09% for 30 mg Q4, 2.46% for 80 mg Q4, And 1.93% for 80 mg Q8. At 20 weeks, the average rate of change from baseline for the DAS28 criterion was 1.02% for 10 mg Q4, 1.03% for 10 mg Q8, 1.62% for 30 mg Q4, 2.00% for 80 mg Q4, And 1.36% for 80 mg Q8.

Overall, the safety profile of ozorarizumab was found to be similar to that of other TNF inhibitor (TNFi) agonists. Reported SAEs also matched the safety profile of other anti-TNF agonists (data not shown).

Example  2: Rheumatism  In arthritis Nano Body ^® ATN -103 vs. five commercially available anti-TNFs

2.1 Purpose:

Nozolarizumab (ATN-103), a novel tumor necrosis factor inhibitor (TNFi), is a humanized, trivalent, bispecific nanobody comprising two human TNF binding domains linked to human serum albumin binding domains. ATN-103 versus five commercially available tumor necrosis factor alpha blocks of various doses / dose regimens with respect to efficacy (ACR20 / 50/70, DAS, HAQ) and safety / tolerability (severe infection rate, SI (%)) Model-based meta analysis (MBMA) was performed to evaluate the comparative efficacy of TNFα) drugs and to evaluate the effects of covariates for content description.

2.2 Method:

Model-based comparative efficacy and safety analyzes between the five comparators (Infliximab, Adalimumab, Etanercept, Sertolizumab Pagol and Golimumab) using published data summarized to study arm levels, and in-house Phase IIa data Was performed.

In particular, the ACR20 / 50/70 dataset included data from 7,474 subjects who participated in 20 trials involving a total of 63 arms. DAS was observed in 21 arms of 8 trials. The severe infection rate dataset included data from 6,209 subjects who participated in 14 trials involving a total of 46 arms. Datasets include drug exposure measures (dose intensity, dosing interval), baseline disease severity covariates (tension and swell joint count, DAS observations, overall assessment of patients and physicians, CRP concentration, disease duration), characteristics of test population (anti-TNFα) % Of subjects who experienced, and region (Asian versus non-Asian subjects).

Three ACR endpoints and a DAS endpoint were described using the joint ACR20 / 50/70 and DAS models, respectively.

2.3 Results :

2.3.1 Simulation capacity—AC20 / 50/70 response

In a typical patient population (TNF experienced average baseline swelling joint counts of 19 and 21% of ACR20 responders in the placebo group) ATN- after 24 weeks of treatment in addition to successive DMARD background medications using the final ACR20 / 50/70 model. The expected dose-response relationships of 103 and 5 comparator drugs were simulated. 4 shows the predicted dose-response relationship of ATN-103 to ACR20 response compared to other anti-TNFα drugs in week 24 population.

Simulation showed that 80 mg Q4W ATN-103 treatment was similar to 95% of the responses to adalimumab and golimumab, whereas 200 mg ATN-103 Q4W was expected to cause a response similar to 95% of the responses to etanercept. .

2.3.2 Simulation dose-DAS response

After an additional 24 weeks of treatment with the ongoing DMARD background drug, the expected dose-response relationship of ATN-103 and five comparator drugs to DAS% CfB was simulated. The DAS placebo response assumed that the rate of change from baseline to DAS was -16%, reflecting the mean of the unstructured mean placebo model parameter predictions (data not shown).

Figure 5 shows the expected dose-response relationship of ATN-103 for the DAS response, as compared to other anti-TNFα drugs. For 200 mg Q4W, simulations showed that ATN-103 was superior to golimumab and sertolizumab for DAS% CfB.

2.3.3 Simulated Severe Infection Rate

The simulated placebo-corrected SI (%) showed that the expected rates of infection of ATN-103 were similar to those of adalimumab and golimumab, whereas sertolizumab, etanercept, and infliximab had higher rates of severe infection (data Not shown).

FIG. 6 provides an integrated overview of the expected efficacy (ACR) and safety (SI (%)) profiles of ATN-103 versus other anti-TNFα drugs. In particular, FIG. 6 shows a 95% predicted interval of the simulated efficacy of anti-TNFα drug (ACR20) in the relevant dosing regimen, and a corresponding simulated 95% predicted interval of severe infection rate. Optimal efficacy / safety availability is shown to progress in the upper left corner direction (high potency, low severe infection rate) while decreasing in the lower right corner direction. Bubble plots indicate significant overlap in utility between anti-TNFα drugs.

By expressing mock efficacy and safety as described above, it was suggested that 200 mg ATN-103 Q4W could be well positioned relative to other anti-TNFα drugs.

2.3.4 Dose regimens of 400 mg Q8W

The model suggested that the response was not dependent on the treatment regimen, and similar effects could be obtained for either the 200 mg Q4W regimen or the 400 mg Q8W.

2.4 Conclusion

The comparative efficacy of ATN-103 versus five anti-TNFα drugs for efficacy (ACR / DAS) and safety / tolerability (severe infection rate) was assessed by model-based meta-analysis. Compared with other anti-TNFα drugs, ATN-103 showed a desirable combination of efficacy and SI effect.

Model based simulations showed that 200 mg ATN-103 Q4W or 400 mg ATN-103 Q8W were similar in terms of efficacy to etanercept, adalimumab and infliximab.

                         SEQUENCE LISTING <110> Ablynx NV   <120> Methods of treating immune disorders with single domain        antibodies <130> P11-021-PCT-1 <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 363 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ser Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu         115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu     130 135 140 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp 145 150 155 160 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser                 165 170 175 Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe             180 185 190 Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn         195 200 205 Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly     210 215 220 Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly 225 230 235 240 Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly                 245 250 255 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala             260 265 270 Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met Tyr Trp Val Arg Gln Ala         275 280 285 Pro Gly Lys Gly Leu Glu Trp Val Ser Glu Ile Asn Thr Asn Gly Leu     290 295 300 Ile Thr Lys Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 305 310 315 320 Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro                 325 330 335 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ser Ser Gly Phe Asn             340 345 350 Arg Gly Gln Gly Thr Leu Val Thr Val Ser Ser         355 360 <210> 2 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser         115 <210> 3 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Glu Pro             20 25 30 Ser Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys         35 40 45 Glu Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Leu Thr Tyr     50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala 65 70 75 80 Lys Asn Thr Val Asp Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr                 85 90 95 Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser             100 105 110 Val Gly Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser      <210> 4 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ser Ala Ser Gly Arg Ser Leu Ser Asn Tyr             20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Leu         35 40 45 Gly Asn Ile Ser Trp Arg Gly Tyr Asn Ile Tyr Tyr Lys Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Ile Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Ser Ile Leu Pro Leu Ser Asp Asp Pro Gly Trp Asn Thr Tyr             100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         115 120 <210> 5 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ser Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser         115 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 7 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 7 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser             20 25 30 <210> 8 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 8 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe             20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val         35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Thr Ile Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser         115 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 9 Ala Val Gln Leu Val Glu Ser Gly Gly Leu Val Gln Gly Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Phe Asp Leu Asn             20 25 30 Leu Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val         35 40 45 Ala Thr Cys Ile Thr Val Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Met Asp Tyr Thr Lys Gln Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys                 85 90 95 Lys Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln Gly Thr Gln             100 105 110 Val Thr Val Ser Ser         115 <210> 10 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe             20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Cys Thr                 85 90 95 Ile Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Gln Gly Thr Leu Val Thr Val             100 105 110 Ser Ser          <210> 11 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 11 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu         115 120 125 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Ala Ala Ser Gly Phe Thr     130 135 140 Phe Ser Asp Tyr Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly 145 150 155 160 Leu Glu Trp Val Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr                 165 170 175 Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys             180 185 190 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala         195 200 205 Leu Tyr Tyr Cys Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly     210 215 220 Thr Gln Val Thr Val Ser Ser 225 230 <210> 12 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 12 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Glu Pro             20 25 30 Ser Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys         35 40 45 Glu Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Leu Thr Tyr     50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala 65 70 75 80 Lys Asn Thr Val Asp Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr                 85 90 95 Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser             100 105 110 Val Gly Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val Glu     130 135 140 Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Ser Glu Pro Ser Gly Tyr Thr Tyr Thr                 165 170 175 Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala             180 185 190 Arg Ile Tyr Trp Ser Ser Gly Leu Thr Tyr Tyr Ala Asp Ser Val Lys         195 200 205 Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Thr Val Asp Leu     210 215 220 Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 225 230 235 240 Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Gly Ser Tyr Asn Tyr                 245 250 255 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser             260 265 <210> 13 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ser Ala Ser Gly Arg Ser Leu Ser Asn Tyr             20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Leu         35 40 45 Gly Asn Ile Ser Trp Arg Gly Tyr Asn Ile Tyr Tyr Lys Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Ile Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Ser Ile Leu Pro Leu Ser Asp Asp Pro Gly Trp Asn Thr Tyr             100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser         115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val     130 135 140 Gln Ala Gly Gly Ser Leu Ser Leu Ser Cys Ser Ala Ser Gly Arg Ser 145 150 155 160 Leu Ser Asn Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu                 165 170 175 Arg Glu Leu Leu Gly Asn Ile Ser Trp Arg Gly Tyr Asn Ile Tyr Tyr             180 185 190 Lys Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys         195 200 205 Asn Thr Ile Tyr Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala     210 215 220 Val Tyr Tyr Cys Ala Ala Ser Ile Leu Pro Leu Ser Asp Asp Pro Gly 225 230 235 240 Trp Asn Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser                 245 250 255 <210> 14 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 14 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys                 85 90 95 Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Gln Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 145 150 155 160 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp                 165 170 175 Tyr Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp             180 185 190 Val Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser         195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu     210 215 220 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 225 230 235 240 Cys Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Gln Val                 245 250 255 Thr Val Ser Ser             260 <210> 15 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Glu Pro             20 25 30 Ser Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys         35 40 45 Glu Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Leu Thr Tyr     50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala 65 70 75 80 Lys Asn Thr Val Asp Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr                 85 90 95 Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser             100 105 110 Val Gly Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser         115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser     130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gln 145 150 155 160 Val Gln Leu Val Glu Ser Gly Gly Lely Val Gln Ala Gly Gly Ser                 165 170 175 Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Glu Pro Ser             180 185 190 Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu         195 200 205 Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Leu Thr Tyr Tyr     210 215 220 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys 225 230 235 240 Asn Thr Val Asp Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala                 245 250 255 Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val             260 265 270 Gly Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser         275 280 285 <210> 16 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ser Ala Ser Gly Arg Ser Leu Ser Asn Tyr             20 25 30 Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Leu         35 40 45 Gly Asn Ile Ser Trp Arg Gly Tyr Asn Ile Tyr Tyr Lys Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Ile Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Ala Ser Ile Leu Pro Leu Ser Asp Asp Pro Gly Trp Asn Thr Tyr             100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser         115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly     130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 145 150 155 160 Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Ser Leu Ser Cys Ser                 165 170 175 Ala Ser Gly Arg Ser Leu Ser Asn Tyr Tyr Met Gly Trp Phe Arg Gln             180 185 190 Ala Pro Gly Lys Glu Arg Glu Leu Leu Gly Asn Ile Ser Trp Arg Gly         195 200 205 Tyr Asn Ile Tyr Tyr Lys Asp Ser Val Lys Gly Arg Phe Thr Ile Ser     210 215 220 Arg Asp Asp Ala Lys Asn Thr Ile Tyr Leu Gln Met Asn Arg Leu Lys 225 230 235 240 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ser Ile Leu Pro Leu                 245 250 255 Ser Asp Asp Pro Gly Trp Asn Thr Tyr Trp Gly Gln Gly Thr Gln Val             260 265 270 Thr Val Ser Ser         275 <210> 17 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ser Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 145 150 155 160 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp                 165 170 175 Tyr Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp             180 185 190 Val Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser         195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu     210 215 220 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val                 245 250 255 Thr Val Ser Cys             260 <210> 18 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr             20 25 30 Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ser Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val Thr             100 105 110 Val Ser Ser Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly         115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly     130 135 140 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 145 150 155 160 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp                 165 170 175 Tyr Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp             180 185 190 Val Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser         195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu     210 215 220 Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ala Arg Ser Pro Ser Gly Phe Asn Arg Gly Gln Gly Thr Leu Val                 245 250 255 Thr Val Ser Ser Gly Gly Gly Cys             260 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 19 Gly Gly Gly Gly Ser 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 20 Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 21 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <220> <221> MISC_FEATURE <222> (6) <223> X = GGGGSGGGGSGGGGSGGGGS <220> <221> MISC_FEATURE <222> (6) <223> X = GGGGSGGGGSGGGGSGGGGSGGGGS <220> <221> MISC_FEATURE <222> (6) <223> X = GGGGSGGGGSGGGGS <400> 21 Gly Gly Gly Gly Ser Xaa 1 5 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 22 Asp Tyr Trp Met Tyr 1 5 <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 23 Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly      <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 24 Ser Pro Ser Gly Phe Asn 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 25 Ser Phe Gly Met Ser 1 5 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 26 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 27 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Nanobody sequence <400> 27 Gly Gly Ser Leu Ser Arg 1 5

Claims (50)

Immunity of such humans by administering multiple doses of 30-400 mg of a polypeptide comprising two anti-tumor necrosis factor alpha (TNFα) SDAB and one anti-human serum albumin (HSA) SDAB to a human in need thereof A polypeptide comprising two antitumor necrosis factor alpha (TNFα) SDABs and one antihuman serum albumin (HSA) SDAB for use in treating a disorder, wherein the dose is administered at a time interval of about every four weeks or more Polypeptide. The method of claim 1, wherein each anti-TNFα SDAB comprises three complementarity determining regions (CDR1, CDR2, and CDR3),
(a) CDR1 is
(i) amino acid sequence DYWMY (SEQ ID NO: 22);
(ii) an amino acid sequence having at least 80% sequence identity with DYWMY (SEQ ID NO: 22); or
(iii) comprises an amino acid sequence that differs by only one amino acid from DYWMY (SEQ ID NO: 22);
(b) CDR2 is
(i) amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 23);
(ii) an amino acid sequence having at least 80%, 90%, or 95% sequence identity with EINTNGLITKYPDSVKG (SEQ ID NO: 23); or
(iii) comprises an amino acid sequence that differs two or one amino acid from EINTNGLITKYPDSVKG (SEQ ID NO: 23);
(c) CDR3 is
(i) amino acid sequence SPSGFN (SEQ ID NO: 24);
(ii) an amino acid sequence having at least 80% sequence identity with SPSGFN (SEQ ID NO: 24); or
(iii) a polypeptide comprising an amino acid sequence that is one amino acid difference from SPSGFN (SEQ ID NO: 24). 3. The amino acid sequence of claim 1, wherein CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 22), CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 23), and CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 24). Polypeptide. 4. The amino acid sequence of claim 1, wherein at least one of the anti-TNFα SDABs comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2 (TNF30). Polypeptide. 5. The method of claim 1, wherein each anti-TNFα SDAB comprises an amino acid sequence having at least 80%, 90%, 95%, or 95% sequence identity with SEQ ID NO: 2 (TNF30). Phosphorous polypeptide. The method according to claim 1, wherein the anti-HSA SDAB comprises three CDRs (CDR1, CDR2, and CDR3),
(a) CDR1 is
(i) amino acid sequence SFGMS (SEQ ID NO: 25);
(ii) an amino acid sequence having at least 80% sequence identity with SFGMS (SEQ ID NO: 25); or
(iii) contains an amino acid sequence that differs by only one amino acid from SFGMS (SEQ ID NO: 25);
(b) CDR2 is
(i) amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 26);
(ii) an amino acid sequence having at least 80%, 90%, or 95% sequence identity with SISGSGSDTLYADSVKG (SEQ ID NO: 26); or
(iii) comprises an amino acid sequence that differs two or one amino acid from SISGSGSDTLYADSVKG (SEQ ID NO: 26);
(c) CDR3 is
(i) amino acid sequence GGSLSR (SEQ ID NO: 27);
(ii) an amino acid sequence having at least 80% sequence identity with GGSLSR (SEQ ID NO: 27); or
(iii) a polypeptide comprising an amino acid sequence that differs by one amino acid from GGSLSR (SEQ ID NO: 27). The polypeptide of claim 6, wherein CDR1 comprises the amino acid sequence SFGMS (SEQ ID NO: 25), CDR2 comprises the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 26), and CDR3 comprises the amino acid sequence GGSLSR (SEQ ID NO: 27). . The polypeptide of claim 6, wherein the anti-HSA SDAB comprises an amino acid sequence having at least 80%, 90%, 95%, or 95% sequence identity with SEQ ID NO: 5 (ALB8). The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 (ozorarizumab). 10. The polypeptide of claim 1, wherein at least one of the SDABs is humanized. 11. The group of claim 1, wherein two anti-TNF SDABs and anti-HSA SDABs are each linked via a linker, wherein each linker consists of the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 7 The polypeptide selected from. The polypeptide of any one of claims 1-11, wherein the doses are administered separately at time intervals of at least about 1 month. The polypeptide of any one of claims 1-36, wherein the doses are administered separately at time intervals of about 6 weeks or more. The polypeptide of any one of claims 1-13, wherein the doses are administered separately at time intervals of at least about 8 weeks. The method of claim 1, wherein the dose is about 10, 30, 80, 100, 120, 140, 160, 180, 200, 250, 275, 300, 320, 350, 375 and 400 mg. A polypeptide selected from the group consisting of SDAB molecules. The polypeptide of claim 1, wherein the polypeptide is administered subcutaneously or intravenously. The polypeptide of claim 1, wherein the human being is treated simultaneously with methotrexate. 18. The polypeptide of any one of the preceding claims, wherein the polypeptide is formulated in a pharmaceutically acceptable formulation. The method of claim 18 wherein the formulation is
(a) the polypeptide according to claim 1 at a concentration of about 10 mg / ml to 250 mg / ml;
(b) a lyophilized protectant selected from sucrose, sorbitol, or trehalose at a concentration of about 5% to about 10%;
(c) a surfactant selected from polysorbate-80 or poloxamer-188 at a concentration of about 0.01% to 0.6%; And
(d) a buffer selected from histidine buffer at a concentration of about 10 to 20 mM or Tris buffer at a concentration of about 20 to 20 so that the pH of the formulation is from about 5.0 to 7.5
Polypeptide comprising a. The method of claim 19, wherein the formulation comprises 100 mg / ml of the polypeptide of claim 1, 20 mM histidine, 7.5% (w / v) sucrose, and 0.01% polysorbate-80 (pH 6.0). Phosphorous polypeptide. 21. The method of any one of claims 1-20, wherein the immune disorder is inflammatory, including arthritis including inflammation, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis and osteoarthritis, COPD, asthma, Crohn's disease and ulcerative colitis Intestinal disease, multiple sclerosis, Addison disease, autoimmune hepatitis, autoimmune mumps, type I diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, male infertility, multiple sclerosis , Polypeptides selected from the group consisting of myasthenia gravis, pemphigus, psoriasis, purulent hanitis, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroid lobe, and vasculitis. The polypeptide of claim 21, wherein the immune disorder is rheumatoid arthritis. Contains the amino acid sequence of SEQ ID NO: 1 for use in treating human rheumatoid arthritis by administering 80 mg of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 about every four weeks to a human being in need of treatment for rheumatoid arthritis Wherein the human is treated concurrently with methotrexate. Contains the amino acid sequence of SEQ ID NO: 1 for use in treating rheumatoid arthritis in humans by administering about 80 weeks of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to a human being in need of treatment for rheumatoid arthritis Wherein the human is treated concurrently with methotrexate. A polypeptide for use in treating an immune disorder in a human in need thereof, wherein said polypeptide competes with a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (ozoralizumab) according to claim 9. Administering a polypeptide comprising two anti-tumor necrosis factor alpha (TNFα) SDAB and one anti-human serum albumin (HSA) SDAB to a human in need of an immune disorder treatment in multiple doses of 30-400 mg A method of treating an immune disorder in humans, wherein the dose is administered at a time interval of about every four weeks or more. The method of claim 26, wherein each anti-TNFα SDAB comprises three complementarity determining regions (CDR1, CDR2, and CDR3),
(a) CDR1 is
(i) amino acid sequence DYWMY (SEQ ID NO: 22);
(ii) an amino acid sequence having at least 80% sequence identity with DYWMY (SEQ ID NO: 22); or
(iii) comprises an amino acid sequence that differs by only one amino acid from DYWMY (SEQ ID NO: 22);
(b) CDR2 is
(i) amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 23);
(ii) an amino acid sequence having at least 80%, 90%, or 95% sequence identity with EINTNGLITKYPDSVKG (SEQ ID NO: 23); or
(iii) comprises an amino acid sequence that differs two or one amino acid from EINTNGLITKYPDSVKG (SEQ ID NO: 23);
(c) CDR3 is
(i) amino acid sequence SPSGFN (SEQ ID NO: 24);
(ii) an amino acid sequence having at least 80% sequence identity with SPSGFN (SEQ ID NO: 24); or
(iii) comprising an amino acid sequence that differs by one amino acid from SPSGFN (SEQ ID NO: 24). 28. The amino acid sequence of claim 26 or 27, wherein CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 22), CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 23), and CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 24). How to do. The method of claim 26, wherein at least one of the anti-TNFα SDABs comprises an amino acid sequence having at least 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 2 (TNF30). How. The method of claim 26, wherein each anti-TNFα SDAB comprises an amino acid sequence having at least 80%, 90%, 95%, or 95% sequence identity with SEQ ID NO: 2 (TNF30). How to be. The method of claim 26, wherein the anti-HSA SDAB comprises three CDRs (CDR1, CDR2, and CDR3),
(a) CDR1 is
(i) amino acid sequence SFGMS (SEQ ID NO: 25);
(ii) an amino acid sequence having at least 80% sequence identity with SFGMS (SEQ ID NO: 25); or
(iii) contains an amino acid sequence that differs by only one amino acid from SFGMS (SEQ ID NO: 25);
(b) CDR2 is
(i) amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 26);
(ii) an amino acid sequence having at least 80%, 90%, or 95% sequence identity with SISGSGSDTLYADSVKG (SEQ ID NO: 26); or
(iii) comprises an amino acid sequence that differs two or one amino acid from SISGSGSDTLYADSVKG (SEQ ID NO: 26);
(c) CDR3 is
(i) amino acid sequence GGSLSR (SEQ ID NO: 27);
(ii) an amino acid sequence having at least 80% sequence identity with GGSLSR (SEQ ID NO: 27); or
(iii) comprising an amino acid sequence that differs by one amino acid from GGSLSR (SEQ ID NO: 27). The method of claim 31, wherein CDR1 comprises the amino acid sequence SFGMS (SEQ ID NO: 25), CDR2 comprises the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 26), and CDR3 comprises the amino acid sequence GGSLSR (SEQ ID NO: 27). Way. 33. The method of claim 31 or 32, wherein the anti-HSA SDAB comprises an amino acid sequence having at least 80%, 90%, 95%, or 95% sequence identity with SEQ ID NO: 5 (ALB8). 34. The method of any one of claims 26-33, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 (ozorarizumab). 35. The method of any one of claims 26-34, wherein at least one of the SDABs is humanized. 36. The group of any one of claims 26-35, wherein two anti-TNF SDABs and anti-HSA SDABs are each linked through a linker, wherein each linker consists of the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 7. Which is selected from. 37. The method of any one of claims 26-36, wherein the doses are administered separately at time intervals of at least about 1 month. 38. The method of any one of claims 26-37, wherein the doses are administered separately at time intervals of at least about 6 weeks. 39. The method of any one of claims 26-38, wherein the doses are administered separately at time intervals of at least about 8 weeks. 40. The dosage form of any one of claims 26-39 wherein the dose is about 10, 30, 80, 100, 120, 140, 160, 180, 200, 250, 275, 300, 320, 350, 375 and 400 mg. The SDAB molecule. 41. The method of any one of claims 26-40, wherein the polypeptide is administered subcutaneously or intravenously. 42. The method of any one of claims 26-41, wherein the human being is treated simultaneously with methotrexate. 43. The method of any one of claims 26-42, wherein the polypeptide is formulated in a pharmaceutically acceptable formulation. The method of claim 43, wherein the formulation is
(a) a polypeptide according to claim 26 at a concentration of about 10 mg / ml to 250 mg / ml;
(b) a lyophilized protectant selected from sucrose, sorbitol, or trehalose at a concentration of about 5% to about 10%;
(c) a surfactant selected from polysorbate-80 or poloxamer-188 at a concentration of about 0.01% to 0.6%; And
(d) a buffer selected from histidine buffer at a concentration of about 10 to 20 mM or Tris buffer at a concentration of about 20 to 20 so that the pH of the formulation is about 5.0 to 7.5
&Lt; / RTI &gt; 45. The method of claim 44, wherein the formulation comprises 100 mg / ml of the polypeptide of claim 1, 20 mM histidine, 7.5% (w / v) sucrose, and 0.01% polysorbate-80 (pH 6.0). How to be. 46. The method of any one of claims 26-45, wherein the immune disorder is inflammatory, including arthritis including inflammation, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis and osteoarthritis, COPD, asthma, Crohn's disease and ulcerative colitis Intestinal disease, multiple sclerosis, Addison disease, autoimmune hepatitis, autoimmune mumps, type I diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, male infertility, multiple sclerosis , Myasthenia gravis, pemphigus, psoriasis, purulent hanitis, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroid lobe, and vasculitis. The method of claim 46, wherein the immune disorder is rheumatoid arthritis. A method of treating rheumatoid arthritis in humans comprising administering about 80 mg of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to a human being in need of treatment for rheumatoid arthritis, wherein the human is simultaneously methotrexate. How to be treated. A method of treating rheumatoid arthritis in humans comprising administering about 80 weeks of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to a human being in need of treatment for rheumatoid arthritis, wherein the human is simultaneously treated with methotrexate. How to be treated. A method of treating an immune disorder in a human comprising administering to the human being in need thereof an polypeptide that competes with the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (ozorarizumab) according to claim 34.

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