WO2024101382A1 - Composition to be used in immune checkpoint regulation - Google Patents
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
Definitions
- compositions for use in regulating immune checkpoints relate to compositions for use in regulating immune checkpoints.
- LSR lymphothelial sarcoma
- gastric cancer breast cancer
- breast cancer endometrial cancer
- colon cancer colon cancer
- bladder cancer bladder cancer
- lung cancer head and neck tumors
- pancreatic cancer pancreatic cancer
- immune checkpoint inhibitors Currently, antibody drugs targeting PD-1, PD-L1, CTLA-4, etc. are being used clinically as immune checkpoint inhibitors. However, these immune checkpoint inhibitors only have a significant therapeutic effect in around 10% to 20% of patients, and there is an urgent need to develop therapeutic drugs for patients in whom these immune checkpoint inhibitors are not effective.
- LSR is a receptor involved in the uptake of lipoproteins into cells in lipid metabolism (Bihain BE, et al. Curr Opin Lipidol. 1998 Jun;9(3):221-4).
- LSR has variable-type (V-type) immunoglobulin (Ig) in the extracellular domain and is classified as the Ig superfamily.
- V-type variable-type immunoglobulin
- PD-L1 molecule belonging to the B7 family is also classified as the Ig superfamily. Therefore, the present inventors suspected that LSR suppresses tumor immunity as an immune checkpoint molecule. Therefore, an anti-LSR antibody developed independently was administered to an LSR-positive mouse breast cancer cell line (4T1) syngenic model mouse.
- anti-LSR antibody exerted an anti-tumor effect on the 4T1 syngenic model mouse, that this anti-tumor effect was lost by the removal of CD8+ T cells, and that the administration of the anti-LSR antibody increased the number and activation rate of CD8+ T cells infiltrating into the tumor.
- the purpose of this disclosure is to provide a treatment using immune checkpoint inhibitors that target lipolysis-stimulated lipoprotein receptors (LSRs) as an effective treatment for intractable solid cancers such as breast cancer and ovarian cancer.
- LSRs lipolysis-stimulated lipoprotein receptors
- the present disclosure provides, for example, the following: (Item 1) A composition for modulating an immune checkpoint comprising a modulator of the lipolysis-stimulating lipoprotein receptor (LSR). (Item 2) The composition described in the preceding item, wherein the LSR regulator is an inhibitor of LSR. (Item 3) The composition according to any one of the preceding items, wherein the inhibitor against LSR is an anti-LSR antibody or an antigen-binding fragment thereof. (Item 4) The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
- the anti-LSR antibody or antigen-binding fragment thereof is (a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively; (b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively; (c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively; (d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153
- a composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells comprising a modulator of an LSR.
- the LSR modulator is an inhibitor of LSR.
- the inhibitor of LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
- the composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
- the anti-LSR antibody or antigen-binding fragment thereof is (a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively; (b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively; (c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively; (d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-
- (Item 13) The composition of any one of the preceding items, wherein the malignant tumor is an LSR-positive malignant tumor.
- (Item 14) The composition of any one of the preceding items, wherein the malignant tumor is breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
- (Item 15) A method for regulating an immune checkpoint using a composition described in any one of the preceding items.
- (Item 16) The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
- (Item 17) The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
- (Item 18) The method of any one of the preceding items, wherein the modulation of an immune checkpoint is inhibition of an immune checkpoint.
- (Item 19) A method for increasing and/or activating tumor-infiltrating CD8+ T cells using the composition according to any one of the preceding items.
- (Item 20) The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
- (Item 21) The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
- (Item 22) A method for treating or preventing malignant tumors using the composition described in any one of the preceding items.
- (Item 23) The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
- (Item 25) A composition according to any one of the preceding items for use in regulating immune checkpoints.
- (Item 27) A composition according to any one of the preceding items for use in the treatment or prevention of malignant tumors.
- the antibody targeting the LSR disclosed herein can be used as an immune checkpoint inhibitor, providing a new treatment for refractory solid cancers that highly express LSR. As a result, this disclosure makes it possible to improve the prognosis of cancer patients.
- FIG. 1 shows the results of sequence analysis of the V-type Ig region of LSR and B7 family molecules.
- FIG. 2 shows the results of ELISA analysis demonstrating that anti-LSR monoclonal antibody #27-6 mF-18 cross-reacts with cynomolgus monkey LSR, rat LSR and mouse LSR with binding affinity equivalent to that of human LSR.
- FIG. 3 shows the results of FACS (FIG. 3, left) and Western blotting (FIG. 3, right) demonstrating the expression of mouse LSR in 4T1 and MC-38-mLSR-13.
- Figure 4 shows a schematic diagram of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using a 4T1 syngenic mouse model (subcutaneous transplantation).
- FIG. 5 shows the results of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using a 4T1 syngenic mouse model (subcutaneous transplantation). Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test.
- FIG. 6 shows a schematic diagram of an in vivo efficacy test verifying the influence of CD8 + T cells on the efficacy of an anti-LSR antibody (#27-6mF18).
- Figure 7 shows the results of a drug efficacy test of anti-LSR monoclonal antibody #27-6 mF-18 using a 4T1 syngenic mouse model (subcutaneous implantation) in which CD8+ T cells were deleted by administration of anti-CD8 antibody.
- Figure 8 shows the results of FACS analysis to confirm the depletion of CD8+ T cells from mice by administration of anti-CD8 antibodies. Data were analyzed using Student's t-test.
- Figure 9 shows the results of FACS analysis of CD8+ T cells infiltrating into the tumors of the anti-LSR monoclonal antibody #27-6 mF-18 treated group. Data were analyzed using Student's t-test.
- Figure 10 shows the results of measuring chemokine concentrations in tumor tissues of a 4T1 syngenic mouse model (subcutaneously implanted) administered with an anti-LSR antibody (#27-6mF18). * indicates P ⁇ 0.05.
- Figure 11 shows the results of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using a 4T1 syngenic mouse model (subcutaneous implantation) lacking T cell function. Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test.
- Figure 12 shows the results of an in vivo efficacy test of an anti-LSR antibody (#27-6mF18) using an MC-38-mLSR syngenic mouse model (subcutaneous implantation). Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test.
- Figure 13 shows the results of a drug efficacy test of anti-LSR monoclonal antibody #27-6 mF-18 using an MC-38-mLSR syngenic mouse model (subcutaneous implantation) in which CD8+ T cells were deleted by administration of an anti-CD8 antibody. Data were analyzed using a one-way ANOVA test followed by Scheffe's post hoc test.
- LSR Low density lipoprotein receptor
- NCBI National Center for Biotechnology Information
- HGNC HUGO Gene Nomenclature Committee
- accession numbers of LSR listed in NCBI are, for example, NP_991403 (amino acid) and /NM_205834.3 (mRNA).
- the amino acid sequence of LSR is, for example, SEQ ID NO: 7.
- the base sequence of LSR mRNA is, for example, SEQ ID NO: 8.
- the amino acid sequence of LSR is not limited as long as it has LSR activity.
- derivatives preferably include, but are not limited to, molecules that contain a region substantially homologous to the protein of interest (e.g., LSR), which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical over the same size amino acid sequence or when compared to sequences aligned by computer homology programs known in the art, or nucleic acids encoding such molecules are hybridizable to sequences encoding the component proteins under (highly) stringent, moderately stringent, or non-stringent conditions.
- LSR substantially homologous to the protein of interest
- polypeptide i.e., a fragment or derivative
- a polypeptide i.e., a fragment or derivative
- a structural, regulatory, or biochemical function of a protein such as biological activity, according to the embodiment to which the polypeptide, i.e., fragment or derivative, of the present disclosure relates.
- LSR is primarily discussed in humans, but since many animals other than humans are known to express LSR, it is understood that these animals, particularly mammals, are also within the scope of the present disclosure.
- the functional domains of the LSR such as the transmembrane domain (positions 260-280), phosphorylation sites (positions 309, 328, 406, 493, 528, 530, 535, 540, 551, 586, 615, 646), are conserved.
- a representative nucleotide sequence of an LSR is: (a) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 7 or a fragment thereof; (b) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof; (c) a polynucleotide encoding a variant polypeptide or a fragment thereof having one or more or one or several amino acids in the amino acid sequence set forth in SEQ ID NO:8, the variant polypeptide having biological activity; (d) a polynucleotide which is a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7, or a fragment thereof; (e) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8, or a fragment thereof; (f) a polynu
- the amino acid sequence of LSR is: (a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof; (b) a polypeptide having one or more amino acid mutations selected from the group consisting of substitutions, additions and deletions in the amino acid sequence set forth in SEQ ID NO:8, and having biological activity; (c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7; (d) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 8; or (e) a polypeptide that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polypeptides (a) to (d) and has biological activity.
- biological activity typically refers to
- LSR regulator refers to a substance that has the effect of regulating the function of LSR.
- LSR is an immune checkpoint molecule, and that LSR suppresses tumor immunity. By regulating the function of LSR, it is possible to regulate tumor immunity.
- immune checkpoint molecule refers to a molecule that is expressed on cells and transmits a signal that inhibits an immune response by binding to a ligand.
- LSR inhibitor refers to a substance or factor that inhibits the biological action of LSR.
- factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, antisense, and nucleic acid forms such as RNAi factors such as siRNA.
- “reduction” or “suppression” of an activity or expression product refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that causes a decrease.
- “elimination” refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as “elimination.” In this specification, “elimination” is encompassed by “reduction” or “suppression.”
- RNA interference or "RNAi” is an abbreviation for RNA interference, which is generally known in the art and is a biological process that inhibits or downregulates gene expression in cells, mediated by factors that cause RNAi.
- RNAi double-stranded RNA
- RNAi double-stranded RNA
- RNAi is understood to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translation inhibition, transcription inhibition, and epigenetics.
- factors that cause RNAi may be anything as long as they cause "RNAi.”
- RNAi agents that cause RNAi
- small interfering nucleic acids include “small interfering nucleic acids,” “siNA,” “small interfering RNA,” “siRNA,” “small interfering nucleic acid molecules,” “small interfering oligonucleotide molecules,” or “chemically modified small interfering nucleic acid molecules,” and these terms refer to any nucleic acid molecule that can inhibit or downregulate gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. These terms can refer to individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. These molecules can be double-stranded nucleic acid molecules that contain self-complementary sense and antisense regions.
- siRNA typically used in this disclosure is a double-stranded RNA that is short, typically about 20 bases long (e.g., typically about 21-23 bases long) or shorter. Such siRNA suppresses gene expression when expressed in cells.
- the siRNA used in this disclosure may take any form as long as it can induce RNAi.
- the antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and the sense region includes a nucleotide sequence or a portion thereof that corresponds to the target nucleic acid sequence.
- each strand includes a nucleotide sequence that is complementary to a nucleotide sequence in the other strand, such that the antisense strand and the sense strand form a duplex or double-stranded structure.
- the double-stranded region can be about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, but can be longer.
- the antisense strand includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule.
- the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the molecule are complementary to the target nucleic acid or a portion thereof).
- these molecules are assembled from a single oligonucleotide, the self-complementary sense and antisense regions of which are linked by a nucleic acid or non-nucleic acid linker.
- the molecules are polynucleotides having double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structures that include self-complementary sense and antisense regions.
- the antisense region may be a circular single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a separate target nucleic acid molecule, and a sense region having a nucleotide sequence or a portion thereof corresponding to the target nucleic acid sequence.
- the molecules may be circular single-stranded polynucleotides having two or more loop structures and a stem including self-complementary sense and antisense regions.
- the antisense region may be a circular single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and a sense region having a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, and the circular polynucleotide may be processed in vivo or in vitro to generate an active molecule capable of mediating RNAi.
- the agents may also include single-stranded polynucleotides having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule (e.g., the agents do not require that a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof be present within the agents).
- the single-stranded polynucleotide may be a 5' phosphate (e.g., Martinez et al., 2002, Cell. , 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10,537-568), and may further comprise a terminal phosphate group, such as 5',3'-diphosphate.
- the LSR inhibitors of the present disclosure comprise separate sense and antisense sequences or regions, where the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or alternately non-covalently linked by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, and/or stacking interactions.
- the LSR inhibitors of the present disclosure comprise a nucleotide sequence that is complementary to the nucleotide sequence of the target gene.
- the LSR inhibitors of the present disclosure interact with the nucleotide sequence of the target gene to inhibit expression of the target gene.
- the LSR inhibitors are not necessarily limited to molecules that comprise only RNA, but also encompass chemically modified nucleotides and non-nucleotides.
- the disclosure when the disclosure is a small interfering nucleic acid molecule, it may lack 2' hydroxy (2'-OH) containing nucleotides.
- the disclosure may be a small interfering nucleic acid that does not require the presence of a nucleotide with a 2' hydroxyl group to mediate RNAi.
- the disclosure when the disclosure is a small interfering nucleic acid molecule, it may not include ribonucleotides (e.g., nucleotides with a 2'-OH group).
- the LSR inhibitor of the disclosure may include ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
- the inhibitor of the LSR may be a nucleic acid molecule capable of mediating sequence-specific RNAi, such as a small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), small interfering oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, or post-transcriptional gene silencing RNA (ptgsRNA).
- siRNA small interfering RNA
- dsRNA double-stranded RNA
- miRNA microRNA
- shRNA short hairpin RNA
- small interfering oligonucleotide small interfering nucleic acid
- small interfering modified oligonucleotide chemically modified siRNA
- ptgsRNA post-transcriptional gene silencing RNA
- factors that induce RNAi include, but are not limited to, RNA or modified forms thereof that contain a double-stranded portion of at least 10 nucleotides in length, including a sequence that has at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions.
- the factor preferably contains a 3' overhanging end, and more preferably, the 3' overhanging end can be DNA that is 2 nucleotides or more in length (e.g., DNA that is 2 to 4 nucleotides in length.
- RNAi used in the present disclosure may include, but is not limited to, a pair of short inverted complementary sequences (e.g., 15 bp or more, e.g., 24 bp, etc.).
- RNAi when a molecule that induces RNAi, such as dsRNA, is introduced into a cell, in the case of a relatively long RNA (e.g., 40 base pairs or more), an RNaseIII-like nuclease called Dicer, which has a helicase domain, cuts out the molecule from the 3' end in units of about 20 base pairs in the presence of ATP, generating short dsRNA (also called siRNA).
- Dicer RNaseIII-like nuclease
- siRNA is an abbreviation for short interfering RNA, and refers to short double-stranded RNA of 10 base pairs or more that is either artificially chemically synthesized or biochemically synthesized, or synthesized in a living organism, or is generated by decomposing double-stranded RNA of about 40 bases or more in the body, and usually has a 5'-phosphate, 3'-OH structure, with the 3' end overhanging by about 2 bases.
- a specific protein binds to this siRNA to form an RNA-induced silencing complex (RISC). This complex recognizes and binds to mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNaseIII-like enzyme activity.
- RISC RNA-induced silencing complex
- the sequence of the siRNA and the sequence of the target mRNA to be cleaved are 100% identical.
- the cleavage activity by RNAi is not completely eliminated, but partial activity remains.
- the effect of base mutations at the center of the siRNA is large, and the cleavage activity of the mRNA by RNAi is extremely reduced.
- the siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA of about 40 bases or more) can be used as such a factor.
- siRNA itself and factors that generate siRNA are also useful.
- in insects for example, 35 dsRNA molecules almost completely degrade 1,000 or more copies of intracellular mRNA, and therefore it is understood that siRNA itself and factors that generate siRNA are useful.
- the factor that causes RNAi of the present disclosure may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3' end.
- shRNA refers to a molecule of about 20 base pairs or more that is a single-stranded RNA that contains a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule and forming a hairpin-like structure.
- shRNA is artificially chemically synthesized.
- such shRNA can be generated by synthesizing RNA in vitro using T7 RNA polymerase from hairpin-structured DNA in which the DNA sequences of the sense strand and the antisense strand are linked in reverse.
- shRNA after being introduced into a cell, is decomposed to a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) in the cell, and causes RNAi in the same way as siRNA, and has the treatment effect of the present disclosure. It should be understood that such an effect is exerted in a wide range of organisms, such as insects, plants, and animals (including mammals).
- shRNA can be used as an active ingredient of the present disclosure since it induces RNAi in the same manner as siRNA.
- shRNA may also preferably have a 3' overhang.
- the length of the double-stranded portion is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more.
- the 3' overhang may preferably be DNA, more preferably at least 2 nucleotides in length, and even more preferably 2 to 4 nucleotides in length.
- the factor that induces RNAi used in the present disclosure may be either artificially synthesized (e.g., chemically or biochemically) or naturally occurring, and there is no essential difference in the effect of the present disclosure between the two. For chemically synthesized factors, it is preferable to purify them by liquid chromatography or the like.
- the factor that induces RNAi used in the present disclosure can also be synthesized in vitro.
- antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and a T7 promoter. When these are annealed in vitro and then introduced into a cell, RNAi is induced through the mechanism described above, and the effect of the present disclosure is achieved.
- RNA can be introduced into a cell by any suitable method, such as the calcium phosphate method.
- the factor that induces RNAi of the present disclosure also includes factors such as single strands that can hybridize with mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present disclosure.
- siDirect2.0 (Naito et al., BMC Bioinformatics. 2009 Nov 30;10:392.) can be used to design RNAi molecules. This can also be outsourced to a contract company (such as Takara Bio Inc.). RNAi action can be confirmed by quantifying the amount of RNA strand expression using real-time RT-PCR. Alternatively, it can be confirmed by analyzing the amount of RNA strand expression using Northern blot, analyzing the amount of protein using Western blot, and observing the phenotype. Plasmids that generate siRNA or shRNA for a specific gene can also be purchased from a contract company (such as Takara Bio Inc.).
- siRNA includes an RNA strand capable of inducing RNAi.
- the two strands of siRNA can be divided into a guide strand and a passenger strand, and the guide strand is incorporated into RISC.
- the guide strand incorporated into RISC is used to recognize the target RNA.
- artificially created ones are mainly used, but some are known to exist endogenously in living organisms.
- the guide strand may be composed of RNA of 15 or more bases. If it is 15 or more bases, the possibility of binding to the target polynucleotide with high accuracy increases.
- the guide strand may also be composed of RNA of 40 or less bases. If it is 40 or less bases, the risk of adverse phenomena such as interferon response is lower.
- shRNA includes a single-stranded RNA strand capable of inducing RNAi and forming a structure folded into a hairpin (hairpin-like structure).
- shRNA is cleaved by Dicer in cells, and siRNA is excised. It is known that this siRNA causes cleavage of the target RNA.
- the above shRNA may be composed of 35 or more nucleotides. If it is 35 or more, the possibility of accurately forming the hairpin-like structure specific to shRNA increases.
- the above shRNA may be composed of RNA of 100 bases or less. If it is 100 bases or less, the risk of adverse phenomena such as interferon response is reduced.
- pre-miRNAs which are generally similar in structure and function to shRNA, are about 100 nucleotides or more in length, it is considered that shRNA can function as an shRNA even if it is not necessarily 100 bases or less in length.
- miRNA includes an RNA strand that has a function similar to that of siRNA, and is known to suppress the translation and degrade the target RNA strand.
- the difference between miRNA and siRNA generally lies in the production pathway and detailed mechanism.
- small RNA refers to a relatively small RNA strand, such as siRNA, shRNA, miRNA, antisense RNA, and single- or double-stranded small RNA.
- RNAi molecule may contain an overhang of 1 to 5 bases at the 5' or 3' end. In this case, it is believed that the efficiency of RNAi increases. This number can be, for example, 5, 4, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 11
- RNAi molecule When the RNAi molecule is double-stranded, mismatched RNA may exist between each RNA strand.
- the number of bases may be, for example, 1, 2, 3, 4, 5, or 10 or less, or within any two of these values.
- the RNAi molecule may also include a hairpin loop, and the number of bases in the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, or within any two of these values.
- the base sequence may have one or more bases deleted, substituted, inserted, or added, as long as it has the desired effect.
- the left side of each base sequence is the 5' end, and the right side is the 3' end.
- the length of the RNAi molecule may be, for example, 15, 18, 20, 25, 30, 40, 50, 60, 80, 100, 200, or 400 bases, or may be within a range of any two of those values.
- the above RNAi molecule contains a base sequence complementary to a portion of the base sequence of the LSR mRNA.
- the above "portion" may be, for example, 5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 50 or more bases, or may be within a range between any two of these values.
- the siRNA contains the base sequence of SEQ ID NO: 9 or 10. These base sequences are complementary to a portion of the LSR mRNA and are considered to function as a guide strand.
- One embodiment of the present disclosure includes an RNAi molecule containing such a base sequence of SEQ ID NO: 9 or 10. This RNAi molecule may further contain a base sequence complementary to the base sequence shown in SEQ ID NO: 9 or 10 (e.g., SEQ ID NO: 11 or 12, respectively).
- a "complementary base sequence” is a base sequence possessed by another polynucleotide that is highly complementary to one polynucleotide and can hybridize with it.
- the full length of the sense strand of the siRNA is the base sequence of SEQ ID NO: 13 or 14, and the full length of the antisense strand is the base sequence of SEQ ID NO: 15 or 16.
- the base sequences listed above may be (i) amino acid sequences in which one or more base sequences are deleted, substituted, inserted, or added in the above base sequences, or (ii) base sequences encoded by polynucleotides that specifically hybridize under stringent conditions to polynucleotides consisting of base sequences complementary to the above base sequences, so long as the LSR siRNA has the desired effect.
- an "LSR-binding substance”, “LSR binding agent” or “LSR-interacting molecule” or “binding factor for LSR” is a molecule or substance that binds to LSR at least temporarily.
- LSR binding agent e.g., be labeled or be capable of being labeled
- the binding e.g., be labeled or be capable of being labeled
- therapeutic agent e.g., an "LSR-binding substance”
- LMWs low molecular weight molecules
- the LSR-binding substance or LSR-interacting molecule can be an inhibitor of LSR, and also includes, for example, binding proteins or binding peptides directed against LSR, in particular against the active site of LSR, as well as nucleic acids directed against the LSR gene.
- Nucleic acids against LSR refer to, for example, double-stranded or single-stranded DNA or RNA, or modifications or derivatives thereof, that inhibit the expression of the LSR gene or the activity of the LSR, and include, but are not limited to, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs), and ribozymes.
- binding protein or “binding peptide” with respect to an LSR refers to any protein or peptide that binds to an LSR, and includes, but is not limited to, antibodies (e.g., polyclonal or monoclonal antibodies), antibody fragments, and functional equivalents directed against an LSR.
- Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly recognized one-letter codes.
- comparisons of amino acid and base sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.13.0 (published 5/17/2022). The identity value in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be regarded as the identity value. If identity is evaluated in multiple regions, the highest value among them shall be regarded as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.
- “several” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values or less. It is known that a polypeptide that has one or several amino acid residues deleted, added, inserted, or substituted with other amino acids maintains its biological activity (Market et al., Proc Natl Acad Sci USA. 1984 Sep; 81(18): 5662-5666.; Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10(20): 6487-6500.; Wang et al., Science. 1984 Jun 29; 224(4656): 1431-1433.).
- Antibodies with deletions or the like can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library.
- the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used for site-specific mutagenesis. It is possible to select antibodies with the same activity as the wild type from mutant antibodies with deletions, etc., by performing various characterizations such as FACS analysis and ELISA.
- a "fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n).
- the length of the fragment can be changed as appropriate depending on the purpose.
- the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit.
- the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit.
- such fragments are understood to be within the scope of the present disclosure, for example, if the full-length fragment functions as a marker or target molecule, as long as the fragment itself also functions as a marker or target molecule.
- the term "activity" as used herein refers to the function of a molecule in its broadest sense. Activity generally includes, but is not intended to be limiting, the biological, biochemical, physical, or chemical functions of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules, stability, ability to localize to a particular subcellular location. Where applicable, the term also relates to the function of protein complexes in their broadest sense.
- biological function refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body, and includes, but is not limited to, the production of specific antibodies, enzyme activity, and the conferring of resistance.
- biological function refers to, but is not limited to, the function of LSR in inhibiting the uptake of VLDL.
- biological activity refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living body, and includes the activity of exerting various functions (e.g., transcription promoting activity), and also includes, for example, the activity of activating or inactivating another molecule by interacting with another molecule.
- a certain factor e.g., polynucleotide, protein, etc.
- various functions e.g., transcription promoting activity
- the biological activity may be the binding between the two molecules and the biological change that occurs as a result of this. For example, when one molecule is precipitated using an antibody and the other molecule is also co-precipitated, the two molecules are considered to be bound.
- a factor is an enzyme
- its biological activity includes its enzymatic activity.
- a factor is a ligand
- its biological activity includes the binding of the ligand to a corresponding receptor. Such biological activity can be measured by techniques well known in the art.
- activity refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.
- the term "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. undergoing a certain action in vivo to become a different form.
- the term refers to the gene, polynucleotide, etc. being transcribed and translated to become a polypeptide, but transcription to produce an mRNA is also an aspect of expression.
- the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, such a polypeptide form may be one that has undergone post-translational processing.
- the expression level of an LSR can be determined by any method.
- the expression level of an LSR can be known by evaluating the amount of LSR mRNA, the amount of LSR protein, and the biological activity of the LSR protein. Such measurements can be used in companion diagnostics.
- the amount of LSR mRNA or protein can be determined by methods detailed elsewhere in this specification or other methods known in the art.
- the term "functional equivalent” refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Therefore, it is understood that the functional equivalent of an "LSR" or its antibody is not the LSR or its antibody itself, but a mutant or modified version of the LSR or its antibody (e.g., an amino acid sequence variant, etc.) that has the biological action of the LSR, as well as an entity that can change into the LSR or its antibody itself or a mutant or modified version of the LSR or its antibody at the time of acting (e.g., a nucleic acid that encodes the LSR or its antibody itself or a mutant or modified version of the LSR or its antibody, and a vector, cell, etc. that contains the nucleic acid).
- a mutant or modified version of the LSR or its antibody e.g., an amino acid sequence variant, etc.
- an LSR or its antibody can be used in the same way as the LSR or its antibody, even if not specifically mentioned.
- the functional equivalent can be found by searching a database, etc.
- search refers to using a certain nucleic acid base sequence to find other nucleic acid base sequences that have a specific function and/or property, electronically, biologically, or by other methods.
- Electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215:403-410(1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad.
- Bio searches include, but are not limited to, stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assays), PCR, and in situ hybridization.
- the gene used in this disclosure should also include corresponding genes identified by such electronic searches and biological searches.
- an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both termini can be used.
- an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both termini means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that may occur naturally.
- the modified amino acid sequence may be, for example, an amino acid sequence in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted or deleted, or added to one or both termini.
- the modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of the LSR or its antibody.
- conservative substitution means replacing one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein.
- a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge.
- non-polar amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine.
- Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
- Positively charged (basic) amino acids include arginine, histidine, and lysine.
- Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- antibody broadly includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotypic antibodies, as well as fragments thereof, such as Fv fragments, Fab' fragments, F(ab') 2 and Fab fragments, and other recombinantly produced conjugates or functional equivalents (e.g., chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc(Fv) 2 (single chain (Fv) 2 ), scFv-Fc).
- antibodies may be covalently linked or recombinantly fused to enzymes, such as alkaline phosphatase, horseradish peroxidase, ⁇ -galactosidase, and the like.
- the anti-LSR antibody used in the present disclosure may be of any origin, type, shape, etc., as long as it binds to an LSR protein.
- known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used.
- monoclonal or polyclonal antibodies can be used as the antibodies, but monoclonal antibodies are preferred.
- the antibody preferably binds specifically to the LSR protein.
- the antibody also includes modified and unmodified antibodies.
- the modified antibody may be bound to various molecules such as polyethylene glycol.
- the modified antibody can be obtained by chemically modifying the antibody using a known method.
- anti-LSR antibody includes an antibody that has binding ability to LSR. There are no particular limitations on the method for producing this anti-LSR antibody, but it may be produced, for example, by immunizing a mammal or bird with LSR.
- an antibody against LSR anti-LSR antibody
- a fragment thereof includes, for example, in the case of antibodies, not only the antibody itself and its fragment having LSR binding activity and, if necessary, inhibitory activity, but also chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-chain antibodies, scFVs, diabodies, sc(Fv) 2 (single chain (Fv) 2 ), scFv-Fc, and the like.
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is preferably an anti-LSR antibody that specifically binds to a specific epitope of LSR, from the viewpoint of particularly strong inhibition of malignant tumor growth.
- the anti-LSR antibody according to one embodiment of the present disclosure may be a monoclonal antibody.
- a monoclonal antibody can act on LSR more efficiently than a polyclonal antibody. From the viewpoint of efficiently producing an anti-LSR monoclonal antibody, it is preferable to immunize chickens with LSR.
- the antibody class of the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is not particularly limited, but may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.
- the anti-LSR antibody according to one embodiment of the present disclosure may be an antibody fragment having antigen-binding activity. In this case, there are effects such as increased stability or antibody production efficiency.
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be a fusion protein.
- This fusion protein may be an anti-LSR antibody with a polypeptide or oligopeptide bound to the N- or C-terminus.
- the oligopeptide may be a His tag.
- the fusion protein may also be a fusion of a mouse, human, or chicken antibody partial sequence. Such fusion proteins are also included in one form of the anti-LSR antibody according to this embodiment.
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be, for example, an antibody obtained through a process of immunizing an organism with purified LSR, LSR-expressing cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization.
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be an antibody having a CDR set of an antibody obtained through a process of immunizing an organism with purified LSR, LSR-expressing cell cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization.
- the CDR set is a set of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3.
- the "LSR-expressing cells” may be obtained, for example, by introducing a polynucleotide encoding LSR into a cell and then expressing LSR.
- the LSR includes an LSR fragment.
- the "LSR-containing lipid membrane” may be obtained, for example, by mixing LSR with a lipid bilayer membrane.
- the LSR includes an LSR fragment.
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is preferably an antibody obtained through a process of immunizing chickens with an antigen, or an antibody having a CDR set of that antibody.
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure may have any binding strength as long as the intended purpose is achieved, and examples thereof include, but are not limited to, at least 1.0 ⁇ 10 or more , 2.0 ⁇ 10 or more, 5.0 ⁇ 10 or more, or 1.0 ⁇ 10 or more , and typically has a K value (kd/ka) of 1.0 ⁇ 10 or less, and may be 1.0 ⁇ 10 or less ( M) or less (M).
- the anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be an antibody that binds to a wild-type or mutant form of LSR. Mutant forms include those resulting from differences in DNA sequences between individuals.
- the amino acid sequence of the wild-type or mutant form of LSR has a homology of preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence shown in SEQ ID NO:8.
- a "polyclonal antibody” can be produced, for example, by administering an immunogen containing an antigen of interest to a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.), bird, etc., in order to induce the production of polyclonal antibodies specific to the antigen.
- the administration of the immunogen may be by injection of one or more immunizing agents and, if desired, an adjuvant.
- Adjuvants may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (e.g., aluminum hydroxide), or surfactants (e.g., lysolecithin), etc.
- Immunization protocols are known in the art and may be performed by any method that induces an immune response, depending on the host organism of choice (Protein Experiment Handbook, Yodosha (2003): 86-91.).
- the term "monoclonal antibody” includes antibodies in which the individual antibodies constituting the population are substantially identical to one another, except for antibodies with small amounts of naturally occurring mutations. Alternatively, the individual antibodies constituting the population may be substantially identical, except for antibodies with small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies that correspond to different epitopes. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins. The term “monoclonal” may indicate the characteristic of being obtained from a substantially homogeneous antibody population, but does not imply that the antibody must be produced by any particular method.
- monoclonal antibodies may be produced by methods similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug7;256(5517):495-497.”
- monoclonal antibodies may be produced by recombinant methods similar to those described in U.S. Pat. No. 4,816,567.
- monoclonal antibodies may be isolated from phage antibody libraries using techniques similar to those described in Clackson et al., Nature. 1991 Aug 15;352(6336):624-628, or Marks et al., J Mol Biol. 1991 Dec 5;222(3):581-597.
- monoclonal antibodies may be produced by methods described in Protein Experiment Handbook, Yodosha (2003):92-96.
- CHO cells are transfected with an H-chain antibody expression vector and an L-chain antibody expression vector, cultured using the selection reagents G418 and Zeocin, and cloned by limiting dilution. After cloning, clones that stably express the antibody are selected by ELISA. The selected CHO cells are expanded and the culture supernatant containing the antibody is collected. The antibody can be purified from the collected culture supernatant by Protein A or Protein G purification.
- an “Fv antibody” is an antibody that contains an antigen recognition site. This region comprises a non-covalent dimer of one heavy chain variable domain and one light chain variable domain. In this configuration, the three CDRs of each variable domain can interact to form an antigen-binding site on the surface of the VH-VL dimer.
- a "Fab antibody” is, for example, an antibody fragment obtained by treating an antibody containing a Fab region and an Fc region with the protease papain, in which approximately the N-terminal half of the H chain and the entire L chain are bound via some disulfide bonds.
- Fab can be obtained, for example, by treating an anti-LSR antibody according to an embodiment of the present disclosure containing a Fab region and an Fc region with the protease papain.
- F(ab') 2 antibody refers to an antibody that contains two sites corresponding to Fab, among fragments obtained by treating an antibody containing a Fab region and an Fc region with the protease pepsin.
- F(ab') 2 can be obtained, for example, by treating an anti-LSR antibody according to an embodiment of the present disclosure that contains a Fab region and an Fc region with the protease pepsin.
- it can be prepared by forming a thioether bond or disulfide bond with the following Fab'.
- a “Fab'antibody” refers to an antibody obtained, for example, by cleaving the disulfide bond in the hinge region of F(ab') 2. For example, it can be obtained by treating F(ab') 2 with a reducing agent, dithiothreitol.
- an "scFv antibody” is an antibody in which VH and VL are linked via a suitable peptide linker.
- an scFv antibody can be produced by obtaining cDNA encoding the VH and VL of an anti-LSR antibody used in a complex according to an embodiment of the present disclosure, constructing a polynucleotide encoding VH-peptide linker-VL, incorporating the polynucleotide into a vector, and using an expression cell.
- a “diabody” is an antibody that has bivalent antigen-binding activity.
- the bivalent antigen-binding activities can be the same, or one of the two can be a different antigen-binding activity.
- a diabody can be produced, for example, by constructing a polynucleotide encoding an scFv such that the amino acid sequence of the peptide linker is 8 residues or less in length, incorporating the resulting polynucleotide into a vector, and using an expression cell.
- dsFv refers to an antibody in which polypeptides with cysteine residues introduced into VH and VL are linked via disulfide bonds between the cysteine residues.
- the position at which the cysteine residue is introduced can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Reiteret et al., Protein Eng. 1994 May;7(5):697-704.)
- a "peptide or polypeptide having antigen-binding activity" refers to an antibody that is composed of an antibody VH, VL, or CDR1, 2, or 3 thereof.
- a peptide containing multiple CDRs can be linked directly or via a suitable peptide linker.
- the method of producing the Fv antibody, Fab antibody, F(ab') 2 antibody, Fab' antibody, scFv antibody, diabody, dsFv antibody, antigen-binding peptide or polypeptide (hereinafter sometimes referred to as "Fv antibody, etc.") used in the present disclosure is not particularly limited.
- DNA encoding a region of the Fv antibody, etc. in the anti-LSR antibody used in the complex according to an embodiment of the present disclosure can be inserted into an expression vector and produced using an expression cell.
- it may be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBOC method (t-butyloxycarbonyl method).
- the antigen-binding fragment according to an embodiment of the present disclosure may be one or more of the above Fv antibodies, etc.
- a "chimeric antibody” refers to, for example, an antibody variable region and an antibody constant region linked between different organisms, and can be constructed by recombinant gene technology.
- Mouse-human chimeric antibodies can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA. 1994 Feb 1;91(3):969-973.”
- the basic method for producing mouse-human chimeric antibodies is, for example, to link mouse leader sequences and variable region sequences present in cloned cDNA to sequences encoding human antibody constant regions already present in a mammalian cell expression vector.
- mouse leader sequences and variable region sequences present in cloned cDNA may be linked to sequences encoding human antibody constant regions, and then linked to a mammalian cell expression vector.
- the fragment of the human antibody constant region can be any human antibody H chain constant region or human antibody L chain constant region, for example, C ⁇ 1, C ⁇ 2, C ⁇ 3, or C ⁇ 4 for the human H chain, and C ⁇ or C ⁇ for the L chain.
- a “humanized antibody” is an antibody that has, for example, one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin, and that binds to a desired antigen.
- Antibody humanization can be performed using various techniques known in the art (Almagro et al., Front Biosci. 2008 Jan 1;13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci U S A.
- FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.) can be used.
- amino acid residues in the human FR region may be replaced with corresponding residues from the CDR donor antibody.
- This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.).
- FR residues important for antigen binding can be identified by modeling the interactions of CDR and FR residues.
- unusual FR residues at specific positions can be identified by sequence comparison.
- a "human antibody” refers to an antibody whose heavy chain variable and constant regions and light chain variable and constant regions are derived from genes encoding human immunoglobulin.
- the main methods for producing human antibodies include the transgenic mouse method for producing human antibodies and the phage display method.
- the transgenic mouse method for producing human antibodies if a functional human Ig gene is introduced into a mouse whose endogenous Ig has been knocked out, human antibodies with diverse antigen-binding abilities are produced instead of mouse antibodies.
- human monoclonal antibodies can be obtained by the conventional hybridoma method. For example, they can be produced by the method described in "Lonberg et al., Int Rev Immunol.
- the phage display method is a system in which a foreign gene is expressed as a fusion protein on the N-terminus of the coat protein (g3p, g10p, etc.) of a filamentous phage, such as M13 or T7, which is typically an E. coli virus, so as not to lose the infectivity of the phage.
- a filamentous phage such as M13 or T7, which is typically an E. coli virus
- the antibody may also be prepared by grafting the heavy chain CDR or light chain CDR of the anti-LSR antibody according to the embodiment of the present disclosure onto any antibody by CDR-grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.).
- the antibody may be obtained by linking DNA encoding the heavy chain CDR or light chain CDR of the anti-LSR antibody according to the embodiment of the present disclosure and DNA encoding a region excluding the heavy chain CDR or light chain CDR of a known antibody derived from a human or non-human organism to a vector according to a method known in the art, and then expressing the vector using a known cell.
- the region excluding the heavy chain CDR or light chain CDR may be optimized using a method known in the art (e.g., a method of randomly mutating amino acid residues of an antibody and screening for one with high reactivity, or a phage display method, etc.).
- the FR region may be optimized, for example, by using FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060.
- Heavy chains are typically the main components of full-length antibodies. Heavy chains are usually bound to light chains by disulfide bonds and non-covalent bonds.
- the N-terminal domain of heavy chains contains a region called a variable region (VH), whose amino acid sequence is not constant even in antibodies of the same species and class, and it is generally known that VH contributes greatly to specificity and affinity for antigens.
- VH variable region
- CDR complementarity determining region
- Fv variable region: including the heavy chain variable region (VH) and the light chain variable region (VL)
- CDR consists of CDR1, CDR2, and CDR3, each consisting of about 5 to 30 amino acid residues. It is known that the CDR of the heavy chain in particular contributes to the binding of the antibody to the antigen. It is also known that among the CDRs, CDR3 contributes most to the binding of the antibody to the antigen.
- the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia's definition (Chothia et al., J. Mol. Biol., 1987;196:901-917) may be adopted.
- the Kabat definition is adopted as a preferred example, but is not necessarily limited to this. In some cases, the definition may be determined taking into consideration both the Kabat definition and the Chothia definition.
- the overlapping portion of the CDR according to each definition, or the portion including both the CDRs according to each definition may be the CDR.
- a specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. USA, 1989;86:9268-9272) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat and Chothia definitions.
- Such CDR information can be used to produce variants that can be used in the present disclosure.
- Such antibody variants can be produced that contain one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions, additions, or deletions in the framework of the original antibody, but do not contain mutations in the CDRs.
- antigen refers to any substance capable of being specifically bound by an antibody molecule.
- immunogen refers to an antigen capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
- epitope or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by one of skill in the art using such well-known and conventional techniques when provided with the primary sequence of nucleic acid or amino acid. It is understood that the antibodies of the present disclosure can be similarly utilized with antibodies having other sequences, so long as the epitopes are the same.
- the antibodies used in this specification may have any specificity as long as false positives are reduced.
- the antibodies used in this disclosure may be polyclonal or monoclonal antibodies.
- the term “means” refers to any tool that can achieve a certain purpose (e.g., detection, diagnosis, treatment), and in particular, in this specification, “selective recognition means” refers to a means that can recognize one object differently from others.
- malignant tumor includes, for example, tumors that develop as a result of mutation of normal cells. Malignant tumors can arise from any organ or tissue in the body. Examples of malignant tumors include lung cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, ovarian cancer, uterine cancer, bladder cancer, prostate cancer, ureter cancer, renal pelvis cancer, ureter cancer, penile cancer, testicular cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, head and neck cancer, glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer, salivary gland cancer, malignant lymphoma, carcinoma, sarcoma, and hematological malignant tumors.
- ovarian cancer includes, for example, ovarian serous, liquid adenocarcinoma, or ovarian clear cell adenocarcinoma.
- Uterine cancer includes, for example, endometrial cancer or cervical cancer.
- Head and neck cancers include, for example, oral cancer, pharyngeal cancer, laryngeal cancer, nasal cancer, paranasal sinus cancer, salivary gland cancer, or thyroid cancer.
- Lung cancers include, for example, non-small cell lung cancer or small cell lung cancer.
- the malignant tumor may also be LSR positive.
- the anti-LSR antibody according to the embodiment of the present disclosure could become a new therapeutic agent for serous adenocarcinoma and clear cell adenocarcinoma.
- LSR-positive malignant tumor includes malignant tumors that significantly or excessively express LSR. Whether a malignant tumor is LSR-positive may be evaluated, for example, by RT-PCR, Western blot, or immunohistochemical staining. In addition, when total protein of malignant tumor cells is subjected to Western blot and a band corresponding to LSR (e.g., a band around 649aa) can be visually confirmed, it may be determined to be LSR-positive. Alternatively, it may be determined to be LSR-positive when the amount of LSR expression in malignant tumor cells derived from a patient is significantly greater than that in normal cells. From the viewpoint of achieving more optimal medication by accurately diagnosing LSR positivity, it is preferable to test the expression of LSR using an anti-LSR antibody.
- the term "subject” refers to an object that is the subject of diagnosis, detection, or treatment according to the present disclosure (e.g., an organism such as a human, or cells, blood, serum, etc. extracted from an organism).
- sample refers to any substance obtained from a subject, etc., including, for example, blood, serum, plasma, saliva, urine, tears, cerebrospinal fluid, etc. Those skilled in the art will be able to select an appropriate and preferred sample based on the description in this specification.
- drug drug
- agent agent
- factor factor
- Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof.
- proteins es, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipid
- Factors specific to a polynucleotide typically include, but are not limited to, polynucleotides that have a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to promoter regions.
- Representative factors specific to a polypeptide include, but are not limited to, an antibody or a derivative or analog thereof (e.g., a single-chain antibody) specifically directed against the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.
- treatment refers to preventing the worsening of a certain disease or disorder (e.g., malignant tumor) when that condition occurs, preferably maintaining the status quo, more preferably alleviating, and even more preferably causing the disease or disorder to recede, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease.
- a certain disease or disorder e.g., malignant tumor
- diagnostic agents used for this purpose are sometimes called “companion diagnostic agents.”
- the term "therapeutic agent” refers broadly to any drug capable of treating a target condition (e.g., a disease such as a malignant tumor).
- the "therapeutic agent” may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers.
- the pharmaceutical composition may be produced, for example, by mixing the active ingredient with the carrier and using any method known in the technical field of pharmaceutical formulations.
- the therapeutic agent may be in any form as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and any other ingredient.
- the shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution).
- the therapeutic agent for malignant tumors includes drugs used for preventing malignant tumors (prophylactic drugs) or agents for suppressing the proliferation of malignant tumor cells.
- prevention refers to preventing a certain disease or disorder (e.g., malignant tumor) from occurring before that state occurs.
- a diagnosis can be made using the drug disclosed herein, and if necessary, the drug disclosed herein can be used to prevent, for example, malignant tumors, or preventive measures can be taken.
- prophylactic drug refers broadly to any drug that can prevent a target condition (e.g., a disease such as a malignant tumor).
- interaction refers to the mutual exertion of force (e.g., intermolecular forces (van der Waals forces), hydrogen bonds, hydrophobic interactions, etc.) between one substance and the other.
- force e.g., intermolecular forces (van der Waals forces), hydrogen bonds, hydrophobic interactions, etc.
- two substances that have interacted are in an associated or bonded state.
- the detection, testing, and diagnosis disclosed herein can be achieved by utilizing such interactions.
- binding refers to a physical or chemical interaction between two substances or a combination thereof. Binding includes ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. Physical interactions (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effect of another protein or compound and does not involve other substantial chemical intermediates.
- an “agent” that "specifically” interacts with (or binds to) a biological agent such as a polynucleotide or polypeptide includes one whose affinity for the biological agent, such as the polynucleotide or polypeptide, is typically equal to or higher than, and preferably significantly (e.g., statistically significantly) higher than, its affinity for other unrelated polynucleotides or polypeptides (e.g., those with less than 30% identity). Such affinity can be measured, for example, by hybridization assays, binding assays, etc.
- a first substance or factor "specifically" interacts with (or binds to) a second substance or factor means that the first substance or factor interacts with (or binds to) the second substance or factor with a higher affinity than it does with substances or factors other than the second substance or factor (particularly other substances or factors present in a sample containing the second substance or factor).
- specific interactions (or binding) for a substance or factor include, but are not limited to, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, and other reactions between nucleic acids and proteins, protein-lipid interactions, and nucleic acid-lipid interactions.
- both substances or factors are nucleic acids
- “specifically interacting" with a first substance or factor includes the first substance or factor having at least partial complementarity to the second substance or factor.
- examples of “specific" interaction (or binding) of a first substance or factor with a second substance or factor include, but are not limited to, interactions due to antigen-antibody reactions, interactions due to receptor-ligand reactions, enzyme-substrate interactions, etc.
- "specific" interaction (or binding) of a first substance or factor with a second substance or factor includes interactions (or binding) between an antibody and its antigen. By utilizing such specific interactions or binding reactions, it is possible to detect or quantify a target substance in a sample.
- detection or “quantification” of polynucleotide or polypeptide expression may be accomplished using any suitable method, including, for example, measurement of mRNA and immunological measurement methods, including binding or interaction with a detection agent, test agent or diagnostic agent.
- molecular biological measurement methods include, for example, Northern blot, dot blot, or PCR.
- immunological measurement methods include, for example, ELISA, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunoturbidimetric (TIA), Western blot, and immunohistochemical staining using microtiter plates.
- quantification methods include ELISA or RIA.
- Genetic analysis may also be performed using arrays (e.g., DNA arrays, protein arrays).
- DNA arrays are broadly reviewed in “DNA Microarrays and the Latest PCR Methods," a special edition of Cell Engineering, edited by Shujunsha. Protein arrays are reviewed in NatGenet. 2002 Dec;32 Suppl:526-532.
- gene expression analysis methods include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like. Such further analysis methods are described, for example, in Genome Analysis Experimental Methods: Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke Yodosha (2002), and all descriptions therein are incorporated by reference.
- expression level refers to the amount of a polypeptide or mRNA, etc., expressed in a cell, tissue, etc. of interest.
- expression level include the expression level at the protein level of the polypeptide of the present disclosure, evaluated by any appropriate method, including immunological measurement methods such as ELISA, RIA, fluorescent antibody technique, Western blotting, and immunohistochemical staining, using an antibody of the present disclosure, or the expression level at the mRNA level of the polypeptide used in the present disclosure, evaluated by any appropriate method, including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR.
- Change in expression level refers to an increase or decrease in the expression level at the protein level or mRNA level of the polypeptide used in the present disclosure, evaluated by any appropriate method, including the immunological measurement method or molecular biological measurement method. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.
- “reduction” or “suppression” of an activity or expression product refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that causes a decrease.
- “elimination” refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as “elimination.”
- “elimination” is encompassed within “reduction” or “suppression.”
- increase or “activation” of an activity or expression product refers to an increase or increasing activity in the amount, quality or effect of a particular activity, transcript or protein.
- label refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others.
- labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc.
- RI radioisotope
- fluorescence method fluorescence method
- biotin method biotin method
- chemiluminescence method etc.
- the labels are labeled with fluorescent substances having mutually different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more.
- Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Examples of combinations of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa TM 555 and Alexa TM 633, and a combination of Alexa TM 488 and Alexa TM 555.
- any dye that can bind to the base moiety can be used, but it is preferable to use cyanine dyes (e.g., Cy3 and Cy5 of the CyDye TM series), rhodamine 6G reagent, 2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), and the like.
- cyanine dyes e.g., Cy3 and Cy5 of the CyDye TM series
- AAF 2-acetylaminofluorene
- AAIF iodine derivative of AAF
- fluorescent substances having a difference in maximum fluorescence wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein.
- such labels can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art
- a "tag” refers to a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a substance that acts as a binding partner for binding a specific substance (e.g., having a relationship such as biotin-avidin or biotin-streptavidin), and may be included in the category of a "label.”
- a specific substance bound to a tag can be selected by contacting the specific substance with a substrate bound to a binding partner of the tag sequence.
- tags or labels are well known in the art. Representative tag sequences include, but are not limited to, myc tags, His tags, HA, and Avi tags.
- in vivo refers to the inside of a living organism. In certain contexts, “within the organism” refers to the location where a substance of interest is to be placed.
- in vitro refers to a state in which a part of a living organism is removed or isolated "outside of a living organism" (e.g., in a test tube) for various research purposes. This term contrasts with in vivo.
- kit refers to a unit in which the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually in two or more compartments.
- This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use.
- Such a kit is advantageously provided with instructions or instructions describing how to use the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, etc.) or how to handle the reagents.
- the kit When the kit is used as a reagent kit in this specification, the kit usually includes instructions describing how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.
- instructions refers to instructions for a physician or other user on how to use the present disclosure.
- the instructions include instructions on how to use the present disclosure's detection method, how to use a diagnostic agent, or how to administer a medicine.
- the instructions may also include instructions for administration via the mouth or esophagus (e.g., by injection, etc.) as the site of administration.
- the instructions are prepared in accordance with a format stipulated by the supervisory authority of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and it is clearly stated that the instructions have been approved by the supervisory authority.
- the instructions are so-called package inserts, and are usually provided in paper form, but are not limited to this, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).
- internalization refers to a cell taking up an antigen-bound substance on the cell surface via endocytosis or phagocytosis, with the substance being mediated by the antigen.
- a substance that binds to an LSR having such activity e.g., an anti-LSR antibody
- target active ingredients include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, etc.
- antibody drug conjugate refers to an antibody or antigen-binding fragment thereof chemically linked to one or more active ingredients of interest.
- the ADC is operably linked via a linker.
- operably linked refers to a relationship in which the linked substances are capable of operating in a predicted manner.
- Active ingredients of interest include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, and the like.
- the linker may be a cleavable linker or a non-cleavable linker.
- cleavable linkers include, but are not limited to, linkers having a sequence cleaved by a protease, acid-labile linkers, disulfide linkers, and the like.
- non-cleavable linkers include, but are not limited to, MCC linkers, and the like.
- cytotoxic activity refers to, for example, causing pathological changes in cells, not only direct trauma, but also any damage to the structure or function of cells, such as DNA breaks, formation of base dimers, chromosome breaks, damage to the cell division system, and reduced activity of various enzymes, thereby directly or indirectly blocking cell function and causing cell death. Therefore, examples of “drugs having cytotoxic activity” include, but are not limited to, alkylating agents, tumor necrosis factor inhibitors, intercalators, microtubule inhibitors, kinase inhibitors, proteasome inhibitors, and topoisomerase inhibitors.
- IC50 50% inhibitory concentration
- the present disclosure provides a composition for regulating immune checkpoints, comprising a regulator of lipolysis-stimulating lipoprotein receptor (LSR).
- LSR lipolysis-stimulating lipoprotein receptor
- LSR lipolysis-stimulating lipoprotein receptor
- LSR expressed on tumor cells interacts with receptors present on CD8+ T cells.
- inhibitory stimuli are transmitted to CD8+ T cells intracellularly, and LSR has the function of suppressing tumor immunity.
- ovarian cancer, gastric cancer, and endometrial cancer with high LSR expression have a significantly poorer prognosis compared to those with low LSR expression, and this can be reasonably explained as being appropriate.
- the modulator of LSR may be an inhibitor of LSR.
- inhibitors of LSR include, but are not limited to, anti-LSR antibodies or antigen-binding fragments thereof, nucleic acids such as antisense nucleic acids or siRNAs, and small molecule compounds.
- siRNAs may include, for example, SEQ ID NOs: 9 to 14.
- the binding agent for LSR or a fragment thereof can be an anti-LSR antibody or an antigen-binding fragment thereof or a functional equivalent.
- the antibody of the present disclosure may be a specific sequence described elsewhere in this disclosure.
- the antibody may be an antibody or antigen-binding fragment thereof that includes any sequence including the CDRs of the full-length sequence, or an antibody or antigen-binding fragment thereof that includes a variable region of the following sequence, and that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, or 20 or more substitutions, additions, or deletions in the framework region.
- the antibody may be produced, etc., using the embodiments described elsewhere in this disclosure and/or techniques known in the art.
- such an antibody or fragment or functional equivalent preferably has an inhibitory activity downstream of the LSR or its signal transduction pathway.
- Such activity may be confirmed by observing the expression level or activity of the LSR, or by directly using a malignant tumor cell line such as ovarian leiomyoma cells to inhibit cell growth, or by transplanting the cell line into a model animal to observe tumor regression.
- a malignant tumor cell line such as ovarian leiomyoma cells to inhibit cell growth
- transplanting the cell line into a model animal to observe tumor regression are well known in the art and may be used in the present disclosure.
- the administration route of the composition of the present disclosure is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration.
- the administration form may be, for example, an injection, capsule, tablet, or granule.
- the aqueous solution for injection may be stored, for example, in a vial or stainless steel container.
- the aqueous solution for injection may also be mixed with, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH.
- the therapeutic agent may also be mixed with, for example, a buffer (for example, a phosphate buffer), a stabilizer, etc.
- compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present disclosure include a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharma- ceutically acceptable carrier or excipient.
- pharmaceutically acceptable means approved by a government regulatory agency or listed in a pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.
- carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered.
- Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like.
- water is the preferred carrier.
- saline and aqueous dextrose are the preferred carriers.
- saline solutions, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for injectable solutions.
- Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc.
- the composition can also contain a small amount of a wetting or emulsifying agent, or a pH buffer, if desired.
- These compositions can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc.
- the composition can also be formulated as a suppository, using traditional binders and carriers, such as triglycerides.
- Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.).
- compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide a form that is properly administered to the patient.
- the formulation should be suitable for the mode of administration.
- surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavorings, etc. may be included.
- various delivery systems are known and such systems can be used to administer the therapeutic agents of the present disclosure to the appropriate site (e.g., esophagus), such as encapsulation in liposomes, microparticles, and microcapsules; using recombinant cells capable of expressing the therapeutic agent (e.g., polypeptides); using receptor-mediated endocytosis; constructing the therapeutic nucleic acid as part of a retroviral or other vector.
- Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
- the pharmaceutical agent can be administered by any suitable route, such as by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), using an inhaler or nebulizer with an aerosolizing agent if necessary, and can be administered together with other biologically active agents. Administration can be systemic or local. When the present disclosure is used in the ovarian region, it may be administered by any suitable route, such as by direct injection into the affected area, such as the ovary.
- the composition can be formulated as a pharmaceutical composition adapted for administration to humans, according to known methods.
- a composition for administration by injection is a solution in a sterile isotonic aqueous buffer.
- the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the site of the injection.
- the ingredients are supplied separately or mixed together in unit dosage form, for example as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachette indicating the quantity of active agent.
- composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
- compositions, pharmaceuticals, therapeutic agents, and prophylactic agents of the present disclosure may be formulated in neutral or salt form or as other prodrugs (e.g., esters, etc.).
- Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxide, etc.
- the amount of the therapeutic agent of the present disclosure effective in treating a particular disorder or condition may vary depending on the nature of the disorder or condition, but can be determined by one of skill in the art using standard clinical techniques based on the description herein. In addition, in vitro assays may be used in some cases to help identify optimal dosage ranges.
- the exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dosage is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dose, or within any two of these values.
- the administration interval is not particularly limited, and may be, for example, 1 or 2 doses per 1, 7, 14, 21, or 28 days, or 1 or 2 doses per two of these values.
- the dosage, administration interval, and administration method may be selected appropriately depending on the age, weight, symptoms, target organ, etc. of the patient.
- the therapeutic agent preferably contains an active ingredient in a therapeutically effective amount, or in an effective amount that exerts a desired effect. If the malignant tumor marker is significantly reduced after administration, it may be determined that the therapeutic effect has been achieved.
- a "patient” includes a human or a non-human mammal (e.g., one or more of mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee).
- the patient may also be a patient who has been determined or diagnosed as having an LSR-positive malignant tumor. In this case, the determination or diagnosis is preferably made by detecting the protein level of LSR.
- composition or therapeutic or prophylactic agent disclosed herein can be provided as a kit.
- the disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of an antibody, conjugate, composition, or medicament of the disclosure.
- containers may bear information indicating approval by a government agency of the manufacture, use, or sale for human administration, in a manner prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products.
- the anti-LSR antibody may be an anti-LSR antibody that specifically binds to an epitope of LSR. More specifically, the antibody may have an epitope at positions 116-135 and/or 216-230 of SEQ ID NO:7.
- One embodiment of the present disclosure relates to an antibody comprising: (a) a heavy chain CDR1, 2, 3, and a light chain CDR1, 2, and 3, each of which comprises the amino acid sequence set forth in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 230 of SEQ ID NO: 1; and (b) a heavy chain CDR1, 2, 3, and a light chain CDR1, 2, and 3, each of which comprises the amino acid sequence set forth in positions 31 to 35, 50 to 66, 99 to 103, 152 to 165, 182 to 188, and 221 to 230 of SEQ ID NO: 2.
- the anti-LSR antibody may be an anti-LSR antibody, in which the amino acid sequences of heavy chain CDR1, 2, and 3 are at least one antibody selected from the group consisting of antibodies comprising the amino acid sequences of positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 6, respectively, or a mutant of the antibody, in which the mutant comprises one or several substitutions, additions, or deletions in the framework of the antibody, but does not comprise a mutation in the CDR.
- an anti-LSR antibody comprising at least one of the sets of amino acid sequences of heavy chain CDR1, 2, and 3 listed above. These antibodies may be antibodies selected from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFVs, diabodies, sc(Fv) 2 (single chain (Fv) 2 ), and scFv-Fc.
- the antibody may contain one or several substitutions, additions or deletions in one, two, three, four, five or six of the six CDRs while maintaining binding to the LSR.
- the antibody may contain up to three, preferably up to two, more preferably up to two, preferably one substitution, addition or deletion in one CDR.
- the substitutions may be conservative substitutions.
- An anti-LSR antibody comprises a set of amino acid sequences of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3, and further, at least one, preferably two, three, four, five, six, seven, or all of the frameworks of heavy chain FR1, 2, 3, and 4, and light chain FR1, 2, 3, and 4 may be identical or substantially identical to any of SEQ ID NOs: 1 to 6, or identical except for conservative substitutions. There may be one or more types of antibodies. Another embodiment of the present disclosure is an anti-LSR antibody comprising at least one of the sets of amino acid sequences of heavy chain FR1, 2, 3, and 4 listed above.
- the anti-LSR antibody according to one embodiment of the present disclosure may be in the form of an scFv, in which case the linker between the heavy and light chains may have the amino acid sequence shown in positions 116 to 132 of SEQ ID NO:1, positions 116 to 132 of SEQ ID NO:2, positions 116 to 132 of SEQ ID NO:3, positions 116 to 132 of SEQ ID NO:4, positions 116 to 132 of SEQ ID NO:5, or positions 116 to 132 of SEQ ID NO:6.
- VHs of #9-7, #16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in the Examples below are positions 1 to 115 of SEQ ID NO:1, positions 1 to 115 of SEQ ID NO:2, positions 1 to 115 of SEQ ID NO:3, positions 1 to 115 of SEQ ID NO:4, positions 1 to 115 of SEQ ID NO:5, and positions 1 to 115 of SEQ ID NO:6, respectively.
- the VLs of #9-7, #16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in the Examples below are positions 133 to 238 of SEQ ID NO:1, positions 133 to 239 of SEQ ID NO:2, positions 133 to 238 of SEQ ID NO:3, positions 133 to 238 of SEQ ID NO:4, positions 133 to 238 of SEQ ID NO:5, and positions 133 to 238 of SEQ ID NO:6, respectively.
- amino acid sequences listed above may be one or more amino acid sequences selected from the group consisting of: (i) the above amino acid sequences in which one or more base sequences have been deleted, substituted, inserted, or added; (ii) amino acid sequences having 90% or more identity (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the above amino acid sequences; and (iii) amino acid sequences encoded by polynucleotides that specifically hybridize under stringent conditions to polynucleotides consisting of a base sequence complementary to the base sequence encoding the above amino acid sequences, so long as the anti-LSR antibody has the desired effect.
- a transformant can be created by introducing a polynucleotide or vector encoding an anti-LSR antibody according to an embodiment of the present disclosure into a cell.
- the transformant can be used to produce an anti-LSR antibody according to an embodiment of the present disclosure.
- the transformant may be a cell of a human or a mammal other than a human (e.g., rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells) and monkey cells COS-7. Alternatively, the transformant may be Escherichia bacteria, yeast, etc.
- the above vectors may be, for example, plasmids derived from E. coli (e.g., pET-Blue), plasmids derived from Bacillus subtilis (e.g., pUB110), yeast-derived plasmids (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5/His-TOPO), bacteriophages such as ⁇ phage, and vectors derived from viruses.
- These vectors may contain components necessary for protein expression, such as a promoter, an origin of replication, or an antibiotic resistance gene.
- the vector may be an expression vector.
- polynucleotides or vectors can be introduced into cells by, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, or microinjection (Revised 4th Edition New Genetic Engineering Handbook, Yodosha (2003): 152-179.).
- the method described in "Protein Experiment Handbook, Yodosha (2003): 128-142.” can be used.
- antibodies for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion and cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography can be used (Protein Experiment Handbook, Yodosha (2003): 27-52.).
- the present disclosure provides a composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells, comprising a modulator of an LSR.
- the CD8+ T cells can be CD69+.
- CD69 is known as an activation marker for CD8+ T cells.
- the compositions and the like of the present disclosure may be for treating or preventing malignant tumors.
- the malignant tumor may be LSR positive.
- the malignant tumor may be breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer, or colon cancer, which may be LSR positive.
- a companion reagent for determining whether or not treatment with the complex, composition, pharmaceutical, etc. of the present disclosure is required, the companion reagent including a detection reagent for LSR.
- kits for treating or preventing malignant tumors comprising an LSR detection reagent, a complex, composition, medicine, etc. of the present disclosure, and instructions.
- Example 1 Sequence analysis of LSR and V-type Ig region of B7 family molecules Sequence analysis of LSR ( Figure 1, left) The identity of the amino acid sequences of human LSR (Q86X29) and mouse LSR (Q99KG5) was analyzed using the Aliment tool of Uniprot.
- V-type Ig region of LSR and B7 family molecules ( Figure 1, right) The amino acid sequence identity of the V-type Ig region of human LSR (Q86X29), human CD80 (P33681), human CD86 (P42081), human ICOSL (O75144), human PDL1 (Q9NZQ7), human PDL2 (Q9BQ51), human CD276 (Q5ZPR3), human VTCN1 (B7-H4) (Q7Z7D3), and human VISTA (Q9H7M9) was analyzed using the Aliment tool of Uniprot.
- the B7 family belongs to the Ig superfamily and is a single-pass transmembrane protein with V-type and constant (C)-type Ig domains. Comparison of amino acid sequences between human LSR and mouse LSR and other B7-like proteins revealed that the DxGxYxC motif in the V-type Ig domain is conserved ( Figure 1). The amino acid sequence identity between human LSR and other B7-like proteins in the V-type Ig domain ranged from 6.77% to 16.9% (CD80: 8.87%, CD86: 6.77%, ICOSL: 10.24%, PD-L1: 11.2%, PD-L2: 8.94%, B7-H3: 7.67%, B7-H4: 8.43%, VISTA: 16.89%).
- LSR Human LSR showed the highest amino acid sequence identity with VISTA, and both proteins lacked the C-type Ig domain.
- the DxGxYxC motif is a typical sequence conserved in Ig domains. Some proteins with Ig domains may have immune checkpoint activity. From the above, it is suggested that LSR is also a B7-like protein, and in the Examples below, we will confirm whether LSR has immune checkpoint activity.
- Example 2 Analysis of cross-reactivity of chicken mouse chimeric anti-LSR antibody (#27-6mF18) with human, cynomolgus monkey, rat, and mouse LSR by ELISA (Materials and Methods) Buffer Preparation
- the buffers used in this example were prepared as follows.
- - Coating buffer 50 mM carbonate-bicarbonate buffer, pH 9.6.
- Dilution buffer The dilution solution used was 10% Block Ace, which was prepared by dissolving 4 g of Block Ace powder in 100 mL of MiliQ and diluting the solution 10-fold with PBS(-).
- Blocking buffer 4 g of Block Ace powder was dissolved in 100 mL of MiliQ, and then diluted 4-fold with PBS (-) to prepare a blocking buffer. The solution obtained by dissolving 4 g of Block Ace powder in 100 mL of MiliQ was used as the stock solution (100% solution). Washing buffer: One packet of PBS-T (Sigma, P3563) was dissolved in 1 L of ultrapure water (0.05% Tween 20/PBS).
- the primary antibody used in this example was prepared.
- Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was serially diluted with dilution buffer to 1000, 100, 10, 1, 0.1, 0.01, and 0.001 nM.
- the antigens used were recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc.
- the anti-LSR monoclonal antibody #27-6 created by the inventors also cross-reacts with mouse LSR (WO/2015/098113).
- the anti-LSR monoclonal antibody #27-6 mF-18 which does not exhibit ADCC activity or CDC activity, was created. This is to demonstrate that the antitumor effect shown in the following examples is not due to ADCC activity or CDC activity.
- ELISA analysis revealed that the anti-LSR monoclonal antibody #27-6 mF-18 reacts with human LSR and mouse LSR ( Figure 2).
- Example 3 LSR expression analysis by FACS and Western blot LSR expression analysis by FACS (Figure 3, left) The expression of LSR was analyzed by FACS analysis for 4T1 (mouse breast cancer cell line), HM-1 (OV2944-HM-1, mouse ovarian cancer cell line), B16F10 (mouse malignant melanoma cell line), CT26 (mouse colon cancer cell line), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mLSR-expressing mouse colon cancer).
- Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was used as the primary antibody, and FITC-labeled Goat Anti-Mouse IgG (H+L chain specific) (southern biotech) was used as the secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using FlowJo (trademark) software (Tree star).
- mouse LSR (result) Expression of mouse LSR was confirmed using 4T1 (mouse breast cancer), HM-1 (OV2944-HM-1, mouse ovarian cancer), B16F10 (mouse melanoma), CT-26 (mouse colon cancer), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mouse colon cancer) cell lines. Expression of mouse LSR was confirmed in 4T1 and MC-38-mLSR-13 by both FACS and Western blotting ( Figure 3).
- Example 4 In vivo efficacy test of anti-LSR antibody (#27-6mF18) using 4T1 syngenic mouse model (subcutaneous transplantation) Paraffin-embedded tissue was sliced, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for LSR was performed using anti-LSR antibody (CST #14804) and ChemMate Envision kit HRP 500T (Dako: K5007).
- FIG. 4 A schematic diagram of the in vivo efficacy test of anti-LSR antibody (#27-6mF18) using the 4T1 syngenic mouse model (subcutaneous transplantation) in this example is shown in Figure 4.
- 2.0 x 10 5 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and the day after transplantation, the mice were divided into 4 groups, and administration of PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) was started.
- Intraperitoneal administration of drugs, measurement of tumor volume, and body weight were performed at the time points described in the experimental outline in Figure 4.
- Example 5 Effect of CD8 + T cells on efficacy of anti-LSR antibody (#27-6mF18) A schematic diagram of the in vivo efficacy test in this example to verify the effect of CD8 + T cells on the efficacy of anti-LSR antibody (#27-6mF18) is shown in FIG.
- mice 2.0x105 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and the day after transplantation, the mice were divided into 4 groups and administered isotype control antibody (5mg/kg), anti-LSR antibody (#27-6mF18) (5mg/kg), anti-CD8 antibody (0.2mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5mg/kg) + anti-CD8 antibody (0.2mg/body).
- Drugs were intraperitoneally administered, tumor volume was measured, and body weight was measured at the time points described in the experimental outline in Figure 6.
- Example 6 Experiment to confirm CD8+ T cell depletion by administration of anti-CD8 antibody An experiment was conducted to confirm that CD8+ T cells in mice were removed by administration of anti-CD8 antibody. 2.0 ⁇ 10 5 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and anti-CD8 antibody (0.2 mg/body) was intraperitoneally administered twice, on the day of transplantation and two days later. On the fourth day, cells were isolated from the spleen, and surface antigens were stained with anti-CD45 (30-F11; catalog No. 103116), anti-CD8 (53-6.7; catalog No. 100734), and anti-CD4 (RM4-5; catalog No. 100516) antibodies at 4°C for 30 minutes. After washing the stained cells, they were measured using FACS CantoII (BD), and the data was analyzed using BD FACS Diva software (BD Biosciences).
- BD FACS CantoII
- Example 7 Analysis of tumor-infiltrating CD8+ T cells To evaluate the number of tumor-infiltrating CD8+ T cells and the rate of activation by anti-LSR antibody administration, tumors were excised on the final day (day 19) for the two groups of isotype control antibody (5 mg/kg) and anti-LSR antibody (#27-6mF18) (5 mg/kg) in Example 5. Single cell suspensions were prepared from the excised tumor tissues using gentleMACS Octo Dissociator (Miltenyi Biotec) and mouse Tumor Dissociation Kit cocktail (Miltenyi Biotec; cat#130-096-730).
- the cell suspension was passed through 100 ⁇ m pre-separation filters (Miltenyi Biotec; cat#130-098-463, cat#130-098-458), and the cell suspension that passed through the filters was collected.
- the cell suspension was reacted with mouse FcR Blocking Reagent (Miltenyi Biotec; catalog No. 130-092-575), and then surface antigens were stained with anti-CD3 (145-2C11; catalog No. 553062, BD Biosciences), anti-CD69 (H1.2F3; catalog No.104508, BioLegend), anti-CD45 (30-F11; catalog No.103116, BioLegend), and anti-CD8 (53-6.7; catalog No.100734, BioLegend) at 4°C for 30 minutes. After washing, the stained cells were measured using a FACS CantoII (BD) and the data were analyzed using FlowJo software (Tree Star).
- BD FACS CantoII
- Example 8 Analysis of chemokine expression in 4T1 tumor tissue 2.0 x 10 5 cells of the 4T1 cell line were subcutaneously transplanted into 8-week-old Balb/c female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. On the 19th day after cell transplantation, tumors were excised from the mice in each group.
- chemokines CXCL9, CXCL10
- CXCL9 and CXCL10 are known to be chemokines that attract CD8+ T cells, so the chemokines (CXCL9, CXCL10) in the tumor tissue were quantified.
- the concentrations of CXCL9 and CXCL10 in the tumor tissue were significantly higher in the anti-LSR monoclonal antibody #27-6 mF-18 administration group than in the control antibody administration group ( Figure 10). Therefore, it is considered that the increase in the concentrations of CXCL9 and CXCL10 in the tumor promotes the infiltration of CD8+ T cells into the tumor in the anti-LSR monoclonal antibody #27-6 mF-18 administration group.
- Example 9 Drug efficacy analysis of anti-LSR antibody (#27-6mF18) using immunodeficient mice 2.0 x 10 5 cells of the 4T1 cell line were subcutaneously transplanted into 7-week-old Balb/c nu/nu female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. At the time points shown in FIG. 11, tumor volume, body weight, and tumor weight were measured.
- Example 10 Analysis of the efficacy of anti-LSR antibody (#27-6mF18) using syngenic model mice created by transplanting MC38-mLSR cells into C57BL/6 mice 5.0 x 10 5 MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. Measurements of tumor volume, body weight, and tumor weight were performed at the time points shown in FIG. 12.
- Example 3 since the expression of LSR is negative in the mouse colon cancer cell line MC-38, MC38-mLSR-13, which stably expresses the mouse LSR gene, was established ( Figure 3). By examining the in vivo antitumor effect of the anti-LSR monoclonal antibody #27-6 mF-18 using MC38-mLSR-13, it was examined whether the antitumor effect was observed as in the case of using 4T1 ( Figure 5).
- the anti-LSR monoclonal antibody #27-6 mF-18 administration group showed a significant antitumor effect at both doses of 5 mg/kg and 20 mg/kg, compared with the PBS administration group and the control antibody administration group (20 mg/kg) (Figure 12).
- Example 11 In vivo efficacy test to verify the influence of CD8 + T cells on the efficacy of anti-LSR antibody (#27-6mF18) in MC38-mLSR syngenic model mice 5.0 ⁇ 10 5 MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and each group was administered isotype control antibody (5 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), anti-CD8 antibody (0.2 mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5 mg/kg) + anti-CD8 antibody (0.2 mg/body). Tumor volume, body weight, and tumor weight were measured at the time points shown in Figure 13.
- anti-LSR antibodies activate tumor immunity by inhibiting the interaction between LSR on tumor cells and an unknown receptor on T cells, and exert an antitumor effect through activation of CD8+ T cells.
- Example 12 Other LSR inhibitors
- tests similar to those in Examples 4 to 11 are performed using anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, and #1-43, siRNA (SEQ ID NO: 15 or 16), or small molecule compounds as LSR inhibitors.
- anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, #1-43, siRNA (SEQ ID NO: 15 or 16), or a small molecule LSR inhibitor will be used to confirm in vivo efficacy tests in a 4T1 syngenic mouse model (subcutaneous implantation). Also, as in Example 7, anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, #1-43, siRNA (SEQ ID NO: 15 or 16), or a small molecule LSR inhibitor will be used to confirm the increase and activation of tumor-infiltrating CD8+ T cells.
- Malignant tumor control technology is provided, and technology that can be used in industries involved in technologies related to the treatment and prevention of malignant tumors (reagents, pharmaceuticals, etc.) is provided.
- SEQ ID NO:1 Anti-LSR antibody 9-7 sequence
- SEQ ID NO:2 Anti-LSR antibody 16-6 sequence
- SEQ ID NO:3 Anti-LSR antibody 26-2 sequence
- SEQ ID NO:4 Anti-LSR antibody 27-6 sequence
- SEQ ID NO:5 Anti-LSR antibody 1-25 sequence
- SEQ ID NO:6 Anti-LSR antibody 1-43 sequence
- SEQ ID NO:7 Human LSR protein sequence (NP_991403.1)
- SEQ ID NO: 8 Human LSR nucleic acid sequence (NM_205834.3)
- SEQ ID NO: 12 Antisense sequence of LSR siRNA 2 core sequence (guide sequence)
- SEQ ID NO: 13 Full-length sense sequence of LSR siRNA 1
- SEQ ID NO: 14 Full-length sense
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Abstract
The present disclosure addresses the problem of providing a novel composition to be used in immune checkpoint regulation. One aspect of the present disclosure provides a composition which is to be used in immune checkpoint regulation and contains a lipolysis-stimulated lipoprotein receptor (LSR) regulator. Another aspect of the present disclosure provides a composition which contains the LSR regulator and activates and/or increases the number of tumor-infiltrating CD8+ T-cells. The LSR regulator in some of the embodiments is an anti-LSR antibody or an LSR inhibitor, which is an antigen-binding fragment thereof.
Description
本開示は、免疫チェックポイントの調節に用いるための組成物に関する。
The present disclosure relates to compositions for use in regulating immune checkpoints.
LSRを高発現する癌としては卵巣癌、胃癌、乳癌、子宮内膜癌、大腸癌、膀胱癌、肺癌、頭頸部腫瘍、膵臓癌などが知られている。いずれの癌も進行期、再発症例においては有効な治療法が確立されておらず、新規治療法の開発が必要とされている。
Cancers that are known to highly express LSR include ovarian cancer, gastric cancer, breast cancer, endometrial cancer, colon cancer, bladder cancer, lung cancer, head and neck tumors, and pancreatic cancer. For any of these cancers, no effective treatment has been established for advanced stages or recurrent cases, and the development of new treatments is needed.
現在PD-1、PD-L1、CTLA-4等を標的とした抗体医薬が免疫チェックポイント阻害剤として臨床で実用化されている。しかしながら、これらの免疫チェックポイント阻害剤に対して優れた薬効が得られるのは10%から20%程度であり、これらの免疫チェックポイント阻害剤による薬効が認められない患者に対する治療薬の開発は急務である。
Currently, antibody drugs targeting PD-1, PD-L1, CTLA-4, etc. are being used clinically as immune checkpoint inhibitors. However, these immune checkpoint inhibitors only have a significant therapeutic effect in around 10% to 20% of patients, and there is an urgent need to develop therapeutic drugs for patients in whom these immune checkpoint inhibitors are not effective.
これまでに我々の研究室では、発明者らは卵巣癌に高発現する新規標的抗原としてLSRを世界に先駆けて同定した(Hiramatsu K, Naka T, et al. Cancer Res. (2018) 78 (2): 516-527、WO2015/098113)。LSRは脂質代謝においてリポタンパク質の細胞内への取り込みに関わる受容体である(Bihain BE, et al. Curr Opin Lipidol. 1998 Jun;9(3):221-4)。
In our laboratory, the inventors were the first in the world to identify LSR as a novel target antigen highly expressed in ovarian cancer (Hiramatsu K, Naka T, et al. Cancer Res. (2018) 78 (2): 516-527, WO2015/098113). LSR is a receptor involved in the uptake of lipoproteins into cells in lipid metabolism (Bihain BE, et al. Curr Opin Lipidol. 1998 Jun;9(3):221-4).
本発明者らの解析では、LSRは細胞外ドメインにvariable-type(V-type)immunoglobulin(Ig)を有しており、Ig superfamilyに分類される。興味深いことに、B7ファミリーに属するPD-L1分子もIg superfamilyに分類されている。そこで、本発明者らはLSRが免疫チェックポイント分子として腫瘍免疫を抑制しているのではないかと考えた。そこで、LSR陽性マウス乳癌細胞株(4T1)シンジェニックモデルマウスに独自に開発した抗LSR抗体を投与した。その結果、抗LSR抗体は4T1シンジェニックモデルマウスに抗腫瘍効果を発揮し、この抗腫瘍効果がCD8+T細胞の除去により失われること、抗LSR抗体の投与により腫瘍内に浸潤したCD8+T細胞の数および活性化の割合の上昇を確認した。これらの結果、抗LSR抗体は腫瘍細胞上のLSRと、CD8+T細胞上の未知の受容体との結合を阻害することでCD8+T細胞の活性化を介して抗腫瘍効果を示していることが示唆された。
In the analysis by the present inventors, LSR has variable-type (V-type) immunoglobulin (Ig) in the extracellular domain and is classified as the Ig superfamily. Interestingly, the PD-L1 molecule belonging to the B7 family is also classified as the Ig superfamily. Therefore, the present inventors suspected that LSR suppresses tumor immunity as an immune checkpoint molecule. Therefore, an anti-LSR antibody developed independently was administered to an LSR-positive mouse breast cancer cell line (4T1) syngenic model mouse. As a result, it was confirmed that the anti-LSR antibody exerted an anti-tumor effect on the 4T1 syngenic model mouse, that this anti-tumor effect was lost by the removal of CD8+ T cells, and that the administration of the anti-LSR antibody increased the number and activation rate of CD8+ T cells infiltrating into the tumor. These results suggest that anti-LSR antibodies exert their antitumor effects through activation of CD8+ T cells by inhibiting the binding of LSR on tumor cells to an unknown receptor on CD8+ T cells.
本開示の目的は、乳癌、卵巣癌などの難治性固形癌に対する有効な治療法としてLipolysis-stimulated lipoprotein receptor(LSR)を標的とした免疫チェックポイント阻害剤による治療法を提供することである。
The purpose of this disclosure is to provide a treatment using immune checkpoint inhibitors that target lipolysis-stimulated lipoprotein receptors (LSRs) as an effective treatment for intractable solid cancers such as breast cancer and ovarian cancer.
本開示は、例えば、以下の項目を提供する。
(項目1)
脂肪分解刺激リポタンパク質受容体(LSR)の調節因子を含む、免疫チェックポイントを調節するための組成物。
(項目2)
前記LSRの調節因子が、LSRに対する抑制因子である、前記項目に記載の組成物。
(項目3)
前記LSRに対する抑制因子が、抗LSR抗体またはその抗原結合フラグメントである、前記項目のいずれか一項に記載の組成物。
(項目4)
前記抗体のエピトープが、配列番号7の116~135位および/または216~230位を含む、前記項目のいずれか一項に記載の組成物。
(項目5)
前記抗LSR抗体またはその抗原結合フラグメントが、以下:
(a)それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(b)それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(c)それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(d)それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(e)それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、または
(f)それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
前記項目のいずれか一項に記載の組成物。
(項目6)
LSRの調節因子を含む、腫瘍浸潤CD8+T細胞の数を増加および/または活性化させるための組成物。
(項目7)
前記腫瘍浸潤CD8+T細胞が、CD69陽性である、前記項目のいずれか一項に記載の組成物。
(項目8)
前記LSRの調節因子が、LSRに対する抑制因子である、前記項目のいずれか一項に記載の組成物。
(項目9)
前記LSRの抑制因子が、抗LSR抗体またはその抗原結合フラグメントである、前記項目のいずれか一項に記載の組成物。
(項目10)
前記抗体のエピトープが、配列番号7の116~135位および/または216~230位を含む、前記項目のいずれか一項に記載の組成物。
(項目11)
前記抗LSR抗体またはその抗原結合フラグメントが、以下:
(a)それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(b)それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(c)それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(d)それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(e)それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、または
(f)それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
からなる群から選択される、前記項目のいずれか一項に記載の組成物。
(項目12)
前記組成物が、悪性腫瘍を治療または予防するためのものである、前記項目のいずれか一項に記載の組成物。
(項目13)
前記悪性腫瘍が、LSR陽性悪性腫瘍である、前記項目のいずれか一項に記載の組成物。
(項目14)
前記悪性腫瘍が、乳癌、卵巣癌、子宮内膜癌、膵臓癌、肺癌、胃癌または大腸癌である、前記項目のいずれか一項に記載の組成物。
(項目15)
前記項目のいずれか一項に記載の組成物を使用する、免疫チェックポイントの調節方法。
(項目16)
in vitroまたはin vivoで使用する、前記項目のいずれか一項に記載の方法。
(項目17)
患者に、治療有効量の組成物を投与する工程を含む、前記項目のいずれか一項に記載の方法。
(項目18)
前記免疫チェックポイントの調節は、免疫チェックポイントの阻害である、前記項目のいずれか一項に記載の方法。
(項目19)
前記項目のいずれか一項に記載の組成物を使用する、腫瘍浸潤CD8+T細胞の数を増加および/または活性化方法。
(項目20)
in vitroまたはin vivoで使用する、前記項目のいずれか一項に記載の方法。
(項目21)
患者に、治療有効量の組成物を投与する工程を含む、前記項目のいずれか一項に記載の方法。
(項目22)
前記項目のいずれか一項に記載の組成物を使用する、悪性腫瘍の治療または予防方法。
(項目23)
in vitroまたはin vivoで使用する、前記項目のいずれか一項に記載の方法。
(項目24)
患者に、治療有効量の組成物を投与する工程を含む、前記項目のいずれか一項に記載の方法。
(項目25)
免疫チェックポイントの調節に用いるための、前記項目のいずれか一項に記載の組成物。
(項目26)
腫瘍浸潤CD8+T細胞の数を増加および/または活性化に用いるための、前記項目のいずれか一項に記載の組成物。
(項目27)
悪性腫瘍の治療または予防に用いるための、前記項目のいずれか一項に記載の組成物。
(項目28)
免疫チェックポイントの調節用組成物の製造ための、前記項目のいずれか一項に記載の組成物の使用。
(項目29)
腫瘍浸潤CD8+T細胞の数を増加および/または活性化用組成物の製造のための、前記項目のいずれか一項に記載の組成物の使用。
(項目30)
悪性腫瘍の治療または予防用組成物の製造のための、前記項目のいずれか一項に記載の組成物の使用。
本開示において、上記1または複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供されうることが意図される。本開示のなおさらなる実施形態および利点は、必要に応じて以下の詳細な説明を読んで理解すれば、当業者に認識される。 The present disclosure provides, for example, the following:
(Item 1)
A composition for modulating an immune checkpoint comprising a modulator of the lipolysis-stimulating lipoprotein receptor (LSR).
(Item 2)
The composition described in the preceding item, wherein the LSR regulator is an inhibitor of LSR.
(Item 3)
The composition according to any one of the preceding items, wherein the inhibitor against LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
(Item 4)
The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
(Item 5)
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition according to any one of the preceding items.
(Item 6)
A composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells comprising a modulator of an LSR.
(Item 7)
The composition of any one of the preceding items, wherein the tumor-infiltrating CD8+ T cells are CD69 positive.
(Item 8)
The composition according to any one of the preceding items, wherein the LSR modulator is an inhibitor of LSR.
(Item 9)
The composition of any one of the preceding items, wherein the inhibitor of LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
(Item 10)
The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
(Item 11)
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition of any one of the preceding claims, selected from the group consisting of:
(Item 12)
The composition according to any one of the preceding items, wherein the composition is for treating or preventing malignant tumors.
(Item 13)
The composition of any one of the preceding items, wherein the malignant tumor is an LSR-positive malignant tumor.
(Item 14)
The composition of any one of the preceding items, wherein the malignant tumor is breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
(Item 15)
A method for regulating an immune checkpoint using a composition described in any one of the preceding items.
(Item 16)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 17)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 18)
The method of any one of the preceding items, wherein the modulation of an immune checkpoint is inhibition of an immune checkpoint.
(Item 19)
A method for increasing and/or activating tumor-infiltrating CD8+ T cells using the composition according to any one of the preceding items.
(Item 20)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 21)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 22)
A method for treating or preventing malignant tumors using the composition described in any one of the preceding items.
(Item 23)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 24)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 25)
A composition according to any one of the preceding items for use in regulating immune checkpoints.
(Item 26)
The composition according to any one of the preceding items for use in increasing the number and/or activating tumor-infiltrating CD8+ T cells.
(Item 27)
A composition according to any one of the preceding items for use in the treatment or prevention of malignant tumors.
(Item 28)
Use of a composition described in any one of the preceding items for the manufacture of a composition for modulating an immune checkpoint.
(Item 29)
Use of a composition according to any one of the preceding items for the manufacture of a composition for increasing and/or activating tumor-infiltrating CD8+ T cells.
(Item 30)
Use of a composition according to any one of the preceding items for the manufacture of a composition for the treatment or prevention of malignant tumors.
It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.
(項目1)
脂肪分解刺激リポタンパク質受容体(LSR)の調節因子を含む、免疫チェックポイントを調節するための組成物。
(項目2)
前記LSRの調節因子が、LSRに対する抑制因子である、前記項目に記載の組成物。
(項目3)
前記LSRに対する抑制因子が、抗LSR抗体またはその抗原結合フラグメントである、前記項目のいずれか一項に記載の組成物。
(項目4)
前記抗体のエピトープが、配列番号7の116~135位および/または216~230位を含む、前記項目のいずれか一項に記載の組成物。
(項目5)
前記抗LSR抗体またはその抗原結合フラグメントが、以下:
(a)それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(b)それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(c)それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(d)それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(e)それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、または
(f)それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
前記項目のいずれか一項に記載の組成物。
(項目6)
LSRの調節因子を含む、腫瘍浸潤CD8+T細胞の数を増加および/または活性化させるための組成物。
(項目7)
前記腫瘍浸潤CD8+T細胞が、CD69陽性である、前記項目のいずれか一項に記載の組成物。
(項目8)
前記LSRの調節因子が、LSRに対する抑制因子である、前記項目のいずれか一項に記載の組成物。
(項目9)
前記LSRの抑制因子が、抗LSR抗体またはその抗原結合フラグメントである、前記項目のいずれか一項に記載の組成物。
(項目10)
前記抗体のエピトープが、配列番号7の116~135位および/または216~230位を含む、前記項目のいずれか一項に記載の組成物。
(項目11)
前記抗LSR抗体またはその抗原結合フラグメントが、以下:
(a)それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(b)それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(c)それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(d)それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(e)それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、または
(f)それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
からなる群から選択される、前記項目のいずれか一項に記載の組成物。
(項目12)
前記組成物が、悪性腫瘍を治療または予防するためのものである、前記項目のいずれか一項に記載の組成物。
(項目13)
前記悪性腫瘍が、LSR陽性悪性腫瘍である、前記項目のいずれか一項に記載の組成物。
(項目14)
前記悪性腫瘍が、乳癌、卵巣癌、子宮内膜癌、膵臓癌、肺癌、胃癌または大腸癌である、前記項目のいずれか一項に記載の組成物。
(項目15)
前記項目のいずれか一項に記載の組成物を使用する、免疫チェックポイントの調節方法。
(項目16)
in vitroまたはin vivoで使用する、前記項目のいずれか一項に記載の方法。
(項目17)
患者に、治療有効量の組成物を投与する工程を含む、前記項目のいずれか一項に記載の方法。
(項目18)
前記免疫チェックポイントの調節は、免疫チェックポイントの阻害である、前記項目のいずれか一項に記載の方法。
(項目19)
前記項目のいずれか一項に記載の組成物を使用する、腫瘍浸潤CD8+T細胞の数を増加および/または活性化方法。
(項目20)
in vitroまたはin vivoで使用する、前記項目のいずれか一項に記載の方法。
(項目21)
患者に、治療有効量の組成物を投与する工程を含む、前記項目のいずれか一項に記載の方法。
(項目22)
前記項目のいずれか一項に記載の組成物を使用する、悪性腫瘍の治療または予防方法。
(項目23)
in vitroまたはin vivoで使用する、前記項目のいずれか一項に記載の方法。
(項目24)
患者に、治療有効量の組成物を投与する工程を含む、前記項目のいずれか一項に記載の方法。
(項目25)
免疫チェックポイントの調節に用いるための、前記項目のいずれか一項に記載の組成物。
(項目26)
腫瘍浸潤CD8+T細胞の数を増加および/または活性化に用いるための、前記項目のいずれか一項に記載の組成物。
(項目27)
悪性腫瘍の治療または予防に用いるための、前記項目のいずれか一項に記載の組成物。
(項目28)
免疫チェックポイントの調節用組成物の製造ための、前記項目のいずれか一項に記載の組成物の使用。
(項目29)
腫瘍浸潤CD8+T細胞の数を増加および/または活性化用組成物の製造のための、前記項目のいずれか一項に記載の組成物の使用。
(項目30)
悪性腫瘍の治療または予防用組成物の製造のための、前記項目のいずれか一項に記載の組成物の使用。
本開示において、上記1または複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供されうることが意図される。本開示のなおさらなる実施形態および利点は、必要に応じて以下の詳細な説明を読んで理解すれば、当業者に認識される。 The present disclosure provides, for example, the following:
(Item 1)
A composition for modulating an immune checkpoint comprising a modulator of the lipolysis-stimulating lipoprotein receptor (LSR).
(Item 2)
The composition described in the preceding item, wherein the LSR regulator is an inhibitor of LSR.
(Item 3)
The composition according to any one of the preceding items, wherein the inhibitor against LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
(Item 4)
The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
(Item 5)
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition according to any one of the preceding items.
(Item 6)
A composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells comprising a modulator of an LSR.
(Item 7)
The composition of any one of the preceding items, wherein the tumor-infiltrating CD8+ T cells are CD69 positive.
(Item 8)
The composition according to any one of the preceding items, wherein the LSR modulator is an inhibitor of LSR.
(Item 9)
The composition of any one of the preceding items, wherein the inhibitor of LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
(Item 10)
The composition of any one of the preceding items, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
(Item 11)
The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition of any one of the preceding claims, selected from the group consisting of:
(Item 12)
The composition according to any one of the preceding items, wherein the composition is for treating or preventing malignant tumors.
(Item 13)
The composition of any one of the preceding items, wherein the malignant tumor is an LSR-positive malignant tumor.
(Item 14)
The composition of any one of the preceding items, wherein the malignant tumor is breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
(Item 15)
A method for regulating an immune checkpoint using a composition described in any one of the preceding items.
(Item 16)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 17)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 18)
The method of any one of the preceding items, wherein the modulation of an immune checkpoint is inhibition of an immune checkpoint.
(Item 19)
A method for increasing and/or activating tumor-infiltrating CD8+ T cells using the composition according to any one of the preceding items.
(Item 20)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 21)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 22)
A method for treating or preventing malignant tumors using the composition described in any one of the preceding items.
(Item 23)
The method according to any one of the preceding paragraphs, for use in vitro or in vivo.
(Item 24)
The method of any one of the preceding claims, comprising administering to a patient a therapeutically effective amount of the composition.
(Item 25)
A composition according to any one of the preceding items for use in regulating immune checkpoints.
(Item 26)
The composition according to any one of the preceding items for use in increasing the number and/or activating tumor-infiltrating CD8+ T cells.
(Item 27)
A composition according to any one of the preceding items for use in the treatment or prevention of malignant tumors.
(Item 28)
Use of a composition described in any one of the preceding items for the manufacture of a composition for modulating an immune checkpoint.
(Item 29)
Use of a composition according to any one of the preceding items for the manufacture of a composition for increasing and/or activating tumor-infiltrating CD8+ T cells.
(Item 30)
Use of a composition according to any one of the preceding items for the manufacture of a composition for the treatment or prevention of malignant tumors.
It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.
本開示のLSRを標的とした抗体は免疫チェックポイント阻害剤として活用出来るため、LSRを高発現する難治性固形癌に対する新規治療法を提供できる。その結果、本開示は、癌患者の予後を改善することが可能となる。
The antibody targeting the LSR disclosed herein can be used as an immune checkpoint inhibitor, providing a new treatment for refractory solid cancers that highly express LSR. As a result, this disclosure makes it possible to improve the prognosis of cancer patients.
以下、本開示を説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形の冠詞(例えば、英語の場合は「a」、「an」、「the」など)は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用されるすべての専門用語および科学技術用語は、本開示の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書(定義を含めて)が優先する。
The present disclosure is described below. Throughout this specification, singular expressions should be understood to include the concept of the plural, unless otherwise specified. Thus, singular articles (e.g., in the case of English, "a," "an," "the," etc.) should be understood to include the concept of the plural, unless otherwise specified. In addition, terms used in this specification should be understood to be used in the sense commonly used in the field, unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present specification (including definitions) will take precedence.
(定義)
最初に本開示において使用される用語および一般的な技術を説明する。 (Definition)
First, terms and general techniques used in this disclosure are explained.
最初に本開示において使用される用語および一般的な技術を説明する。 (Definition)
First, terms and general techniques used in this disclosure are explained.
本明細書において、「約」とは、示される値の±10%を意味する。
In this specification, "about" means ±10% of the indicated value.
本明細書において「LSR(Lipolysis stimulated lipoprotein receptor)」とは、一般的に、低密度リポタンパク質(LDL)の代謝に関わる分子として知られている。LSRのアミノ酸配列等の詳細は、NCBI(NationalCenter for Biotechnology Information)、またはHGNC(HUGO Gene Nomenclature Committee)等のWEBサイトから見ることができる。NCBIに記載されているLSRのアクセッションナンバーは、例えば、NP_991403(アミノ酸)、/NM_205834.3(mRNA)である。LSRのアミノ酸配列は、例えば、配列番号7である。LSR mRNAの塩基配列は、例えば、配列番号8である。LSRは、LSR活性を有していれば、そのアミノ酸配列は限定されない。したがって、本開示の具体的な目的に合致する限り、特定の配列番号またはアクセッション番号に記載されるアミノ酸配列を有するタンパク質(あるいはそれをコードする核酸)のみならず、機能的に活性なその類似体もしくは誘導体、または機能的に活性なそのフラグメント、またはその相同体、または高ストリンジェンシー条件または低ストリンジェンシー条件下で、このタンパク質をコードする核酸にハイブリダイズする核酸にコードされる変異体もまた、本開示において用いることができることが理解される。
In this specification, "LSR (Lipolysis stimulated lipoprotein receptor)" is generally known as a molecule involved in the metabolism of low density lipoprotein (LDL). Details of the amino acid sequence of LSR can be found on websites such as NCBI (National Center for Biotechnology Information) or HGNC (HUGO Gene Nomenclature Committee). The accession numbers of LSR listed in NCBI are, for example, NP_991403 (amino acid) and /NM_205834.3 (mRNA). The amino acid sequence of LSR is, for example, SEQ ID NO: 7. The base sequence of LSR mRNA is, for example, SEQ ID NO: 8. The amino acid sequence of LSR is not limited as long as it has LSR activity. Therefore, as long as it meets the specific objectives of this disclosure, it is understood that not only a protein having an amino acid sequence set forth in a particular SEQ ID NO or accession number (or a nucleic acid encoding the same), but also a functionally active analog or derivative thereof, or a functionally active fragment thereof, or a homolog thereof, or a mutant encoded by a nucleic acid that hybridizes to the nucleic acid encoding the protein under high or low stringency conditions, can be used in this disclosure.
本明細書で使用される「誘導体」、「類似体」または「変異体」は、好ましくは、限定を意図するものではないが、対象となるタンパク質(例えば、LSR)に実質的に相同な領域を含む分子を含み、このような分子は、種々の実施形態において、同一サイズのアミノ酸配列にわたり、または当該分野で公知のコンピュータ相同性プログラムによってアラインメントを行ってアラインされる配列と比較した際、少なくとも30%、40%、50%、60%、70%、80%、90%、95%または99%同一であるか、あるいはこのような分子をコードする核酸は、(高度に)ストリンジェントな条件、中程度にストリンジェントな条件、またはストリンジェントでない条件下で、構成要素タンパク質をコードする配列にハイブリダイズ可能である。これは、それぞれ、アミノ酸置換、欠失および付加によって、天然存在タンパク質を改変した産物であり、その誘導体がなお天然存在タンパク質の生物学的機能を、必ずしも同じ度合いでなくてもよいが示すタンパク質を意味する。例えば、本明細書において記載されあるいは当該分野で公知の適切で利用可能なin vitroアッセイによって、このようなタンパク質の生物学的機能を調べることも可能である。本明細書で使用される「機能的に活性な」は、本明細書において、本開示のポリペプチド、すなわちフラグメントまたは誘導体が関連する態様に従って、生物学的活性などの、タンパク質の構造的機能、制御機能、または生化学的機能を有する、ポリペプチド、すなわちフラグメントまたは誘導体を指す。本開示では、LSRについてヒトが主に論じられるが、ヒト以外の多くの動物がLSRを発現していることが知られているため、これらの動物、特に哺乳動物についても、本開示の範囲内に入ることが理解される。好ましくは、LSRの機能的ドメイン、例えば、膜貫通ドメイン(260-280位)、リン酸化部位(309位、328位、406位、493位、528位、530位、535位、540位、551位、586位、615位、646位)は保存されていることが好ましい。
As used herein, "derivatives," "analogs," or "variants" preferably include, but are not limited to, molecules that contain a region substantially homologous to the protein of interest (e.g., LSR), which in various embodiments are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical over the same size amino acid sequence or when compared to sequences aligned by computer homology programs known in the art, or nucleic acids encoding such molecules are hybridizable to sequences encoding the component proteins under (highly) stringent, moderately stringent, or non-stringent conditions. This refers to proteins that are the product of modification of naturally occurring proteins by amino acid substitutions, deletions, and additions, respectively, and whose derivatives still exhibit the biological functions of the naturally occurring proteins, although not necessarily to the same degree. For example, the biological functions of such proteins can be examined by suitable available in vitro assays described herein or known in the art. As used herein, "functionally active" refers to a polypeptide, i.e., a fragment or derivative, that has a structural, regulatory, or biochemical function of a protein, such as biological activity, according to the embodiment to which the polypeptide, i.e., fragment or derivative, of the present disclosure relates. In this disclosure, LSR is primarily discussed in humans, but since many animals other than humans are known to express LSR, it is understood that these animals, particularly mammals, are also within the scope of the present disclosure. Preferably, the functional domains of the LSR, such as the transmembrane domain (positions 260-280), phosphorylation sites (positions 309, 328, 406, 493, 528, 530, 535, 540, 551, 586, 615, 646), are conserved.
LSRの代表的なヌクレオチド配列は、
(a)配列番号7記載の塩基配列またはそのフラグメント配列を有するポリヌクレオチ
ド;
(b)配列番号8に記載のアミノ酸配列からなるポリペプチドまたはそのフラグメント
をコードするポリヌクレオチド;
(c)配列番号8に記載のアミノ酸配列において、1以上または1もしくは数個のアミノ酸が、置換、付加および欠失からなる群より選択される1つの変異を有する改変体ポリペプチドまたはそのフラグメントであって、生物学的活性を有する改変体ポリペプチドをコードする、ポリヌクレオチド;
(d)配列番号7に記載の塩基配列のスプライス変異体もしくは対立遺伝子変異体またはそのフラグメントである、ポリヌクレオチド;
(e)配列番号8に記載のアミノ酸配列からなるポリペプチドの種相同体またはそのフラグメントをコードする、ポリヌクレオチド;
(f)(a)~(e)のいずれか1つのポリヌクレオチドにストリンジェント条件下でハイブリダイズし、かつ生物学的活性を有するポリペプチドをコードするポリヌクレオチド;または
(g)(a)~(e)のいずれか1つのポリヌクレオチドまたはその相補配列に対する同一性が少なくとも70%、少なくとも75%、少なくとも80%、少なくとも85%、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%である塩基配列からなり、かつ、生物学的活性を有するポリペプチドをコードするポリヌクレオチドであり得る。ここで、生物学的活性とは、代表的に、LSRの有する活性またはマーカーとして同じ生物内に存在する他のタンパク質から識別し得ることをいう。 A representative nucleotide sequence of an LSR is:
(a) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 7 or a fragment thereof;
(b) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof;
(c) a polynucleotide encoding a variant polypeptide or a fragment thereof having one or more or one or several amino acids in the amino acid sequence set forth in SEQ ID NO:8, the variant polypeptide having biological activity;
(d) a polynucleotide which is a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7, or a fragment thereof;
(e) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8, or a fragment thereof;
(f) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (a) to (e) and encodes a polypeptide having biological activity; or (g) a polynucleotide that has a base sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotides (a) to (e) or their complementary sequences and encodes a polypeptide having biological activity. Here, biological activity typically refers to the activity of an LSR or the ability to be distinguished from other proteins present in the same organism as a marker.
(a)配列番号7記載の塩基配列またはそのフラグメント配列を有するポリヌクレオチ
ド;
(b)配列番号8に記載のアミノ酸配列からなるポリペプチドまたはそのフラグメント
をコードするポリヌクレオチド;
(c)配列番号8に記載のアミノ酸配列において、1以上または1もしくは数個のアミノ酸が、置換、付加および欠失からなる群より選択される1つの変異を有する改変体ポリペプチドまたはそのフラグメントであって、生物学的活性を有する改変体ポリペプチドをコードする、ポリヌクレオチド;
(d)配列番号7に記載の塩基配列のスプライス変異体もしくは対立遺伝子変異体またはそのフラグメントである、ポリヌクレオチド;
(e)配列番号8に記載のアミノ酸配列からなるポリペプチドの種相同体またはそのフラグメントをコードする、ポリヌクレオチド;
(f)(a)~(e)のいずれか1つのポリヌクレオチドにストリンジェント条件下でハイブリダイズし、かつ生物学的活性を有するポリペプチドをコードするポリヌクレオチド;または
(g)(a)~(e)のいずれか1つのポリヌクレオチドまたはその相補配列に対する同一性が少なくとも70%、少なくとも75%、少なくとも80%、少なくとも85%、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%である塩基配列からなり、かつ、生物学的活性を有するポリペプチドをコードするポリヌクレオチドであり得る。ここで、生物学的活性とは、代表的に、LSRの有する活性またはマーカーとして同じ生物内に存在する他のタンパク質から識別し得ることをいう。 A representative nucleotide sequence of an LSR is:
(a) a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 7 or a fragment thereof;
(b) a polynucleotide encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof;
(c) a polynucleotide encoding a variant polypeptide or a fragment thereof having one or more or one or several amino acids in the amino acid sequence set forth in SEQ ID NO:8, the variant polypeptide having biological activity;
(d) a polynucleotide which is a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7, or a fragment thereof;
(e) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8, or a fragment thereof;
(f) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides (a) to (e) and encodes a polypeptide having biological activity; or (g) a polynucleotide that has a base sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polynucleotides (a) to (e) or their complementary sequences and encodes a polypeptide having biological activity. Here, biological activity typically refers to the activity of an LSR or the ability to be distinguished from other proteins present in the same organism as a marker.
LSRのアミノ酸配列としては、
(a)配列番号8に記載のアミノ酸配列またはそのフラグメントからなる、ポリペプチド;
(b)配列番号8に記載のアミノ酸配列において、1以上のアミノ酸が置換、付加および欠失からなる群より選択される1つの変異を有し、かつ、生物学的活性を有する、ポリペプチド;
(c)配列番号7に記載の塩基配列のスプライス変異体または対立遺伝子変異体によってコードされる、ポリペプチド;
(d)配列番号8に記載のアミノ酸配列の種相同体である、ポリペプチド;または
(e)(a)~(d)のいずれか1つのポリペプチドに対する同一性が少なくとも70%、少なくとも75%、少なくとも80%、少なくとも85%、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%であるアミノ酸配列を有し、かつ、生物学的活性を有する、ポリペプチド、であり得る。ここで、生物学的活性とは、代表的に、LSRの有する活性またはマーカーとして同じ生物内に存在する他のタンパク質から識別し得ること(例えば、抗原として用いられる場合特異的エピトープとして機能し得る領域を含むこと)をいう。 The amino acid sequence of LSR is:
(a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof;
(b) a polypeptide having one or more amino acid mutations selected from the group consisting of substitutions, additions and deletions in the amino acid sequence set forth in SEQ ID NO:8, and having biological activity;
(c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7;
(d) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 8; or (e) a polypeptide that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polypeptides (a) to (d) and has biological activity. Here, biological activity typically refers to the ability to distinguish the LSR activity or marker from other proteins present in the same organism (e.g., the inclusion of a region that can function as a specific epitope when used as an antigen).
(a)配列番号8に記載のアミノ酸配列またはそのフラグメントからなる、ポリペプチド;
(b)配列番号8に記載のアミノ酸配列において、1以上のアミノ酸が置換、付加および欠失からなる群より選択される1つの変異を有し、かつ、生物学的活性を有する、ポリペプチド;
(c)配列番号7に記載の塩基配列のスプライス変異体または対立遺伝子変異体によってコードされる、ポリペプチド;
(d)配列番号8に記載のアミノ酸配列の種相同体である、ポリペプチド;または
(e)(a)~(d)のいずれか1つのポリペプチドに対する同一性が少なくとも70%、少なくとも75%、少なくとも80%、少なくとも85%、少なくとも90%、少なくとも95%、少なくとも96%、少なくとも97%、少なくとも98%、または少なくとも99%であるアミノ酸配列を有し、かつ、生物学的活性を有する、ポリペプチド、であり得る。ここで、生物学的活性とは、代表的に、LSRの有する活性またはマーカーとして同じ生物内に存在する他のタンパク質から識別し得ること(例えば、抗原として用いられる場合特異的エピトープとして機能し得る領域を含むこと)をいう。 The amino acid sequence of LSR is:
(a) a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:8 or a fragment thereof;
(b) a polypeptide having one or more amino acid mutations selected from the group consisting of substitutions, additions and deletions in the amino acid sequence set forth in SEQ ID NO:8, and having biological activity;
(c) a polypeptide encoded by a splice variant or allelic variant of the nucleotide sequence set forth in SEQ ID NO: 7;
(d) a polypeptide that is a species homologue of the amino acid sequence set forth in SEQ ID NO: 8; or (e) a polypeptide that has an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the polypeptides (a) to (d) and has biological activity. Here, biological activity typically refers to the ability to distinguish the LSR activity or marker from other proteins present in the same organism (e.g., the inclusion of a region that can function as a specific epitope when used as an antigen).
本開示において、「LSRの調節因子」とは、LSRの機能を調節する作用を有する物質を指す。本開示において、LSRが免疫チェックポイント分子であり、LSRが腫瘍免疫を抑制していることが示唆された。LSRの機能を調節することにより、腫瘍免疫を調節することが可能である。
In this disclosure, "LSR regulator" refers to a substance that has the effect of regulating the function of LSR. In this disclosure, it has been suggested that LSR is an immune checkpoint molecule, and that LSR suppresses tumor immunity. By regulating the function of LSR, it is possible to regulate tumor immunity.
本開示において、「免疫チェックポイント分子」とは、細胞上に発現し、リガンドと結合することによって、免疫応答を阻害するシグナルを伝達する分子をいう。
In this disclosure, "immune checkpoint molecule" refers to a molecule that is expressed on cells and transmits a signal that inhibits an immune response by binding to a ligand.
本開示において、「LSRの抑制因子」とは、LSRの生物学的作用を阻害する物質または因子をいう。このような因子には、抗体、その抗原結合フラグメント、それらの誘導体、機能的等価物、アンチセンス、siRNA等のRNAi因子等の核酸の形態のもの等を挙げることができるがこれらに限定されない。
In this disclosure, "LSR inhibitor" refers to a substance or factor that inhibits the biological action of LSR. Such factors include, but are not limited to, antibodies, antigen-binding fragments thereof, derivatives thereof, functional equivalents, antisense, and nucleic acid forms such as RNAi factors such as siRNA.
本開示において、活性、発現産物(例えば、タンパク質、転写物(RNAなど))の「減少」または「抑制」あるいはその類義語は、特定の活性、転写物またはタンパク質の量、質または効果における減少、または減少させる活性をいう。減少のうち「消失」した場合は、活性、発現産物等が検出限界未満になることをいい、特に「消失」ということがある。本明細書では、「消失」は、「減少」または「抑制」に包含される。
In this disclosure, "reduction" or "suppression" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that causes a decrease. When "elimination" is used as a reduction, it refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as "elimination." In this specification, "elimination" is encompassed by "reduction" or "suppression."
本明細書において「RNA干渉」または「RNAi」とは、RNA interferenceの略称で、当該分野で一般に知られており、RNAiを引き起こす因子によって媒介される、細胞における遺伝子発現を阻害または下方制御する生物学的プロセスである。例えば、二本鎖RNA(dsRNAともいう)のようなRNAiを引き起こす因子を細胞に導入することにより、相同なmRNAが特異的に分解され、遺伝子産物の合成が抑制される現象およびそれに用いられる技術をいう。本明細書において「RNAi」はまた、場合によっては、「RNAiを引き起こす因子」、「RNAiを起こす因子」、「RNAi因子」などと同義に用いられ得る。また、本開示では、RNAiという用語は、転写後遺伝子サイレンシング、翻訳阻害、転写阻害、エピジェネティクスなどの配列特異的RNA干渉の記述に用いられる他の用語と同義のものを示すものとして理解される。本明細書では、「RNAiを起こす因子」は「RNAi」を起こす限りどのようなものであってもよい。
As used herein, "RNA interference" or "RNAi" is an abbreviation for RNA interference, which is generally known in the art and is a biological process that inhibits or downregulates gene expression in cells, mediated by factors that cause RNAi. For example, it refers to the phenomenon in which homologous mRNA is specifically degraded and synthesis of gene products is suppressed by introducing factors that cause RNAi, such as double-stranded RNA (also called dsRNA), into cells, and the technology used therefor. In this specification, "RNAi" may also be used synonymously with "factors that cause RNAi," "factors that cause RNAi," "RNAi factors," and the like, in some cases. In addition, in this disclosure, the term RNAi is understood to be synonymous with other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translation inhibition, transcription inhibition, and epigenetics. In this specification, "factors that cause RNAi" may be anything as long as they cause "RNAi."
本明細書では「RNAiを起こす因子」としては、「低分子干渉核酸」、「siNA」、「低分子干渉RNA」、「siRNA」、「低分子干渉核酸分子」、「低分子干渉オリゴヌクレオチド分子」または「化学修飾低分子干渉核酸分子」等が挙げられ、これらの用語は、RNA干渉「RNAi」または遺伝子サイレンシングを配列特異的に媒介することによって、遺伝子発現またはウイルス複製を阻害または下方制御することができる任意の核酸分子を指す。これらの用語は、個々の核酸分子、複数のかかる核酸分子、またはかかる核酸分子のプールも表し得る。これらの分子は、自己相補的なセンス領域とアンチセンス領域を含む二本鎖核酸分子であり得る。
As used herein, "agents that cause RNAi" include "small interfering nucleic acids," "siNA," "small interfering RNA," "siRNA," "small interfering nucleic acid molecules," "small interfering oligonucleotide molecules," or "chemically modified small interfering nucleic acid molecules," and these terms refer to any nucleic acid molecule that can inhibit or downregulate gene expression or viral replication by mediating RNA interference "RNAi" or gene silencing in a sequence-specific manner. These terms can refer to individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. These molecules can be double-stranded nucleic acid molecules that contain self-complementary sense and antisense regions.
本開示で代表的に用いられる「siRNA」は、短い長さ、通常、約20塩基前後(例えば、代表的には約21~23塩基長)またはそれ未満の長さの二本鎖RNAである。このようなsiRNAは、細胞に発現させることにより遺伝子発現を抑制する。本開示において用いられるsiRNAは、RNAiを引き起こすことができる限り、どのような形態を採っていてもよい。
The "siRNA" typically used in this disclosure is a double-stranded RNA that is short, typically about 20 bases long (e.g., typically about 21-23 bases long) or shorter. Such siRNA suppresses gene expression when expressed in cells. The siRNA used in this disclosure may take any form as long as it can induce RNAi.
本開示において、siRNA等のRNAiを起こす因子では、アンチセンス領域は、標的核酸分子中のヌクレオチド配列またはその一部に相補的であるヌクレオチド配列、および標的核酸配列に対応するヌクレオチド配列またはその一部を有するセンス領域を含む。これらの分子は、一方の鎖がセンス鎖であり、他方がアンチセンス鎖である、2個の別々のオリゴヌクレオチドから組み立てることができる。ここで、アンチセンス鎖とセンス鎖は自己相補的である(すなわち、アンチセンス鎖とセンス鎖が二本鎖または二本鎖構造を形成するなど、各鎖は、他方の鎖中のヌクレオチド配列に相補的であるヌクレオチド配列を含む。ここで、例えば、二本鎖領域は、約15から約30、例えば、約15、16、17、18、19、20、21、22、23、24、25、26、27、28、29または30塩基対でありうるが、これらより長くてもよい。アンチセンス鎖は、標的核酸分子中のヌクレオチド配列またはその一部に相補的であるヌクレオチド配列を含み、センス鎖は標的核酸配列またはその一部に対応するヌクレオチド配列を含む(例えば、その分子の約15から約25個またはそれを超えるヌクレオチドは、標的核酸またはその一部に相補的である)。あるいは、これらの分子は、単一のオリゴヌクレオチドから組み立てられ、これらの分子の自己相補的なセンス領域とアンチセンス領域は、核酸リンカーまたは非核酸リンカーによって連結されている。これらの分子は、自己相補的なセンス領域とアンチセンス領域を含む、二本鎖、非対称二本鎖、ヘアピンまたは非対称ヘアピン二次構造を有するポリヌクレオチドであり得る。ここで、アンチセンス領域は、別個の標的核酸分子中のヌクレオチド配列またはその一部に相補的であるヌクレオチド配列、および標的核酸配列に対応するヌクレオチド配列またはその一部を有するセンス領域を含む。これらの分子は、2個以上のループ構造と、自己相補的なセンス領域とアンチセンス領域を含む軸(stem)とを有する、環状一本鎖ポリヌクレオチドであり得る。ここで、アンチセンス領域は、標的核酸分子中のヌクレオチド配列またはその一部に相補的であるヌクレオチド配列、および標的核酸配列またはその一部に対応するヌクレオチド配列を有するセンス領域を含み、環状ポリヌクレオチドは、インビボまたはインビトロでプロセシングを受けて、RNAiを媒介し得る活性な分子を生成し得る。これらの因子は、標的核酸分子中のヌクレオチド配列またはその一部に相補的であるヌクレオチド配列を有する一本鎖ポリヌクレオチドも含み得る(例えば、これらの因子は、標的核酸配列またはその一部に対応するヌクレオチド配列がこれらの因子内に存在する必要がない。)。一本鎖ポリヌクレオチドは、5’リン酸(例えば、Martinez et Al.,2002,Cell.,110,563-574及びSchwarz et al.,2002,Molecular Cell,10,537-568参照)、5’,3’-二リン酸などの末端リン酸基を更に含み得る。ある実施形態においては、本開示のLSRの抑制因子は、別々のセンスおよびアンチセンス配列または領域を含む。ここで、センス領域とアンチセンス領域は、当該分野で公知のヌクレオチドまたは非ヌクレオチドリンカー分子によって共有結合しており、またはイオン相互作用、水素結合、ファンデルワールス相互作用、疎水的相互作用および/またはスタッキング相互作用によって交互に非共有結合している。ある実施形態においては、本開示のLSRの抑制因子は、標的遺伝子のヌクレオチド配列に相補的であるヌクレオチド配列を含む。別の実施形態においては、本開示のLSRの抑制因子は、標的遺伝子の発現を阻害するように、標的遺伝子のヌクレオチド配列と相互作用する。本明細書では、LSRの抑制因子は、RNAのみを含む分子に必ずしも限定されず、化学修飾ヌクレオチドおよび非ヌクレオチドも包含する。ある実施形態においては、本開示が低分子干渉核酸分子である場合は、2’ヒドロキシ(2’-OH)含有ヌクレオチドを欠いていてもよい。ある実施形態において、本開示はRNAiを媒介するのに2’ヒドロキシル基を有するヌクレオチドの存在が不要である低分子干渉核酸でありうる。したがって、本開示が低分子干渉核酸分子である場合は、リボヌクレオチド(例えば、2’-OH基を有するヌクレオチド)を含まなくてもよい。しかし、RNAiを維持するのにLSRの抑制因子内のリボヌクレオチドの存在が不要である場合は、2’-OH基を有する1個以上のヌクレオチドを含む、結合したリンカー、または他の結合若しくは会合した基、部分若しくは鎖を有し得る。場合によっては、本開示のLSRを抑制する因子は、ヌクレオチド位置の約5、10、20、30、40または50%においてリボヌクレオチドを含み得る。本開示ではLSRの抑制因子は、配列特異的RNAiを媒介し得る核酸分子、例えば、低分子干渉RNA(siRNA)、二本鎖RNA(dsRNA)、マイクロRNA(miRNA)、短鎖ヘアピンRNA(shRNA)、低分子干渉オリゴヌクレオチド、低分子干渉核酸、低分子干渉修飾オリゴヌクレオチド、化学修飾siRNA、転写後遺伝子サイレンシングRNA(ptgsRNA)であってもよい。
In the present disclosure, in agents that cause RNAi, such as siRNA, the antisense region includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and the sense region includes a nucleotide sequence or a portion thereof that corresponds to the target nucleic acid sequence. These molecules can be assembled from two separate oligonucleotides, one strand being the sense strand and the other being the antisense strand, where the antisense strand and the sense strand are self-complementary (i.e., each strand includes a nucleotide sequence that is complementary to a nucleotide sequence in the other strand, such that the antisense strand and the sense strand form a duplex or double-stranded structure. Here, for example, the double-stranded region can be about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, but can be longer. The antisense strand includes a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule. The sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, these molecules are assembled from a single oligonucleotide, the self-complementary sense and antisense regions of which are linked by a nucleic acid or non-nucleic acid linker. These molecules are polynucleotides having double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structures that include self-complementary sense and antisense regions. The antisense region may be a circular single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a separate target nucleic acid molecule, and a sense region having a nucleotide sequence or a portion thereof corresponding to the target nucleic acid sequence. The molecules may be circular single-stranded polynucleotides having two or more loop structures and a stem including self-complementary sense and antisense regions. The antisense region may be a circular single-stranded polynucleotide having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule, and a sense region having a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, and the circular polynucleotide may be processed in vivo or in vitro to generate an active molecule capable of mediating RNAi. The agents may also include single-stranded polynucleotides having a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in a target nucleic acid molecule (e.g., the agents do not require that a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof be present within the agents). The single-stranded polynucleotide may be a 5' phosphate (e.g., Martinez et al., 2002, Cell. , 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10,537-568), and may further comprise a terminal phosphate group, such as 5',3'-diphosphate. In certain embodiments, the LSR inhibitors of the present disclosure comprise separate sense and antisense sequences or regions, where the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules known in the art, or alternately non-covalently linked by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the LSR inhibitors of the present disclosure comprise a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, the LSR inhibitors of the present disclosure interact with the nucleotide sequence of the target gene to inhibit expression of the target gene. As used herein, the LSR inhibitors are not necessarily limited to molecules that comprise only RNA, but also encompass chemically modified nucleotides and non-nucleotides. In some embodiments, when the disclosure is a small interfering nucleic acid molecule, it may lack 2' hydroxy (2'-OH) containing nucleotides. In some embodiments, the disclosure may be a small interfering nucleic acid that does not require the presence of a nucleotide with a 2' hydroxyl group to mediate RNAi. Thus, when the disclosure is a small interfering nucleic acid molecule, it may not include ribonucleotides (e.g., nucleotides with a 2'-OH group). However, when the presence of ribonucleotides in the LSR inhibitor is not required to maintain RNAi, it may have an attached linker or other linked or associated group, moiety, or strand that includes one or more nucleotides with a 2'-OH group. In some cases, the LSR inhibitor of the disclosure may include ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. In the present disclosure, the inhibitor of the LSR may be a nucleic acid molecule capable of mediating sequence-specific RNAi, such as a small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), small interfering oligonucleotide, small interfering nucleic acid, small interfering modified oligonucleotide, chemically modified siRNA, or post-transcriptional gene silencing RNA (ptgsRNA).
本明細書においてRNAiを引き起こす因子としては、例えば、標的遺伝子の核酸配列の一部に対して少なくとも約70%の相同性を有する配列またはストリンジェントな条件下でハイブリダイズする配列を含む、少なくとも10ヌクレオチド長の二本鎖部分を含むRNAまたはその改変体が挙げられるがそれに限定されない。ここで、この因子は、好ましくは、3’突出末端を含み、より好ましくは、3’突出末端は、2ヌクレオチド長以上のDNA(例えば、2~4ヌクレオチド長のDNAであり得る。
As used herein, factors that induce RNAi include, but are not limited to, RNA or modified forms thereof that contain a double-stranded portion of at least 10 nucleotides in length, including a sequence that has at least about 70% homology to a portion of the nucleic acid sequence of a target gene or a sequence that hybridizes under stringent conditions. Here, the factor preferably contains a 3' overhanging end, and more preferably, the 3' overhanging end can be DNA that is 2 nucleotides or more in length (e.g., DNA that is 2 to 4 nucleotides in length.
あるいは、本開示において用いられるRNAiとしては、例えば、短い逆向きの相補的配列(例えば、15bp以上であり、例えば、24bpなど)のペアが挙げられるがそれらに限定されない。
Alternatively, the RNAi used in the present disclosure may include, but is not limited to, a pair of short inverted complementary sequences (e.g., 15 bp or more, e.g., 24 bp, etc.).
理論に束縛されないが、RNAiが働く機構として考えられるものの一つとして、dsRNAのようなRNAiを引き起こす分子が細胞に導入されると、比較的長い(例えば、40塩基対以上)RNAの場合、ヘリカーゼドメインを持つダイサー(Dicer)と呼ばれるRNaseIII様のヌクレアーゼがATP存在下で、その分子を3’末端から約20塩基対ずつ切り出し、短鎖dsRNA(siRNAとも呼ばれる)を生じる。本明細書において「siRNA」とは、short interfering RNAの略称であり、人工的に化学合成されるかまたは生化学的に合成されたものか、あるいは生物体内で合成されたものか、あるいは約40塩基以上の二本鎖RNAが体内で分解されてできた10塩基対以上の短鎖二本鎖RNAをいい、通常、5’-リン酸、3’-OHの構造を有しており、3’末端は約2塩基突出している。このsiRNAに特異的なタンパク質が結合して、RISC(RNA-induced-silencing-complex)が形成される。この複合体は、siRNAと同じ配列を有するmRNAを認識して結合し、RNaseIII様の酵素活性によってsiRNAの中央部でmRNAを切断する。siRNAの配列と標的として切断するmRNAの配列の関係については、100%一致することが好ましい。しかし、siRNAの中央から外れた位置についての塩基の変異については、完全にRNAiによる切断活性がなくなるのではなく、部分的な活性が残存する。他方、siRNAの中央部の塩基の変異は影響が大きく、RNAiによるmRNAの切断活性が極度に低下する。このような性質を利用して、変異をもつmRNAについては、その変異を中央に配したsiRNAを合成し、細胞内に導入することで特異的に変異を含むmRNAだけを分解することができる。従って、本開示では、siRNAそのものを、RNAiを引き起こす因子として用いることができるし、siRNAを生成するような因子(例えば、代表的に約40塩基以上のdsRNA)をそのような因子として用いることができる。
Without being bound by theory, one of the possible mechanisms by which RNAi works is that when a molecule that induces RNAi, such as dsRNA, is introduced into a cell, in the case of a relatively long RNA (e.g., 40 base pairs or more), an RNaseIII-like nuclease called Dicer, which has a helicase domain, cuts out the molecule from the 3' end in units of about 20 base pairs in the presence of ATP, generating short dsRNA (also called siRNA). In this specification, "siRNA" is an abbreviation for short interfering RNA, and refers to short double-stranded RNA of 10 base pairs or more that is either artificially chemically synthesized or biochemically synthesized, or synthesized in a living organism, or is generated by decomposing double-stranded RNA of about 40 bases or more in the body, and usually has a 5'-phosphate, 3'-OH structure, with the 3' end overhanging by about 2 bases. A specific protein binds to this siRNA to form an RNA-induced silencing complex (RISC). This complex recognizes and binds to mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNaseIII-like enzyme activity. It is preferable that the sequence of the siRNA and the sequence of the target mRNA to be cleaved are 100% identical. However, for base mutations at positions away from the center of the siRNA, the cleavage activity by RNAi is not completely eliminated, but partial activity remains. On the other hand, the effect of base mutations at the center of the siRNA is large, and the cleavage activity of the mRNA by RNAi is extremely reduced. By utilizing such properties, for mRNA having a mutation, an siRNA with the mutation located at the center can be synthesized and introduced into cells to specifically degrade only the mRNA containing the mutation. Therefore, in the present disclosure, the siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, a dsRNA of about 40 bases or more) can be used as such a factor.
また、理論に束縛されることを希望しないが、siRNAは、上記経路とは別に、siRNAのアンチセンス鎖がmRNAに結合してRNA依存性RNAポリメラーゼ(RdRP)のプライマーとして作用し、dsRNAが合成され、このdsRNAが再びダイサーの基質となり、新たなsiRNAを生じて作用を増幅することも企図される。従って、本開示では、siRNA自体およびsiRNAが生じるような因子もまた、有用である。実際に、昆虫などでは、例えば35分子のdsRNA分子が、1,000コピー以上ある細胞内のmRNAをほぼ完全に分解することから、siRNA自体およびsiRNAが生じるような因子が有用であることが理解される。
Furthermore, without wishing to be bound by theory, it is also contemplated that, apart from the above-mentioned pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RdRP), synthesizing dsRNA, which then serves as a substrate for Dicer again, generating new siRNA and amplifying the effect. Therefore, in the present disclosure, siRNA itself and factors that generate siRNA are also useful. In fact, in insects, for example, 35 dsRNA molecules almost completely degrade 1,000 or more copies of intracellular mRNA, and therefore it is understood that siRNA itself and factors that generate siRNA are useful.
別の実施形態において、本開示のRNAiを引き起こす因子は、3’末端に突出部を有する短いヘアピン構造(shRNA;short hairpin RNA)であり得る。本明細書において「shRNA」とは、一本鎖RNAで部分的に回文状の塩基配列を含むことにより、分子内で二本鎖構造をとり、ヘアピンのような構造となる約20塩基対以上の分子をいう。そのようなshRNAは、人工的に化学合成される。あるいは、そのようなshRNAは、センス鎖およびアンチセンス鎖のDNA配列を逆向きに連結したヘアピン構造のDNAをT7RNAポリメラーゼによりインビトロでRNAを合成することによって生成することができる。理論に束縛されることは希望しないが、そのようなshRNAは、細胞内に導入された後、細胞内で約20塩基(代表的には例えば、21塩基、22塩基、23塩基)の長さに分解され、siRNAと同様にRNAiを引き起こし、本開示の処置効果があることが理解されるべきである。このような効果は、昆虫、植物、動物(哺乳動物を含む)など広汎な生物において発揮されることが理解されるべきである。このように、shRNAは、siRNAと同様にRNAiを引き起こすことから、本開示の有効成分として用いることができる。shRNAはまた、好ましくは、3’突出末端を有し得る。二本鎖部分の長さは特に限定されないが、好ましくは約10ヌクレオチド長以上、より好ましくは約20ヌクレオチド長以上であり得る。ここで、3’突出末端は、好ましくはDNAであり得、より好ましくは少なくとも2ヌクレオチド長以上のDNAであり得、さらに好ましくは2~4ヌクレオチド長のDNAであり得る。本開示において用いられるRNAiを引き起こす因子は、人工的に合成した(例えば、化学的または生化学的)ものでも、天然に存在するものでも用いることができ、この両者の間で本開示の効果に本質的な違いは生じない。化学的に合成したものでは、液体クロマトグラフィーなどにより精製をすることが好ましい。
In another embodiment, the factor that causes RNAi of the present disclosure may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3' end. In this specification, "shRNA" refers to a molecule of about 20 base pairs or more that is a single-stranded RNA that contains a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule and forming a hairpin-like structure. Such shRNA is artificially chemically synthesized. Alternatively, such shRNA can be generated by synthesizing RNA in vitro using T7 RNA polymerase from hairpin-structured DNA in which the DNA sequences of the sense strand and the antisense strand are linked in reverse. Although not wishing to be bound by theory, it should be understood that such shRNA, after being introduced into a cell, is decomposed to a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) in the cell, and causes RNAi in the same way as siRNA, and has the treatment effect of the present disclosure. It should be understood that such an effect is exerted in a wide range of organisms, such as insects, plants, and animals (including mammals). Thus, shRNA can be used as an active ingredient of the present disclosure since it induces RNAi in the same manner as siRNA. shRNA may also preferably have a 3' overhang. The length of the double-stranded portion is not particularly limited, but may preferably be about 10 nucleotides or more, more preferably about 20 nucleotides or more. Here, the 3' overhang may preferably be DNA, more preferably at least 2 nucleotides in length, and even more preferably 2 to 4 nucleotides in length. The factor that induces RNAi used in the present disclosure may be either artificially synthesized (e.g., chemically or biochemically) or naturally occurring, and there is no essential difference in the effect of the present disclosure between the two. For chemically synthesized factors, it is preferable to purify them by liquid chromatography or the like.
本開示において用いられるRNAiを引き起こす因子は、インビトロで合成することもできる。この合成系において、T7RNAポリメラーゼおよびT7プロモーターを用いて、鋳型DNAからアンチセンスおよびセンスのRNAを合成する。これらをインビトロでアニーリングした後、細胞に導入すると、上述のような機構を通じてRNAiが引き起こされ、本開示の効果が達成される。ここでは、例えば、リン酸カルシウム法等の任意の適切な方法でそのようなRNAを細胞内に導入することができる。本開示のRNAiを引き起こす因子としてはまた、mRNAとハイブリダイズし得る一本鎖、あるいはそれらのすべての類似の核酸アナログのような因子も挙げられる。そのような因子もまた、本開示において有用である。
The factor that induces RNAi used in the present disclosure can also be synthesized in vitro. In this synthesis system, antisense and sense RNAs are synthesized from template DNA using T7 RNA polymerase and a T7 promoter. When these are annealed in vitro and then introduced into a cell, RNAi is induced through the mechanism described above, and the effect of the present disclosure is achieved. Here, such RNA can be introduced into a cell by any suitable method, such as the calcium phosphate method. The factor that induces RNAi of the present disclosure also includes factors such as single strands that can hybridize with mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the present disclosure.
RNAi分子のデザインには、例えば、siDirect2.0(Naito et al., BMC Bioinformatics. 2009 Nov 30;10:392.)等を使用できる。また、受託会社(例えば、タカラバイオ(株)等)に委託してもよい。RNAi作用の確認は、リアルタイムRT-PCRによるRNA鎖発現量の定量によって行なうことができる。または、ノーザンブロットによるRNA鎖発現量の解析や、ウェスタンブロットによる蛋白量の解析・表現型の観察等の方法でも行うことができる。また、特定の遺伝子に対するsiRNAまたはshRNAを生成するプラスミドは、例えば、受託会社(例えば、タカラバイオ(株)等)から購入することができる。
For example, siDirect2.0 (Naito et al., BMC Bioinformatics. 2009 Nov 30;10:392.) can be used to design RNAi molecules. This can also be outsourced to a contract company (such as Takara Bio Inc.). RNAi action can be confirmed by quantifying the amount of RNA strand expression using real-time RT-PCR. Alternatively, it can be confirmed by analyzing the amount of RNA strand expression using Northern blot, analyzing the amount of protein using Western blot, and observing the phenotype. Plasmids that generate siRNA or shRNA for a specific gene can also be purchased from a contract company (such as Takara Bio Inc.).
本開示の一実施形態において「siRNA」は、RNAiを誘導可能なRNA鎖を含む。一般的にsiRNAの2本鎖はガイド鎖とパッセンジャー鎖に分けることができ、ガイド鎖がRISCに取り込まれる。RISCに取り込まれたガイド鎖は、標的RNAを認識するために使われる。RNAi研究では主に人工的に作成したものが使用されるが、生体内において内在的に存在するものも知られている。上記ガイド鎖は15塩基以上のRNAから構成されていてもよい。15塩基以上であれば、標的のポリヌクレオチドに対して精度よく結合できる可能性が高まる。また、そのガイド鎖は40塩基以下のRNAから構成されていてもよい。40塩基以下であれば、インターフェロン応答等の不利益な現象が生じるリスクがより低くなる。
In one embodiment of the present disclosure, "siRNA" includes an RNA strand capable of inducing RNAi. In general, the two strands of siRNA can be divided into a guide strand and a passenger strand, and the guide strand is incorporated into RISC. The guide strand incorporated into RISC is used to recognize the target RNA. In RNAi research, artificially created ones are mainly used, but some are known to exist endogenously in living organisms. The guide strand may be composed of RNA of 15 or more bases. If it is 15 or more bases, the possibility of binding to the target polynucleotide with high accuracy increases. The guide strand may also be composed of RNA of 40 or less bases. If it is 40 or less bases, the risk of adverse phenomena such as interferon response is lower.
本開示の一実施形態において「shRNA」は、RNAiを誘導可能で、且つヘアピン状に折りたたまれた構造(ヘアピン様構造)を形成可能な1本鎖のRNA鎖を含む。典型的には、shRNAは細胞内でDicerによって切断され、siRNAが切り出される。このsiRNAによって標的RNAの切断が生じることが知られている。上記shRNAは35以上のヌクレオチドから構成されていてもよい。35以上であれば、shRNAに特有のへアピン様構造を精度よく形成できる可能性が高まる。また、上記shRNAは100塩基以下のRNAから構成されていてもよい。100塩基以下であれば、インターフェロン応答等の不利益な現象が生じるリスクが低くなる。但し、一般的にshRNAと構造および機能が類似しているpre-miRNAの多くが、100ヌクレオチド程度またはそれ以上の長さを有していることから、shRNAの長さは必ずしも100塩基以下でなくても、shRNAとして機能できると考えられる。
In one embodiment of the present disclosure, "shRNA" includes a single-stranded RNA strand capable of inducing RNAi and forming a structure folded into a hairpin (hairpin-like structure). Typically, shRNA is cleaved by Dicer in cells, and siRNA is excised. It is known that this siRNA causes cleavage of the target RNA. The above shRNA may be composed of 35 or more nucleotides. If it is 35 or more, the possibility of accurately forming the hairpin-like structure specific to shRNA increases. In addition, the above shRNA may be composed of RNA of 100 bases or less. If it is 100 bases or less, the risk of adverse phenomena such as interferon response is reduced. However, since many pre-miRNAs, which are generally similar in structure and function to shRNA, are about 100 nucleotides or more in length, it is considered that shRNA can function as an shRNA even if it is not necessarily 100 bases or less in length.
本開示の一実施形態において「miRNA」は、siRNAと類似の機能を有しているRNA鎖を含み、標的RNA鎖の翻訳抑制や分解をすることが知られている。miRNAとsiRNAとの違いは、一般的に生成経路と、詳細なメカニズムにある。
In one embodiment of the present disclosure, "miRNA" includes an RNA strand that has a function similar to that of siRNA, and is known to suppress the translation and degrade the target RNA strand. The difference between miRNA and siRNA generally lies in the production pathway and detailed mechanism.
本開示の一実施形態において「small RNA」とは、比較的小さいRNA鎖をいい、例えば、siRNA、shRNA、miRNA、アンチセンスRNA、1または2本鎖の低分子RNAなどを挙げることができる。
In one embodiment of the present disclosure, "small RNA" refers to a relatively small RNA strand, such as siRNA, shRNA, miRNA, antisense RNA, and single- or double-stranded small RNA.
上記RNAi分子は、5’末端または3’末端に1~5塩基からなるオーバーハングを含んでいてもよい。この場合、RNAiの効率が上昇すると考えられる。この数は、例えば、5、4、3
、2、または1塩基であってもよく、それらいずれか2つの値の範囲内であってもよい。ま
た上記RNAi分子が2本鎖のとき、各RNA鎖間にミスマッチRNAが存在していてもよい。その数は、例えば、1、2、3、4、5、または10個以下であってもよく、それらいずれか2つの値の範囲内であってもよい。また上記RNAi分子は、ヘアピンループを含んでいてもよい、ヘアピンループの塩基数は、例えば、10、8、6、5、4、または3塩基であってもよく、それらいずれか2つの値の範囲内であってもよい。塩基配列は、所望の効果を有する限り、1または複数個の塩基配列が欠失、置換、挿入、もしくは付加していてもよい。なお、各塩基配列の表記は、左側が5’末端、右側が3’末端である。 The RNAi molecule may contain an overhang of 1 to 5 bases at the 5' or 3' end. In this case, it is believed that the efficiency of RNAi increases. This number can be, for example, 5, 4, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 123, 134, 140, 145, 150, 165, 170, 186, 197, 198, 199
, 2, or 1 base, or within any two of these values. When the RNAi molecule is double-stranded, mismatched RNA may exist between each RNA strand. The number of bases may be, for example, 1, 2, 3, 4, 5, or 10 or less, or within any two of these values. The RNAi molecule may also include a hairpin loop, and the number of bases in the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, or within any two of these values. The base sequence may have one or more bases deleted, substituted, inserted, or added, as long as it has the desired effect. In addition, the left side of each base sequence is the 5' end, and the right side is the 3' end.
、2、または1塩基であってもよく、それらいずれか2つの値の範囲内であってもよい。ま
た上記RNAi分子が2本鎖のとき、各RNA鎖間にミスマッチRNAが存在していてもよい。その数は、例えば、1、2、3、4、5、または10個以下であってもよく、それらいずれか2つの値の範囲内であってもよい。また上記RNAi分子は、ヘアピンループを含んでいてもよい、ヘアピンループの塩基数は、例えば、10、8、6、5、4、または3塩基であってもよく、それらいずれか2つの値の範囲内であってもよい。塩基配列は、所望の効果を有する限り、1または複数個の塩基配列が欠失、置換、挿入、もしくは付加していてもよい。なお、各塩基配列の表記は、左側が5’末端、右側が3’末端である。 The RNAi molecule may contain an overhang of 1 to 5 bases at the 5' or 3' end. In this case, it is believed that the efficiency of RNAi increases. This number can be, for example, 5, 4, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 123, 134, 140, 145, 150, 165, 170, 186, 197, 198, 199
, 2, or 1 base, or within any two of these values. When the RNAi molecule is double-stranded, mismatched RNA may exist between each RNA strand. The number of bases may be, for example, 1, 2, 3, 4, 5, or 10 or less, or within any two of these values. The RNAi molecule may also include a hairpin loop, and the number of bases in the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, or within any two of these values. The base sequence may have one or more bases deleted, substituted, inserted, or added, as long as it has the desired effect. In addition, the left side of each base sequence is the 5' end, and the right side is the 3' end.
上記RNAi分子の長さは、例えば、15、18、20、25、30、40、50、60、80、100、200、または400塩基であってもよく、それらいずれか2つの値の範囲内であってもよい。
The length of the RNAi molecule may be, for example, 15, 18, 20, 25, 30, 40, 50, 60, 80, 100, 200, or 400 bases, or may be within a range of any two of those values.
上記RNAi分子は、安定的にRNAi作用を発揮する観点からは、LSR mRNAの塩基配列の一部に対して、相補的な塩基配列を含むことが好ましい。上記「一部」は、例えば、5、10、15、18、20、22、24、26、28、30、35、40、または50塩基以上であってもよく、それらいずれか2つの値の範囲内であってもよい。
From the viewpoint of stably exerting the RNAi effect, it is preferable that the above RNAi molecule contains a base sequence complementary to a portion of the base sequence of the LSR mRNA. The above "portion" may be, for example, 5, 10, 15, 18, 20, 22, 24, 26, 28, 30, 35, 40, or 50 or more bases, or may be within a range between any two of these values.
siRNAは、配列番号9または10の塩基配列を含む。これらの塩基配列は、LSR mRNAの一部に相補的な塩基配列であり、ガイド鎖としての機能を担う部分であると考えられる。本開示の一実施形態は、そのような、配列番号9または10の塩基配列を含むRNAi分子を含む。またこのRNAi分子は、配列番号9または10で示される塩基配列に対して相補的な塩基配列(例えば、それぞれ配列番号11、12)をさらに含んでいてもよい。本開示の一実施形態において「相補的な塩基配列」とは、一つのポリヌクレオチドに対して、ハイブリダイズすることが可能な相補性の高い他のポリヌクレオチドが有している塩基配列である。なお、siRNAのセンス鎖全長は配列番号13または14の塩基配列であり、アンチセンス鎖全長は配列番号15または16の塩基配列である。
The siRNA contains the base sequence of SEQ ID NO: 9 or 10. These base sequences are complementary to a portion of the LSR mRNA and are considered to function as a guide strand. One embodiment of the present disclosure includes an RNAi molecule containing such a base sequence of SEQ ID NO: 9 or 10. This RNAi molecule may further contain a base sequence complementary to the base sequence shown in SEQ ID NO: 9 or 10 (e.g., SEQ ID NO: 11 or 12, respectively). In one embodiment of the present disclosure, a "complementary base sequence" is a base sequence possessed by another polynucleotide that is highly complementary to one polynucleotide and can hybridize with it. The full length of the sense strand of the siRNA is the base sequence of SEQ ID NO: 13 or 14, and the full length of the antisense strand is the base sequence of SEQ ID NO: 15 or 16.
上に列挙した塩基配列は、LSR siRNAが所望の効果を有する限り、(i)上記の塩基酸配列において、1または数個の塩基配列が欠失、置換、挿入、もしくは付加しているアミノ酸配列、または(ii)上記の塩基配列に相補的な塩基配列からなるポリヌクレオチドに、ストリンジェントな条件下で特異的にハイブリダイズするポリヌクレオチドがコードする塩基配列であってもよい。
The base sequences listed above may be (i) amino acid sequences in which one or more base sequences are deleted, substituted, inserted, or added in the above base sequences, or (ii) base sequences encoded by polynucleotides that specifically hybridize under stringent conditions to polynucleotides consisting of base sequences complementary to the above base sequences, so long as the LSR siRNA has the desired effect.
本開示において、「LSRに結合する物質」、「LSR(の)結合剤」または「LSR相互作用分子」、「LSRに対する結合因子」は、少なくとも一時的にLSRに結合する分子または物質である。検出目的では好ましくは、結合したことを表示しうる(例えば標識されるか標識可能な状態である)ことが有利であり、治療目的では、さらに治療用薬剤が結合していることが有利である。これらは、例としては、抗体、アンチセンス・オリゴヌクレオチド、siRNA、低分子量分子(LMW)、結合性ペプチド、アプタマー、リボザイムおよびペプチド模倣体(peptidomimetic)等を挙げることができる。LSRに結合する物質またはLSR相互作用分子は、LSRの阻害剤であってもよく、例えばLSRに対して向けられる、特にLSRの活性部位に対して向けられる、結合性タンパク質または結合性ペプチド、並びにLSR遺伝子に対して向けられる核酸も含まれる。LSRに対する核酸は、例えばLSR遺伝子の発現またはLSRの活性を阻害する、二本鎖または一本鎖DNAまたはRNA、あるいはその修飾物または誘導体を指し、そしてアンチセンス核酸、アプタマー、siRNA(低分子干渉RNA)およびリボザイムを含むがこれらに限定されない。本明細書において、LSRについて「結合タンパク質」または「結合ペプチド」とは、LSRに結合する任意のタンパク質またはペプチドを指し、そしてLSRに対して指向される抗体(例えば、ポリクローナル抗体またはモノクローナル抗体)、抗体フラグメントおよび機能的等価物を含むがこれらに限定されない。
In the present disclosure, an "LSR-binding substance", "LSR binding agent" or "LSR-interacting molecule" or "binding factor for LSR" is a molecule or substance that binds to LSR at least temporarily. For detection purposes, it is preferably advantageous to be able to indicate the binding (e.g., be labeled or be capable of being labeled), and for therapeutic purposes, it is advantageous to further bind a therapeutic agent. These can be, for example, antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules (LMWs), binding peptides, aptamers, ribozymes, and peptidomimetics. The LSR-binding substance or LSR-interacting molecule can be an inhibitor of LSR, and also includes, for example, binding proteins or binding peptides directed against LSR, in particular against the active site of LSR, as well as nucleic acids directed against the LSR gene. Nucleic acids against LSR refer to, for example, double-stranded or single-stranded DNA or RNA, or modifications or derivatives thereof, that inhibit the expression of the LSR gene or the activity of the LSR, and include, but are not limited to, antisense nucleic acids, aptamers, siRNAs (small interfering RNAs), and ribozymes. As used herein, "binding protein" or "binding peptide" with respect to an LSR refers to any protein or peptide that binds to an LSR, and includes, but is not limited to, antibodies (e.g., polyclonal or monoclonal antibodies), antibody fragments, and functional equivalents directed against an LSR.
アミノ酸は、その一般に公知の3文字記号か、またはIUPAC-IUB Biochemical Nomenclature Commissionにより推奨される1文字記号のいずれかにより、本明細書中で言及され得る。ヌクレオチドも同様に、一般に認知された1文字コードにより言及され得る。本明細書では、アミノ酸配列および塩基配列の類似性、同一性および相同性の比較は、配列分析用ツールであるBLASTを用いてデフォルトパラメータを用いて算出される。同一性の検索は例えば、NCBIのBLAST2.13.0(2022/5/17発行)を用いて行うことができる。本明細書における同一性の値は通常は上記BLASTを用い、デフォルトの条件でアラインした際の値をいう。ただし、パラメータの変更により、より高い値が出る場合は、最も高い値を同一性の値とする。複数の領域で同一性が評価される場合はそのうちの最も高い値を同一性の値とする。類似性は、同一性に加え、類似のアミノ酸についても計算に入れた数値である。
Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly recognized one-letter codes. In this specification, comparisons of amino acid and base sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.13.0 (published 5/17/2022). The identity value in this specification usually refers to the value when aligned under default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be regarded as the identity value. If identity is evaluated in multiple regions, the highest value among them shall be regarded as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity.
本開示の一実施形態において「数個」は、例えば、10、8、6、5、4、3、または2個であってもよく、それらいずれかの値以下であってもよい。1または数個のアミノ酸残基の欠失、付加、挿入、または他のアミノ酸による置換を受けたポリペプチドが、その生物学的活性を維持することは知られている(Market al., Proc Natl Acad Sci USA.1984 Sep;81(18): 5662-5666.、Zoller et al.,Nucleic Acids Res. 1982 Oct 25;10(20): 6487-6500.、Wang et al., Science. 1984 Jun 29;224(4656): 1431-1433.)。欠失等がなされた抗体は、例えば、部位特異的変異導入法、ランダム変異導入法、または抗体ファージライブラリを用いたバイオパニング等によって作製できる。部位特異的変異導入法としては、例えばKOD-Plus- Mutagenesis Kit (TOYOBO CO., LTD.)を使用できる。欠失等を導入した変異型抗体から、野生型と同様の活性のある抗体を選択することは、FACS解析やELISA等の各種キャラクタリゼーションを行うことで可能である。
In one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values or less. It is known that a polypeptide that has one or several amino acid residues deleted, added, inserted, or substituted with other amino acids maintains its biological activity (Market et al., Proc Natl Acad Sci USA. 1984 Sep; 81(18): 5662-5666.; Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10(20): 6487-6500.; Wang et al., Science. 1984 Jun 29; 224(4656): 1431-1433.). Antibodies with deletions or the like can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. For example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used for site-specific mutagenesis. It is possible to select antibodies with the same activity as the wild type from mutant antibodies with deletions, etc., by performing various characterizations such as FACS analysis and ELISA.
本明細書において「フラグメント」とは、全長のポリペプチドまたはポリヌクレオチド(長さがn)に対して、1~n-1までの配列長さを有するポリペプチドまたはポリヌクレオチドをいう。フラグメントの長さは、その目的に応じて、適宜変更することができ、例えば、その長さの下限としては、ポリペプチドの場合、3、4、5、6、7、8、9、10、15、20、25、30、40、50およびそれ以上のアミノ酸が挙げられ、ここの具体的に列挙していない整数で表される長さ(例えば、11など)もまた、下限として適切であり得る。また、ポリヌクレオチドの場合、5、6、7、8、9、10、15、20、25、30、40、50、75、100およびそれ以上のヌクレオチドが挙げられ、ここの具体的に列挙していない整数で表される長さ(例えば、11など)もまた、下限として適切であり得る。本明細書において、このようなフラグメントは、例えば、全長のものがマーカーまたは標的分子として機能する場合、そのフラグメント自体もまたマーカーまたは標的分子としての機能を有する限り、本開示の範囲内に入ることが理解される。
As used herein, a "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of the fragment can be changed as appropriate depending on the purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In addition, for a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. As used herein, such fragments are understood to be within the scope of the present disclosure, for example, if the full-length fragment functions as a marker or target molecule, as long as the fragment itself also functions as a marker or target molecule.
本開示に従って、用語「活性」は、本明細書において、最も広い意味での分子の機能を指す。活性は、限定を意図するものではないが、概して、分子の生物学的機能、生化学的機能、物理的機能または化学的機能を含む。活性は、例えば、酵素活性、他の分子と相互作用する能力、および他の分子の機能を活性化するか、促進するか、安定化するか、阻害するか、抑制するか、または不安定化する能力、安定性、特定の細胞内位置に局在する能力を含む。適用可能な場合、この用語はまた、最も広い意味でのタンパク質複合体の機能にも関する。
In accordance with the present disclosure, the term "activity" as used herein refers to the function of a molecule in its broadest sense. Activity generally includes, but is not intended to be limiting, the biological, biochemical, physical, or chemical functions of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules, stability, ability to localize to a particular subcellular location. Where applicable, the term also relates to the function of protein complexes in their broadest sense.
本明細書において「生物学的機能」とは、ある遺伝子またはそれに関する核酸分子もしくはポリペプチドについて言及するとき、その遺伝子、核酸分子またはポリペプチドが生体内において有し得る特定の機能をいい、これには、例えば、特異的な抗体の生成、酵素活性、抵抗性の付与等を挙げることができるがそれらに限定されない。本開示においては、例えば、LSRがVLDLの取り込みの阻害等に関与する機能などを挙げることができるがそれらに限定されない。本明細書において、生物学的機能は、「生物学的活性」によって発揮され得る。本明細書において「生物学的活性」とは、ある因子(例えば、ポリヌクレオチド、タンパク質など)が、生体内において有し得る活性のことをいい、種々の機能(例えば、転写促進活性)を発揮する活性が包含され、例えば、ある分子との相互作用によって別の分子が活性化または不活化される活性も包含される。2つの因子が相互作用する場合、その生物学的活性は、その二分子の間の結合およびそれによって生じる生物学的変化であり得、そして、例えば、一つの分子を抗体を用いて沈降させたときに他の分子も共沈するとき、2分子は結合していると考えられる。従って、そのような共沈を見ることが一つの判断手法として挙げられる。例えば、ある因子が酵素である場合、その生物学的活性は、その酵素活性を包含する。別の例では、ある因子がリガンドである場合、そのリガンドが対応するレセプターへの結合を包含する。そのような生物学的活性は、当該分野において周知の技術によって測定することができる。従って、「活性」は、結合(直接的または間接的のいずれか)を示すかまたは明らかにするか;応答に影響する(すなわち、いくらかの曝露または刺激に応答する測定可能な影響を有する)、種々の測定可能な指標をいい、例えば、本開示のポリペプチドまたはポリヌクレオチドに直接結合する化合物の親和性、または例えば、いくつかの刺激後または事象後の上流または下流のタンパク質の量あるいは他の類似の機能の尺度が挙げられる。
In this specification, the term "biological function" refers to a specific function that a gene, a nucleic acid molecule, or a polypeptide related thereto may have in a living body, and includes, but is not limited to, the production of specific antibodies, enzyme activity, and the conferring of resistance. In the present disclosure, the term "biological function" refers to, but is not limited to, the function of LSR in inhibiting the uptake of VLDL. In this specification, the biological function may be exerted by "biological activity." In this specification, "biological activity" refers to the activity that a certain factor (e.g., polynucleotide, protein, etc.) may have in a living body, and includes the activity of exerting various functions (e.g., transcription promoting activity), and also includes, for example, the activity of activating or inactivating another molecule by interacting with another molecule. When two factors interact with each other, the biological activity may be the binding between the two molecules and the biological change that occurs as a result of this. For example, when one molecule is precipitated using an antibody and the other molecule is also co-precipitated, the two molecules are considered to be bound. Therefore, observing such co-precipitation is one method of judgment. For example, if a factor is an enzyme, its biological activity includes its enzymatic activity. In another example, if a factor is a ligand, its biological activity includes the binding of the ligand to a corresponding receptor. Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that indicate or reveal binding (either directly or indirectly); affect a response (i.e., have a measurable effect in response to some exposure or stimulus), such as the affinity of a compound that binds directly to a polypeptide or polynucleotide of the present disclosure, or, for example, a measure of the amount of an upstream or downstream protein or other similar function after some stimulus or event.
本明細書において遺伝子、ポリヌクレオチド、ポリペプチドなどの「発現」とは、その遺伝子などがインビボで一定の作用を受けて、別の形態になることをいう。好ましくは、遺伝子、ポリヌクレオチドなどが、転写および翻訳されて、ポリペプチドの形態になることをいうが、転写されてmRNAが作製されることもまた発現の一態様である。したがって、本明細書において「発現産物」とは、このようなポリペプチドもしくはタンパク質、またはmRNAを含む。より好ましくは、そのようなポリペプチドの形態は、翻訳後プロセシン
グを受けたものであり得る。例えば、LSRの発現レベルは、任意の方法によって決定することができる。具体的には、LSRのmRNAの量、LSRタンパク質の量、そしてLSRタンパク質の生物学的な活性を評価することによって、LSRの発現レベルを知ることができる。このような測定値はコンパニオン診断において使用し得る。LSRのmRNAやタンパク質の量は、本明細書の他の箇所に詳述したような方法あるいは他の当該分野において公知の方法によって決定することができる。 As used herein, the term "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. undergoing a certain action in vivo to become a different form. Preferably, the term refers to the gene, polynucleotide, etc. being transcribed and translated to become a polypeptide, but transcription to produce an mRNA is also an aspect of expression. Thus, as used herein, the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, such a polypeptide form may be one that has undergone post-translational processing. For example, the expression level of an LSR can be determined by any method. Specifically, the expression level of an LSR can be known by evaluating the amount of LSR mRNA, the amount of LSR protein, and the biological activity of the LSR protein. Such measurements can be used in companion diagnostics. The amount of LSR mRNA or protein can be determined by methods detailed elsewhere in this specification or other methods known in the art.
グを受けたものであり得る。例えば、LSRの発現レベルは、任意の方法によって決定することができる。具体的には、LSRのmRNAの量、LSRタンパク質の量、そしてLSRタンパク質の生物学的な活性を評価することによって、LSRの発現レベルを知ることができる。このような測定値はコンパニオン診断において使用し得る。LSRのmRNAやタンパク質の量は、本明細書の他の箇所に詳述したような方法あるいは他の当該分野において公知の方法によって決定することができる。 As used herein, the term "expression" of a gene, polynucleotide, polypeptide, etc. refers to the gene, etc. undergoing a certain action in vivo to become a different form. Preferably, the term refers to the gene, polynucleotide, etc. being transcribed and translated to become a polypeptide, but transcription to produce an mRNA is also an aspect of expression. Thus, as used herein, the term "expression product" includes such a polypeptide or protein, or mRNA. More preferably, such a polypeptide form may be one that has undergone post-translational processing. For example, the expression level of an LSR can be determined by any method. Specifically, the expression level of an LSR can be known by evaluating the amount of LSR mRNA, the amount of LSR protein, and the biological activity of the LSR protein. Such measurements can be used in companion diagnostics. The amount of LSR mRNA or protein can be determined by methods detailed elsewhere in this specification or other methods known in the art.
本明細書において「機能的等価物」とは、対象となるもとの実体に対して、目的となる機能が同じであるが構造が異なる任意のものをいう。従って、「LSR」またはその抗体の機能的等価物は、LSRまたはその抗体自体ではないが、LSRまたはその抗体の変異体または改変体(例えば、アミノ酸配列改変体等)であって、LSRの持つ生物学的作用を有するもの、ならびに、作用する時点において、LSRまたはその抗体自体またはこのLSRまたはその抗体の変異体もしくは改変体に変化することができるもの(例えば、LSRまたはその抗体自体またはLSRまたはその抗体の変異体もしくは改変体をコードする核酸、およびその核酸を含むベクター、細胞等を含む)が包含されることが理解される。本開示において、LSRまたはその抗体の機能的等価物は、格別に言及していなくても、LSRまたはその抗体と同様に用いられうることが理解される。機能的等価物は、データベース等を検索することによって、見出すことができる。本明細書において「検索」とは、電子的にまたは生物学的あるいは他の方法により、ある核酸塩基配列を利用して、特定の機能および/または性質を有する他の核酸塩基配列を見出すことをいう。電子的な検索としては、BLAST(Altschul et al.,J.Mol.Biol. 215:403-410(1990))、FASTA (Pearson & Lipman, Proc.Natl.Acad.Sci.,USA 85:2444-2448(1988))、Smith and Waterman法(Smith and Waterman, J.Mol.Biol.147: 195-197(1981))、およびNeedleman and Wunsch法(Needleman and Wunsch, J.Mol.Biol. 48:443-453(1970))などが挙げられるがそれらに限定されない。生物学的な検索としては、ストリンジェントハイブリダイゼーション、ゲノムDNAをナイロンメンブレン等に貼り付けたマクロアレイまたはガラス板に貼り付けたマイクロアレイ(マイクロアレイアッセイ)、PCRおよびin situハイブリダイゼーションなどが挙げられるがそれらに限定されない。本明細書において、本開示において使用される遺伝子には、このような電子的検索、生物学的検索によって同定された対応遺伝子も含まれるべきであることが意図される。
In this specification, the term "functional equivalent" refers to any entity that has the same intended function but a different structure compared to the original entity of interest. Therefore, it is understood that the functional equivalent of an "LSR" or its antibody is not the LSR or its antibody itself, but a mutant or modified version of the LSR or its antibody (e.g., an amino acid sequence variant, etc.) that has the biological action of the LSR, as well as an entity that can change into the LSR or its antibody itself or a mutant or modified version of the LSR or its antibody at the time of acting (e.g., a nucleic acid that encodes the LSR or its antibody itself or a mutant or modified version of the LSR or its antibody, and a vector, cell, etc. that contains the nucleic acid). In this disclosure, it is understood that the functional equivalent of an LSR or its antibody can be used in the same way as the LSR or its antibody, even if not specifically mentioned. The functional equivalent can be found by searching a database, etc. In this specification, "search" refers to using a certain nucleic acid base sequence to find other nucleic acid base sequences that have a specific function and/or property, electronically, biologically, or by other methods. Electronic searches include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215:403-410(1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85:2444-2448(1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147:195-197(1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970)). Biological searches include, but are not limited to, stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane or the like, or microarrays in which genomic DNA is attached to a glass plate (microarray assays), PCR, and in situ hybridization. In this specification, it is intended that the gene used in this disclosure should also include corresponding genes identified by such electronic searches and biological searches.
本開示の機能的等価物としては、アミノ酸配列において、1もしくは複数個のアミノ酸の挿入、置換もしくは欠失、またはその一方もしくは両末端への付加されたものを用いることができる。本明細書において、「アミノ酸配列において、1もしくは複数個のアミノ酸の挿入、置換もしくは欠失、またはその一方もしくは両末端への付加」とは、部位特異的突然変異誘発法等の周知の技術的方法により、あるいは天然の変異により、天然に生じ得る程度の複数個の数のアミノ酸の置換等により改変がなされていることを意味する。改変アミノ酸配列は、例えば1~30個、好ましくは1~20個、より好ましくは1~9個、さらに好ましくは1~5個、特に好ましくは1~2個のアミノ酸の挿入、置換、もしくは欠失、またはその一方もしくは両末端への付加がなされたものであることができる。改変アミノ酸配列は、好ましくは、そのアミノ酸配列が、LSRまたはその抗体のアミノ酸配列において1または複数個(好ましくは、1もしくは数個または1、2、3、もしくは4個)の保存的置換を有するアミノ酸配列であってもよい。ここで「保存的置換」とは、タンパク質の機能を実質的に改変しないように、1または複数個のアミノ酸残基を、別の化学的に類似したアミノ酸残基で置換えることを意味する。例えば、ある疎水性残基を別の疎水性残基によって置換する場合、ある極性残基を同じ電荷を有する別の極性残基によって置換する場合などが挙げられる。このような置換を行うことができる機能的に類似のアミノ酸は、アミノ酸毎に当該分野において公知である。具体例を挙げると、非極性(疎水性)アミノ酸としては、アラニン、バリン、イソロイシン、ロイシン、プロリン、トリプトファン、フェニルアラニン、メチオニンなどが挙げられる。極性(中性)アミノ酸としては、グリシン、セリン、スレオニン、チロシン、グルタミン、アスパラギン、システインなどが挙げられる。陽電荷をもつ(塩基性)アミノ酸としては、アルギニン、ヒスチジン、リジンなどが挙げられる。また、負電荷をもつ(酸性)アミノ酸としては、アスパラギン酸、グルタミン酸などが挙げられる。
As the functional equivalent of the present disclosure, an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both termini, can be used. In this specification, "an amino acid sequence in which one or more amino acids have been inserted, substituted or deleted, or added to one or both termini" means that the amino acid sequence has been modified by a well-known technical method such as site-directed mutagenesis, or by natural mutation, by substitution of a number of amino acids to the extent that may occur naturally. The modified amino acid sequence may be, for example, an amino acid sequence in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted or deleted, or added to one or both termini. The modified amino acid sequence may preferably be an amino acid sequence having one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of the LSR or its antibody. Here, "conservative substitution" means replacing one or more amino acid residues with another chemically similar amino acid residue so as not to substantially alter the function of the protein. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally similar amino acids that can be used for such substitutions are known in the art for each amino acid. Specific examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
本明細書において「抗体」は、広義にはポリクローナル抗体、モノクローナル抗体、多重特異性抗体、キメラ抗体、および抗イディオタイプ抗体、ならびにそれらのフラグメント、例えばFvフラグメント、Fab’フラグメント、F(ab’)2およびFabフラグメント、ならびにその他の組換えにより生産された結合体または機能的等価物(例えば、キメラ抗体、ヒト化抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー(diabody)、sc(Fv)2(singlechain(Fv)2)、scFv-Fc)を含む。さらにこのような抗体を、酵素、例えばアルカリホスファターゼ、西洋ワサビペルオキシダーゼ、αガラクトシダーゼなど、に共有結合させまたは組換えにより融合させてよい。本開示で用いられる抗LSR抗体は、LSRのタンパク質に結合すればよく、その由来、種類、形状などは問われない。具体的には、非ヒト動物の抗体(例えば、マウス抗体、ラット抗体、ラクダ抗体)、ヒト抗体、キメラ抗体、ヒト化抗体などの公知の抗体が使用できる。本開示においては、モノクローナル、あるいはポリクローナルを抗体として利用することができるが好ましくはモノクローナル抗体である。抗体のLSRタンパク質への結合は特異的な結合であることが好ましい。また抗体は、抗体修飾物または抗体非修飾物を含む。抗体修飾物は、抗体と、例えばポリエチレングリコール等の各種分子が結合していてもよい。抗体修飾物は、抗体に公知の手法を用いて化学的な修飾を施すことによって得ることができる。
In the present specification, the term "antibody" broadly includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotypic antibodies, as well as fragments thereof, such as Fv fragments, Fab' fragments, F(ab') 2 and Fab fragments, and other recombinantly produced conjugates or functional equivalents (e.g., chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc(Fv) 2 (single chain (Fv) 2 ), scFv-Fc). Furthermore, such antibodies may be covalently linked or recombinantly fused to enzymes, such as alkaline phosphatase, horseradish peroxidase, α-galactosidase, and the like. The anti-LSR antibody used in the present disclosure may be of any origin, type, shape, etc., as long as it binds to an LSR protein. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present disclosure, monoclonal or polyclonal antibodies can be used as the antibodies, but monoclonal antibodies are preferred. The antibody preferably binds specifically to the LSR protein. The antibody also includes modified and unmodified antibodies. The modified antibody may be bound to various molecules such as polyethylene glycol. The modified antibody can be obtained by chemically modifying the antibody using a known method.
本明細書において、「抗LSR抗体」は、LSRに結合性を有する抗体を含む。この抗LSR抗体の生産方法は特に限定されないが、例えば、LSRを哺乳類または鳥類に免疫することによって生産してもよい。
In this specification, "anti-LSR antibody" includes an antibody that has binding ability to LSR. There are no particular limitations on the method for producing this anti-LSR antibody, but it may be produced, for example, by immunizing a mammal or bird with LSR.
また、「LSRに対する抗体(抗LSR抗体)、または、そのフラグメント」の「機能的等価物」は、例えば、抗体の場合、LSRの結合活性、必要であれば抑制活性を有する抗体自体およびそのフラグメント自体のほか、キメラ抗体、ヒト化抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(singlechain(Fv)2)、scFv-Fcなども包含されることが理解される。
It is further understood that the "functional equivalent" of "an antibody against LSR (anti-LSR antibody) or a fragment thereof" includes, for example, in the case of antibodies, not only the antibody itself and its fragment having LSR binding activity and, if necessary, inhibitory activity, but also chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-chain antibodies, scFVs, diabodies, sc(Fv) 2 (single chain (Fv) 2 ), scFv-Fc, and the like.
本開示の一実施形態に係る複合体において使用される抗LSR抗体は、悪性腫瘍の増殖が特に強く抑制される観点からは、LSRの特定のエピトープに特異的に結合する抗LSR抗体であることが好ましい。
The anti-LSR antibody used in the complex according to one embodiment of the present disclosure is preferably an anti-LSR antibody that specifically binds to a specific epitope of LSR, from the viewpoint of particularly strong inhibition of malignant tumor growth.
本開示の一実施形態に係る抗LSR抗体は、モノクローナル抗体であってもよい。モノクローナル抗体であれば、ポリクローナル抗体に比べて、効率的にLSRに対して作用させることができる。抗LSRモノクローナル抗体を効率的に生産する観点からは、LSRをニワトリに免疫することが好ましい。
The anti-LSR antibody according to one embodiment of the present disclosure may be a monoclonal antibody. A monoclonal antibody can act on LSR more efficiently than a polyclonal antibody. From the viewpoint of efficiently producing an anti-LSR monoclonal antibody, it is preferable to immunize chickens with LSR.
本開示の一実施形態に係る複合体において使用される抗LSR抗体の抗体クラスは特に限定されないが、例えばIgM、IgD、IgG、IgA、IgE、またはIgYであってもよい。
The antibody class of the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is not particularly limited, but may be, for example, IgM, IgD, IgG, IgA, IgE, or IgY.
本開示の一実施形態に係る抗LSR抗体は、抗原結合活性を有する抗体フラグメントであっても良い。この場合、安定性または抗体の生産効率が上昇する等の効果がある。
The anti-LSR antibody according to one embodiment of the present disclosure may be an antibody fragment having antigen-binding activity. In this case, there are effects such as increased stability or antibody production efficiency.
本開示の一実施形態に係る複合体において使用される抗LSR抗体は、融合タンパク質であってもよい。この融合タンパク質は、抗LSR抗体のNまたはC末端に、ポリペプチドまたはオリゴペプチドが結合したものであってもよい。ここで、オリゴペプチドは、Hisタグであってもよい。また融合タンパク質は、マウス、ヒト、またはニワトリの抗体部分配列を融合したものであってもよい。それらのような融合タンパク質も、本実施形態に係る抗LSR抗体の一形態に含まれる。
The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be a fusion protein. This fusion protein may be an anti-LSR antibody with a polypeptide or oligopeptide bound to the N- or C-terminus. Here, the oligopeptide may be a His tag. The fusion protein may also be a fusion of a mouse, human, or chicken antibody partial sequence. Such fusion proteins are also included in one form of the anti-LSR antibody according to this embodiment.
本開示の一実施形態に係る複合体において使用される抗LSR抗体は、例えば、精製LSR、LSR発現細胞、またはLSR含有脂質膜で生物を免疫する工程を経て得られる抗体であってもよい。LSR陽性悪性腫瘍に対する治療効果を高める観点からは、LSR発現細胞を免疫に使用することが好ましい。
The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be, for example, an antibody obtained through a process of immunizing an organism with purified LSR, LSR-expressing cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization.
本開示の一実施形態に係る複合体において使用される抗LSR抗体は、精製LSR、LSR発現細胞胞またはLSR含有脂質膜で生物を免疫する工程を経て得られる抗体の、CDRセットを有する抗体であってもよい。LSR陽性悪性腫瘍に対する治療効果を高める観点からは、LSR発現細胞を免疫に使用することが好ましい。CDRセットとは、重鎖CDR1、2、および3、ならびに、軽鎖CDR1、2、および3のセットである。
The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be an antibody having a CDR set of an antibody obtained through a process of immunizing an organism with purified LSR, LSR-expressing cell cells, or an LSR-containing lipid membrane. From the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, it is preferable to use LSR-expressing cells for immunization. The CDR set is a set of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3.
本開示の一実施形態において「LSR発現細胞」は、例えば、LSRをコードするポリヌクレオチドを細胞に導入後、LSRを発現させることによって得てもよい。ここでLSRは、LSRフラグメントを含む。また本開示の一実施形態において「LSR含有脂質膜」は、例えば、LSRと脂質二重膜を混合することによって得てもよい。ここでLSRは、LSRフラグメントを含む。また本開示の一実施形態に係る複合体において使用される抗LSR抗体は、LSR陽性悪性腫瘍に対する治療効果を高める観点からは、抗原をニワトリに免疫する工程を経て得られる抗体、またはその抗体のCDRセットを有する抗体が好ましい。
In one embodiment of the present disclosure, the "LSR-expressing cells" may be obtained, for example, by introducing a polynucleotide encoding LSR into a cell and then expressing LSR. Here, the LSR includes an LSR fragment. In another embodiment of the present disclosure, the "LSR-containing lipid membrane" may be obtained, for example, by mixing LSR with a lipid bilayer membrane. Here, the LSR includes an LSR fragment. In addition, from the viewpoint of enhancing the therapeutic effect against LSR-positive malignant tumors, the anti-LSR antibody used in the complex according to one embodiment of the present disclosure is preferably an antibody obtained through a process of immunizing chickens with an antigen, or an antibody having a CDR set of that antibody.
本開示の一実施形態に係る複合体において使用される抗LSR抗体は、目的を達成する限り、どのような結合力を有していてもよく、例えば、少なくとも1.0×106以上、2.0×106以上、5.0×106以上、1.0×107以上を挙げることができるがこれらに限定されず、通常は、KD値(kd/ka)が、1.0×10-7以下であってもよく、1.0×10-9(M)あるいは1.0×10-10(M)以下であり得る。
The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may have any binding strength as long as the intended purpose is achieved, and examples thereof include, but are not limited to, at least 1.0×10 or more , 2.0× 10 or more, 5.0×10 or more, or 1.0×10 or more , and typically has a K value (kd/ka) of 1.0× 10 or less, and may be 1.0× 10 or less ( M) or less (M).
本開示の一実施形態に係る複合体において使用される抗LSR抗体は、LSRの野生型または変異型に結合する抗体であってもよい。変異型とは、個体間のDNA配列の差異に起因するものを含む。野生型または変異型のLSRのアミノ酸配列は、配列番号8に示すアミノ酸配列に対し、好ましくは80%以上、より好ましくは90%以上、より好ましくは95%以上、特に好ましくは98%以上の相同性を有している。
The anti-LSR antibody used in the complex according to one embodiment of the present disclosure may be an antibody that binds to a wild-type or mutant form of LSR. Mutant forms include those resulting from differences in DNA sequences between individuals. The amino acid sequence of the wild-type or mutant form of LSR has a homology of preferably 80% or more, more preferably 90% or more, more preferably 95% or more, and particularly preferably 98% or more to the amino acid sequence shown in SEQ ID NO:8.
本明細書において「ポリクローナル抗体」は、例えば、抗原に特異的なポリクローナル抗体の産生を誘導するために、哺乳類(例えば、ラット、マウス、ウサギ、ウシ、サル等)、鳥類等に、目的の抗原を含む免疫原を投与することによって生成することが可能である。免疫原の投与は、1つ以上の免疫剤、および所望の場合にはアジュバントの注入をしてもよい。アジュバントは、免疫応答を増加させるために使用されることもあり、フロイントアジュバント(完全または不完全)、ミネラルゲル(水酸化アルミニウム等)、または界面活性物質(リゾレシチン等)等を含んでいてもよい。免疫プロトコールは、当該技術分野で公知であり、選択する宿主生物に合わせて、免疫応答を誘発する任意の方法によって実施される場合がある(タンパク質実験ハンドブック,羊土社(2003):86-91.)。
As used herein, a "polyclonal antibody" can be produced, for example, by administering an immunogen containing an antigen of interest to a mammal (e.g., rat, mouse, rabbit, cow, monkey, etc.), bird, etc., in order to induce the production of polyclonal antibodies specific to the antigen. The administration of the immunogen may be by injection of one or more immunizing agents and, if desired, an adjuvant. Adjuvants may be used to increase the immune response and may include Freund's adjuvant (complete or incomplete), mineral gels (e.g., aluminum hydroxide), or surfactants (e.g., lysolecithin), etc. Immunization protocols are known in the art and may be performed by any method that induces an immune response, depending on the host organism of choice (Protein Experiment Handbook, Yodosha (2003): 86-91.).
本明細書において「モノクローナル抗体」は、集団を構成する個々の抗体が、少量自然に生じることが可能な突然変異を有する抗体を除いて、実質的に単一のエピトープに対応する抗体である場合を含む。または、集団を構成する個々の抗体が、少量自然に生じることが可能な突然変異を有する抗体を除いて、実質的に同一である抗体であってもよい。モノクローナル抗体は高度に特異的であり、異なるエピトープに対応する異なる抗体を典型的に含むような、通常のポリクローナル抗体とは異なる。その特異性に加えて、モノクローナル抗体は、他の免疫グロブリンによって汚染されていないハイブリドーマ培養から合成できる点で有用である。「モノクローナル」という形容は、実質的に均一な抗体集団から得られるという特徴を示していてもよいが、抗体を何か特定の方法で生産しなければならないことを意味するものではない。例えば、モノクローナル抗体は、"Kohler G, Milstein C., Nature. 1975 Aug7;256(5517):495-497."に掲載されているようなハイブリドーマ法と同様の方法によって作製してもよい。あるいは、モノクローナル抗体は、米国特許第4816567号に記載されているような組換え法と同様の方法によって作製してもよい。または、モノクローナル抗体は、"Clackson et al., Nature. 1991 Aug 15;352(6336): 624-628."、または"Marks et al., J Mol Biol. 1991 Dec 5;222(3): 581-597."に記載されているような技術と同様の方法を用いてファージ抗体ライブラリーから単離してもよい。または、"タンパク質実験ハンドブック,羊土社(2003):92-96."に掲載されている方法でよって作製してもよい。
As used herein, the term "monoclonal antibody" includes antibodies in which the individual antibodies constituting the population are substantially identical to one another, except for antibodies with small amounts of naturally occurring mutations. Alternatively, the individual antibodies constituting the population may be substantially identical, except for antibodies with small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific and differ from conventional polyclonal antibodies, which typically contain different antibodies that correspond to different epitopes. In addition to their specificity, monoclonal antibodies are useful in that they can be synthesized from hybridoma cultures that are not contaminated by other immunoglobulins. The term "monoclonal" may indicate the characteristic of being obtained from a substantially homogeneous antibody population, but does not imply that the antibody must be produced by any particular method. For example, monoclonal antibodies may be produced by methods similar to the hybridoma method described in "Kohler G, Milstein C., Nature. 1975 Aug7;256(5517):495-497." Alternatively, monoclonal antibodies may be produced by recombinant methods similar to those described in U.S. Pat. No. 4,816,567. Alternatively, monoclonal antibodies may be isolated from phage antibody libraries using techniques similar to those described in Clackson et al., Nature. 1991 Aug 15;352(6336):624-628, or Marks et al., J Mol Biol. 1991 Dec 5;222(3):581-597. Alternatively, monoclonal antibodies may be produced by methods described in Protein Experiment Handbook, Yodosha (2003):92-96.
抗体の大量生産については、当該分野で公知の任意の手法を用いることができるが、例えば、代表的な抗体の大量生産系の構築および抗体製造としては、以下を例示することができる。すなわち、CHO細胞にH鎖抗体発現ベクターおよびL鎖抗体発現ベクターをトランスフェクションし、選択試薬であるG418およびZeocinを用いて培養を行い、限界希釈法によるクローニングを行う。クローニング後、安定的に抗体を発現しているクローンをELISA法により選択する。選択したCHO細胞を用いて拡大培養し、抗体を含む培養上清を回収する。回収した培養上清からProtein AもしくはProtein G精製により抗体を精製することができる。
Any method known in the art can be used for the mass production of antibodies, but the following is an example of a typical method for constructing a mass production system for antibodies and producing antibodies. That is, CHO cells are transfected with an H-chain antibody expression vector and an L-chain antibody expression vector, cultured using the selection reagents G418 and Zeocin, and cloned by limiting dilution. After cloning, clones that stably express the antibody are selected by ELISA. The selected CHO cells are expanded and the culture supernatant containing the antibody is collected. The antibody can be purified from the collected culture supernatant by Protein A or Protein G purification.
本明細書において「Fv抗体」は、抗原認識部位を含む抗体である。この領域は、非共有結合による1つの重鎖可変ドメインおよび1つの軽鎖可変ドメインの二量体を含む。この構成において、各可変ドメインの3つのCDRは相互に作用してVH-VL二量体の表面に抗原結合部位を形成することができる。
As used herein, an "Fv antibody" is an antibody that contains an antigen recognition site. This region comprises a non-covalent dimer of one heavy chain variable domain and one light chain variable domain. In this configuration, the three CDRs of each variable domain can interact to form an antigen-binding site on the surface of the VH-VL dimer.
本明細書において「Fab抗体」は、例えば、Fab領域およびFc領域を含む抗体をタンパク質分解酵素パパインで処理して得られる断片のうち、H鎖のN末端側約半分とL鎖全体が一部のジスルフィド結合を介して結合した抗体である。Fabは、例えば、Fab領域およびFc領域を含む本開示の実施形態に係る抗LSR抗体を、タンパク質分解酵素パパインで処理して得ることができる。
In this specification, a "Fab antibody" is, for example, an antibody fragment obtained by treating an antibody containing a Fab region and an Fc region with the protease papain, in which approximately the N-terminal half of the H chain and the entire L chain are bound via some disulfide bonds. Fab can be obtained, for example, by treating an anti-LSR antibody according to an embodiment of the present disclosure containing a Fab region and an Fc region with the protease papain.
本明細書において「F(ab’)2抗体」は、例えば、Fab領域およびFc領域を含む抗体をタンパク質分解酵素ペプシンで処理して得られる断片のうち、Fabに相当する部位を2つ含む抗体である。F(ab’)2は、例えば、Fab領域およびFc領域を含む本開示の実施形態に係る抗LSR抗体を、タンパク質分解酵素ペプシンで処理して得ることができる。また、例えば、下記のFab’をチオエーテル結合あるいはジスルフィド結合させることで、作製することができる。
As used herein, the term "F(ab') 2 antibody" refers to an antibody that contains two sites corresponding to Fab, among fragments obtained by treating an antibody containing a Fab region and an Fc region with the protease pepsin. F(ab') 2 can be obtained, for example, by treating an anti-LSR antibody according to an embodiment of the present disclosure that contains a Fab region and an Fc region with the protease pepsin. In addition, for example, it can be prepared by forming a thioether bond or disulfide bond with the following Fab'.
本明細書において「Fab’抗体」は、例えば、F(ab’)2のヒンジ領域のジスルフィド結合を切断して得られる抗体である。例えば、F(ab’)2を還元剤ジチオスレイトール処理して得ることができる。
As used herein, a "Fab'antibody" refers to an antibody obtained, for example, by cleaving the disulfide bond in the hinge region of F(ab') 2. For example, it can be obtained by treating F(ab') 2 with a reducing agent, dithiothreitol.
本明細書において「scFv抗体」は、VHとVLとが適当なペプチドリンカーを介して連結した抗体である。scFv抗体は、例えば、本開示の実施形態に係る複合体において使用される抗LSR抗体のVHおよびVLをコードするcDNAを取得し、VH-ペプチドリンカー-VLをコードするポリヌクレオチドを構築し、そのポリヌクレオチドをベクターに組み込み、発現用の細胞を用いて生産できる。
As used herein, an "scFv antibody" is an antibody in which VH and VL are linked via a suitable peptide linker. For example, an scFv antibody can be produced by obtaining cDNA encoding the VH and VL of an anti-LSR antibody used in a complex according to an embodiment of the present disclosure, constructing a polynucleotide encoding VH-peptide linker-VL, incorporating the polynucleotide into a vector, and using an expression cell.
本明細書において「ダイアボディー(diabody)」は、二価の抗原結合活性を有する抗体である。二価の抗原結合活性は、同一であることもできるし、一方を異なる抗原結合活性とすることもできる。ダイアボディー(diabody)は、例えば、scFvをコードするポリヌクレオチドをペプチドリンカーのアミノ酸配列の長さが8残基以下となるように構築し、得られたポリヌクレオチドをベクターに組み込み、発現用の細胞を用いて生産できる。
As used herein, a "diabody" is an antibody that has bivalent antigen-binding activity. The bivalent antigen-binding activities can be the same, or one of the two can be a different antigen-binding activity. A diabody can be produced, for example, by constructing a polynucleotide encoding an scFv such that the amino acid sequence of the peptide linker is 8 residues or less in length, incorporating the resulting polynucleotide into a vector, and using an expression cell.
本明細書において「dsFv」は、VHおよびVL中にシステイン残基を導入したポリペプチドを、システイン残基間のジスルフィド結合を介して結合させた抗体である。システイン残基に導入する位置はReiterらにより示された方法(Reiteret al., Protein Eng. 1994 May;7(5):697-704.)に従って、抗体の立体構造予測に基づいて選択することができる。
As used herein, "dsFv" refers to an antibody in which polypeptides with cysteine residues introduced into VH and VL are linked via disulfide bonds between the cysteine residues. The position at which the cysteine residue is introduced can be selected based on the prediction of the three-dimensional structure of the antibody according to the method shown by Reiter et al. (Reiteret et al., Protein Eng. 1994 May;7(5):697-704.)
本明細書において「抗原結合性を有するペプチドまたはポリペプチド」は、抗体のVH、VL、またはそれらのCDR1、2、もしくは3を含んで構成される抗体である。複数のCDRを含むペプチドは、直接または適当なペプチドリンカーを介して結合させることができる。
As used herein, a "peptide or polypeptide having antigen-binding activity" refers to an antibody that is composed of an antibody VH, VL, or CDR1, 2, or 3 thereof. A peptide containing multiple CDRs can be linked directly or via a suitable peptide linker.
本開示において使用されるFv抗体、Fab抗体、F(ab’)2抗体、Fab’抗体、scFv抗体、ダイアボディー(diabody)、dsFv抗体、抗原結合性を有するペプチドまたはポリペプチド(以下、「Fv抗体等」と称することもある)の生産方法は特に限定しない。例えば、本開示の実施形態に係る複合体において使用される抗LSR抗体におけるFv抗体等の領域をコードするDNAを発現用ベクターに組み込み、発現用細胞を用いて生産できる。または、Fmoc法(フルオレニルメチルオキシカルボニル法)、tBOC法(t-ブチルオキシカルボニル法)などの化学合成法によって生産してもよい。なお本開示の一実施形態に係る抗原結合性断片は、上記Fv抗体等の1種以上であってもよい。
The method of producing the Fv antibody, Fab antibody, F(ab') 2 antibody, Fab' antibody, scFv antibody, diabody, dsFv antibody, antigen-binding peptide or polypeptide (hereinafter sometimes referred to as "Fv antibody, etc.") used in the present disclosure is not particularly limited. For example, DNA encoding a region of the Fv antibody, etc. in the anti-LSR antibody used in the complex according to an embodiment of the present disclosure can be inserted into an expression vector and produced using an expression cell. Alternatively, it may be produced by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBOC method (t-butyloxycarbonyl method). The antigen-binding fragment according to an embodiment of the present disclosure may be one or more of the above Fv antibodies, etc.
本明細書において「キメラ抗体」は、例えば、異種生物間における抗体の可変領域と、抗体の定常領域とを連結したもので、遺伝子組換え技術によって構築できる。マウス-ヒトキメラ抗体は、例えば、"Roguska et al., Proc Natl Acad Sci USA. 1994 Feb 1;91(3):969-973."に記載の方法で作製できる。マウス-ヒトキメラ抗体を作製するための基本的な方法は、例えば、クローン化されたcDNAに存在するマウスリーダー配列および可変領域配列を、哺乳類細胞の発現ベクター中にすでに存在するヒト抗体定常領域をコードする配列に連結する。または、クローン化されたcDNAに存在するマウスリーダー配列および可変領域配列をヒト抗体定常領域をコードする配列に連結した後、哺乳類細胞発現ベクターに連結してもよい。ヒト抗体定常領域の断片は、任意のヒト抗体のH鎖定常領域およびヒト抗体のL鎖定常領域のものとすることができ、例えばヒトH鎖のものについてはCγ1、Cγ2、Cγ3またはCγ4を、L鎖のものについてはCλまたはCκを各々挙げることができる。
As used herein, a "chimeric antibody" refers to, for example, an antibody variable region and an antibody constant region linked between different organisms, and can be constructed by recombinant gene technology. Mouse-human chimeric antibodies can be produced, for example, by the method described in "Roguska et al., Proc Natl Acad Sci USA. 1994 Feb 1;91(3):969-973." The basic method for producing mouse-human chimeric antibodies is, for example, to link mouse leader sequences and variable region sequences present in cloned cDNA to sequences encoding human antibody constant regions already present in a mammalian cell expression vector. Alternatively, mouse leader sequences and variable region sequences present in cloned cDNA may be linked to sequences encoding human antibody constant regions, and then linked to a mammalian cell expression vector. The fragment of the human antibody constant region can be any human antibody H chain constant region or human antibody L chain constant region, for example, Cγ1, Cγ2, Cγ3, or Cγ4 for the human H chain, and Cλ or Cκ for the L chain.
本明細書において「ヒト化抗体」は、例えば、非ヒト種由来の1つ以上のCDR、およびヒト免疫グロブリン由来のフレームワーク領域(FR)、さらにヒト免疫グロブリン由来の定常領域を有し、所望の抗原に結合する抗体である。抗体のヒト化は、当該技術分野で既知の種々の手法を使用して実施可能である(Almagro et al., Front Biosci. 2008 Jan 1;13: 1619-1633.)。例えば、CDRグラフティング(Ozaki et al.,Blood. 1999 Jun 1;93(11): 3922-3930.)、Re-surfacing(Roguska et al., Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):969-973.)、またはFRシャッフル(Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.)などが挙げられる。抗原結合を改変するために(好ましくは改善するために)、ヒトFR領域のアミノ酸残基は、CDRドナー抗体からの対応する残基と置換してもよい。このFR置換は、当該技術分野で周知の方法によって実施可能である(Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.)。例えば、CDRとFR残基の相互作用のモデリングによって抗原結合に重要なFR残基を同定してもよい。または、配列比較によって、特定の位置で異常なFR残基を同定してもよい。
As used herein, a "humanized antibody" is an antibody that has, for example, one or more CDRs from a non-human species, a framework region (FR) from a human immunoglobulin, and a constant region from a human immunoglobulin, and that binds to a desired antigen. Antibody humanization can be performed using various techniques known in the art (Almagro et al., Front Biosci. 2008 Jan 1;13: 1619-1633.). For example, CDR grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):969-973.), or FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.) can be used. To alter (preferably improve) antigen binding, amino acid residues in the human FR region may be replaced with corresponding residues from the CDR donor antibody. This FR substitution can be performed by methods well known in the art (Riechmann et al., Nature. 1988 Mar 24;332(6162):323-327.). For example, FR residues important for antigen binding can be identified by modeling the interactions of CDR and FR residues. Alternatively, unusual FR residues at specific positions can be identified by sequence comparison.
本明細書において「ヒト抗体」は、例えば、抗体を構成する重鎖の可変領域および定常領域、軽鎖の可変領域および定常領域を含む領域が、ヒトイムノグロブリンをコードする遺伝子に由来する抗体である。主な作製方法としてはヒト抗体作製用トランスジェニックマウス法、ファージディスプレイ法などがある。ヒト抗体作製用トランスジェニックマウス法では、内因性Igをノックアウトしたマウスに機能的なヒトのIg遺伝子を導入すれば、マウス抗体の代わりに多様な抗原結合能を持つヒト抗体が産生される。さらにこのマウスを免疫すればヒトモノクローナル抗体を従来のハイブリドーマ法で得ることが可能である。例えば、"Lonberg et al., Int Rev Immunol. 1995;13(1):65-93."に記載の方法で作製できる。ファージディスプレイ法は、典型的には大腸菌ウイルスの一つであるM13やT7などの繊維状ファージのコートタンパク質(g3p、g10p等)のN末端側にファージの感染性を失わないよう外来遺伝子を融合タンパク質として発現させるシステムである。例えば、"Vaughan et al., Nat Biotechnol. 1996 Mar;14(3):309-314."に記載の方法で作製できる。
In this specification, a "human antibody" refers to an antibody whose heavy chain variable and constant regions and light chain variable and constant regions are derived from genes encoding human immunoglobulin. The main methods for producing human antibodies include the transgenic mouse method for producing human antibodies and the phage display method. In the transgenic mouse method for producing human antibodies, if a functional human Ig gene is introduced into a mouse whose endogenous Ig has been knocked out, human antibodies with diverse antigen-binding abilities are produced instead of mouse antibodies. Furthermore, if this mouse is immunized, human monoclonal antibodies can be obtained by the conventional hybridoma method. For example, they can be produced by the method described in "Lonberg et al., Int Rev Immunol. 1995;13(1):65-93." The phage display method is a system in which a foreign gene is expressed as a fusion protein on the N-terminus of the coat protein (g3p, g10p, etc.) of a filamentous phage, such as M13 or T7, which is typically an E. coli virus, so as not to lose the infectivity of the phage. For example, it can be produced by the method described in "Vaughan et al., Nat Biotechnol. 1996 Mar;14(3):309-314."
また抗体は、CDR-grafting(Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.)によって任意の抗体に本開示の実施形態に係る抗LSR抗体の重鎖CDRまたは軽鎖CDRをグラフティングすることで作製してもよい。または、本開示の実施形態に係る抗LSR抗体の重鎖CDRまたは軽鎖CDRをコードするDNAと、公知のヒトまたはヒト以外の生物由来の抗体の、重鎖CDRまたは軽鎖CDRを除く領域をコードするDNAとを、当該技術分野で公知の方法に従ってベクターに連結後、公知の細胞を使用して発現させることによって得ることができる。このとき、抗LSR抗体の標的抗原への作用効率を上げるために、当該分野で公知の方法(例えば、抗体のアミノ酸残基をランダムに変異させ、反応性の高いものをスクリーニングする方法、またはファージディスプレイ法等)を用いて、重鎖CDRまたは軽鎖CDRを除く領域を最適化してもよい。また、例えば、FRシャッフル(Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.)、またはバーニヤゾーンのアミノ酸残基またはパッケージング残基を置換する方法(特開2006-241026、またはFooteet al., J Mol Biol.1992 Mar 20;224(2):487-499.)を用いて、FR領域を最適化してもよい。
The antibody may also be prepared by grafting the heavy chain CDR or light chain CDR of the anti-LSR antibody according to the embodiment of the present disclosure onto any antibody by CDR-grafting (Ozaki et al., Blood. 1999 Jun 1;93(11):3922-3930.). Alternatively, the antibody may be obtained by linking DNA encoding the heavy chain CDR or light chain CDR of the anti-LSR antibody according to the embodiment of the present disclosure and DNA encoding a region excluding the heavy chain CDR or light chain CDR of a known antibody derived from a human or non-human organism to a vector according to a method known in the art, and then expressing the vector using a known cell. In this case, in order to increase the efficiency of the action of the anti-LSR antibody on the target antigen, the region excluding the heavy chain CDR or light chain CDR may be optimized using a method known in the art (e.g., a method of randomly mutating amino acid residues of an antibody and screening for one with high reactivity, or a phage display method, etc.). In addition, the FR region may be optimized, for example, by using FR shuffling (Damschroder et al., Mol Immunol. 2007 Apr;44(11):3049-3060. Epub 2007 Jan 22.) or a method of substituting amino acid residues or packaging residues in the vernier zone (JP Patent Publication 2006-241026, or Footeet et al., J Mol Biol. 1992 Mar 20;224(2):487-499.).
本明細書において「重鎖」は、典型的には、全長抗体の主な構成要素である。重鎖は、通常、軽鎖とジスルフィド結合および非共有結合によって結合している。重鎖のN末端側のドメインには、同種の同一クラスの抗体でもアミノ酸配列が一定しない可変領域(VH)と呼ばれる領域が存在し、一般的に、VHが抗原に対する特異性、親和性に大きく寄与していることが知られている。例えば、"Reiter et al., J Mol Biol. 1999 Jul 16;290(3):685-98."にはVHのみの分子を作製したところ、抗原と特異的に、高い親和性で結合したことが記載されている。さらに、"Wolfson W, Chem Biol. 2006 Dec;13(12):1243-1244."には、ラクダの抗体の中には、軽鎖を持たない重鎖のみの抗体が存在していることが記載されている。
Heavy chains, as used herein, are typically the main components of full-length antibodies. Heavy chains are usually bound to light chains by disulfide bonds and non-covalent bonds. The N-terminal domain of heavy chains contains a region called a variable region (VH), whose amino acid sequence is not constant even in antibodies of the same species and class, and it is generally known that VH contributes greatly to specificity and affinity for antigens. For example, "Reiter et al., J Mol Biol. 1999 Jul 16;290(3):685-98." describes that when a molecule consisting of only VH was produced, it bound specifically to antigens with high affinity. Furthermore, "Wolfson W, Chem Biol. 2006 Dec;13(12):1243-1244." describes that camel antibodies contain only heavy chains and no light chains.
本明細書において「CDR(相補性決定領域)」は、抗体において、実際に抗原に接触して結合部位を形成している領域である。一般的にCDRは、抗体のFv(可変領域:重鎖可変領域(VH)および軽鎖可変領域(VL)を含む)上に位置している。また一般的にCDRは、5~30アミノ酸残基程度からなるCDR1、CDR2、CDR3が存在する。そして、特に重鎖のCDRが抗体の抗原への結合に寄与していることが知られている。またCDRの中でも、CDR3が抗体の抗原への結合における寄与が最も高いことが知られている。例えば、"Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128"には、重鎖CDR3を改変させることで抗体の結合能を上昇させたことが記載されている。CDRは1または数個(例えば、2個、3個等)の改変(例えば、保存的改変)をしても結合力が保持されることが知られており、本開示においてこれらの改変体も特定のCDRの範囲内であることが理解される。CDR以外のFv領域はフレームワーク領域(FR)と呼ばれ、FR1、FR2、FR3およびFR4からなり、抗体間で比較的よく保存されている(Kabatet al.,「Sequence of Proteins of Immunological Interest」US Dept. Health and Human Services,1983.)。即ち、抗体の反応性を特徴付ける要因はCDRにあり、特に重鎖CDRにあるといえる。
In this specification, "CDR (complementarity determining region)" refers to the region of an antibody that actually contacts an antigen and forms a binding site. Generally, CDR is located on the Fv (variable region: including the heavy chain variable region (VH) and the light chain variable region (VL)) of an antibody. Generally, CDR consists of CDR1, CDR2, and CDR3, each consisting of about 5 to 30 amino acid residues. It is known that the CDR of the heavy chain in particular contributes to the binding of the antibody to the antigen. It is also known that among the CDRs, CDR3 contributes most to the binding of the antibody to the antigen. For example, "Willy et al., Biochemical and Biophysical Research Communications Volume 356, Issue 1, 27 April 2007, Pages 124-128" describes that the binding ability of an antibody was increased by modifying the heavy chain CDR3. It is known that one or several (e.g., two, three, etc.) CDRs can be modified (e.g., conservatively modified) while retaining binding ability, and in this disclosure, these modifications are also understood to be within the scope of a particular CDR. The Fv region other than the CDRs is called the framework region (FR), which consists of FR1, FR2, FR3, and FR4, and is relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest," US Dept. Health and Human Services, 1983.). In other words, it can be said that the factors that characterize the reactivity of an antibody are the CDRs, and in particular the heavy chain CDRs.
CDRの定義およびその位置を決定する方法は複数報告されている。例えば、Kabatの定義(Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991))、またはChothiaの定義(Chothiaet al., J. Mol. Biol.,1987;196:901-917)を採用してもよい。本開示の一実施形態においては、Kabatの定義を好適な例として採用するが、必ずしもこれに限定されない。また、場合によっては、Kabatの定義とChothiaの定義の両方を考慮して決定しても良く、例えば、各々の定義によるCDRの重複部分を、または各々の定義によるCDRの両方を含んだ部分をCDRとすることもできる。そのような方法の具体例としては、Kabatの定義とChothiaの定義の折衷案である、Oxford Molecular's AbM antibody modeling softwareを用いたMartinらの方法(Proc.Natl.Acad.Sci.USA, 1989;86:9268-9272)がある。このようなCDRの情報を用いて、本開示に使用されうる変異体を生産することができる。このような抗体の変異体では、もとの抗体のフレームワークに1または数個(例えば、2個、3個、4個、5個、6個、7個、8個、9個、10個)の置換、付加もしくは欠失を含むが、該CDRには変異を含まないように生産することができる。
Several methods for determining the definition and location of CDRs have been reported. For example, the Kabat definition (Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) or Chothia's definition (Chothia et al., J. Mol. Biol., 1987;196:901-917) may be adopted. In one embodiment of the present disclosure, the Kabat definition is adopted as a preferred example, but is not necessarily limited to this. In some cases, the definition may be determined taking into consideration both the Kabat definition and the Chothia definition. For example, the overlapping portion of the CDR according to each definition, or the portion including both the CDRs according to each definition, may be the CDR. A specific example of such a method is the method of Martin et al. (Proc. Natl. Acad. Sci. USA, 1989;86:9268-9272) using Oxford Molecular's AbM antibody modeling software, which is a compromise between the Kabat and Chothia definitions. Such CDR information can be used to produce variants that can be used in the present disclosure. Such antibody variants can be produced that contain one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) substitutions, additions, or deletions in the framework of the original antibody, but do not contain mutations in the CDRs.
本明細書において「抗原」(antigen)とは、抗体分子によって特異的に結合され得る任意の基質をいう。本明細書において「免疫原」(immunogen)とは、抗原特異的免疫応答を生じるリンパ球活性化を開始し得る抗原をいう。本明細書において「エピトープ」または「抗原決定基」とは、抗体またはリンパ球レセプターが結合する抗原分子中の部位をいう。エピトープを決定する方法は、当該分野において周知であり、そのようなエピトープは、核酸またはアミノ酸の一次配列が提供されると、当業者はそのような周知慣用技術を用いて決定することができる。本開示の抗体は、エピトープが同じであれば、他の配列を有する抗体であっても同様に利用することができることが理解される。
As used herein, "antigen" refers to any substance capable of being specifically bound by an antibody molecule. As used herein, "immunogen" refers to an antigen capable of initiating lymphocyte activation resulting in an antigen-specific immune response. As used herein, "epitope" or "antigenic determinant" refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by one of skill in the art using such well-known and conventional techniques when provided with the primary sequence of nucleic acid or amino acid. It is understood that the antibodies of the present disclosure can be similarly utilized with antibodies having other sequences, so long as the epitopes are the same.
本明細書において使用される抗体は、擬陽性が減じられる限り、どのような特異性の抗体を用いても良いことが理解される。従って、本開示において用いられる抗体は、ポリクローナル抗体であってもよく、モノクローナル抗体であってもよい。
It is understood that the antibodies used in this specification may have any specificity as long as false positives are reduced. Thus, the antibodies used in this disclosure may be polyclonal or monoclonal antibodies.
本明細書において「手段」とは、ある目的(例えば、検出、診断、治療)を達成する任意の道具となり得るものをいい、特に、本明細書では、「選択的に認識する手段」とは、ある対象を他のものとは異なって認識することができる手段をいう。
In this specification, the term "means" refers to any tool that can achieve a certain purpose (e.g., detection, diagnosis, treatment), and in particular, in this specification, "selective recognition means" refers to a means that can recognize one object differently from others.
本明細書において使用される「悪性腫瘍」は、例えば、正常な細胞が突然変異を起こして発生する腫瘍を含む。悪性腫瘍は全身のあらゆる臓器や組織から生じ得る。この悪性腫瘍は、例えば、肺癌、食道癌、胃癌、肝臓癌、膵臓癌、腎臓癌、副腎癌、胆道癌、乳癌、大腸癌、小腸癌、卵巣癌、子宮癌、膀胱癌、前立腺癌、尿管癌、腎盂癌、尿管癌、陰茎癌、精巣癌、脳腫瘍、中枢神経系の癌、末梢神経系の癌、頭頸部癌、グリオーマ、多形性膠芽腫、皮膚癌、メラノーマ、甲状腺癌、唾液腺癌、悪性リンパ腫、癌腫、肉腫、および血液悪性腫瘍からなる群から選ばれる1種以上を含む。ここで、卵巣癌は、例えば、卵巣漿、液性腺癌、または卵巣明細胞線癌を含む。子宮癌は、例えば、子宮内膜癌、または子宮頸癌を含む。頭頸部癌は、例えば、口腔癌、咽頭癌、喉頭癌、鼻腔癌、副鼻腔癌、唾液腺癌、または甲状腺癌を含む。肺癌は、例えば、非小細胞肺癌、または小細胞肺癌を含む。また悪性腫瘍は、LSR陽性であってもよい。
As used herein, the term "malignant tumor" includes, for example, tumors that develop as a result of mutation of normal cells. Malignant tumors can arise from any organ or tissue in the body. Examples of malignant tumors include lung cancer, esophageal cancer, gastric cancer, liver cancer, pancreatic cancer, kidney cancer, adrenal cancer, biliary tract cancer, breast cancer, colon cancer, small intestine cancer, ovarian cancer, uterine cancer, bladder cancer, prostate cancer, ureter cancer, renal pelvis cancer, ureter cancer, penile cancer, testicular cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, head and neck cancer, glioma, glioblastoma multiforme, skin cancer, melanoma, thyroid cancer, salivary gland cancer, malignant lymphoma, carcinoma, sarcoma, and hematological malignant tumors. Here, ovarian cancer includes, for example, ovarian serous, liquid adenocarcinoma, or ovarian clear cell adenocarcinoma. Uterine cancer includes, for example, endometrial cancer or cervical cancer. Head and neck cancers include, for example, oral cancer, pharyngeal cancer, laryngeal cancer, nasal cancer, paranasal sinus cancer, salivary gland cancer, or thyroid cancer. Lung cancers include, for example, non-small cell lung cancer or small cell lung cancer. The malignant tumor may also be LSR positive.
悪性腫瘍の中でも漿液性腺癌は、非常に進行の早い癌であり、市販の抗癌剤でも癌を全て消し去るのは難しい。さらに、再発した場合は、市販の抗癌剤がほとんど効かない。また、明細胞線癌は、市販の抗癌剤ではほとんど治療効果が期待できない。一方で、本開示の実施形態に係る抗LSR抗体は、漿液性腺癌および明細胞線癌の新しい治療薬となり得る。
Among malignant tumors, serous adenocarcinoma progresses very quickly, and it is difficult to completely eliminate the cancer even with commercially available anticancer drugs. Furthermore, in cases of recurrence, commercially available anticancer drugs are almost ineffective. Furthermore, for clear cell adenocarcinoma, commercially available anticancer drugs are unlikely to have any therapeutic effect. On the other hand, the anti-LSR antibody according to the embodiment of the present disclosure could become a new therapeutic agent for serous adenocarcinoma and clear cell adenocarcinoma.
本開示の一実施形態において「LSR陽性悪性腫瘍」は、LSRを有意または過剰に発現している悪性腫瘍を含む。悪性腫瘍がLSR陽性かどうかは、例えば、RT-PCR、ウェスタンブロット、または免疫組織化学染色法で評価してもよい。また、ウェスタンブロットに悪性腫瘍細胞の総タンパク質を供し、目視でLSRに相当するバンド(例えば、649aa付近のバンド)が確認できた場合に、LSR陽性と判断してもよい。または、患者由来の悪性腫瘍細胞のLSR発現量が、正常細胞の場合に比べて有意に大きい場合に、LSR陽性と判断してもよい。LSR陽性であることを正確に診断することによって、より最適な投薬を実現する観点からは、抗LSR抗体を使用してLSRの発現を検査することが好ましい。
In one embodiment of the present disclosure, "LSR-positive malignant tumor" includes malignant tumors that significantly or excessively express LSR. Whether a malignant tumor is LSR-positive may be evaluated, for example, by RT-PCR, Western blot, or immunohistochemical staining. In addition, when total protein of malignant tumor cells is subjected to Western blot and a band corresponding to LSR (e.g., a band around 649aa) can be visually confirmed, it may be determined to be LSR-positive. Alternatively, it may be determined to be LSR-positive when the amount of LSR expression in malignant tumor cells derived from a patient is significantly greater than that in normal cells. From the viewpoint of achieving more optimal medication by accurately diagnosing LSR positivity, it is preferable to test the expression of LSR using an anti-LSR antibody.
本明細書において「被験体(者)」とは、本開示の診断または検出、あるいは治療等の対象となる対象(例えば、ヒト等の生物または生物から取り出した細胞、血液、血清等)をいう。
As used herein, the term "subject" refers to an object that is the subject of diagnosis, detection, or treatment according to the present disclosure (e.g., an organism such as a human, or cells, blood, serum, etc. extracted from an organism).
本明細書において「試料」とは、被験体等から得られた任意の物質をいい、例えば、血液、血清、血漿、唾液、尿、涙液、脳脊髄液等が含まれる。当業者は本明細書の記載をもとに適宜好ましい試料を選択することができる。
As used herein, the term "sample" refers to any substance obtained from a subject, etc., including, for example, blood, serum, plasma, saliva, urine, tears, cerebrospinal fluid, etc. Those skilled in the art will be able to select an appropriate and preferred sample based on the description in this specification.
本明細書において「薬剤」、「剤」または「因子」(いずれも英語ではagentに相当する)は、広義には、交換可能に使用され、意図する目的を達成することができる限りどのような物質または他の要素(例えば、光、放射能、熱、電気などのエネルギー)でもあってもよい。そのような物質としては、例えば、タンパク質、ポリペプチド、オリゴペプチド、ペプチド、ポリヌクレオチド、オリゴヌクレオチド、ヌクレオチド、核酸(例えば、cDNA、ゲノムDNAのようなDNA、mRNAのようなRNAを含む)、ポリサッカリド、オリゴサッカリド、脂質、有機低分子(例えば、ホルモン、リガンド、情報伝達物質、有機低分子、コンビナトリアルケミストリで合成された分子、医薬品として利用され得る低分子(例えば、低分子リガンドなど)など)、これらの複合分子が挙げられるがそれらに限定されない。ポリヌクレオチドに対して特異的な因子としては、代表的には、そのポリヌクレオチドの配列に対して一定の配列相同性を(例えば、70%以上の配列同一性)もって相補性を有するポリヌクレオチド、プロモーター領域に結合する転写因子のようなポリペプチドなどが挙げられるがそれらに限定されない。ポリペプチドに対して特異的な因子としては、代表的には、そのポリペプチドに対して特異的に指向された抗体またはその誘導体あるいはその類似物(例えば、単鎖抗体)、そのポリペプチドがレセプターまたはリガンドである場合の特異的なリガンドまたはレセプター、そのポリペプチドが酵素である場合、その基質などが挙げられるがそれらに限定されない。
In this specification, the terms "drug", "agent" or "factor" (all of which correspond to the English term agent) are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as it can achieve the intended purpose. Such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic small molecules (e.g., hormones, ligands, signaling substances, organic small molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands, etc.)), and composite molecules thereof. Factors specific to a polynucleotide typically include, but are not limited to, polynucleotides that have a certain sequence homology (e.g., 70% or more sequence identity) with respect to the sequence of the polynucleotide and polypeptides such as transcription factors that bind to promoter regions. Representative factors specific to a polypeptide include, but are not limited to, an antibody or a derivative or analog thereof (e.g., a single-chain antibody) specifically directed against the polypeptide, a specific ligand or receptor when the polypeptide is a receptor or ligand, and a substrate when the polypeptide is an enzyme.
本明細書において「治療」とは、ある疾患または障害(例えば、悪性腫瘍)について、そのような状態になった場合に、そのような疾患または障害の悪化を防止、好ましくは、現状維持、より好ましくは、軽減、さらに好ましくは消退させることをいい、患者の疾患、もしくは疾患に伴う1つ以上の症状の、症状改善効果あるいは予防効果を発揮しうることを含む。事前に診断を行って適切な治療を行うことは「コンパニオン治療」といい、そのための診断薬を「コンパニオン診断薬」ということがある。
In this specification, "treatment" refers to preventing the worsening of a certain disease or disorder (e.g., malignant tumor) when that condition occurs, preferably maintaining the status quo, more preferably alleviating, and even more preferably causing the disease or disorder to recede, and includes exerting a symptom-improving or preventive effect on the patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is called "companion treatment," and diagnostic agents used for this purpose are sometimes called "companion diagnostic agents."
本明細書において「治療薬(剤)」とは、広義には、目的の状態(例えば、悪性腫瘍等の疾患など)を治療できるあらゆる薬剤をいう。本開示の一実施形態において「治療薬」は、有効成分と、薬理学的に許容される1つもしくはそれ以上の担体とを含む医薬組成物であってもよい。医薬組成物は、例えば有効成分と上記担体とを混合し、製剤学の技術分野において知られる任意の方法により製造できる。また治療薬は、治療のために用いられる物であれば使用形態は限定されず、有効成分単独であってもよいし、有効成分と任意の成分との混合物であってもよい。また上記担体の形状は特に限定されず、例えば、固体または液体(例えば、緩衝液)であってもよい。なお悪性腫瘍の治療薬は、悪性腫瘍の予防のために用いられる薬物(予防薬)、または悪性腫瘍細胞の増殖抑制剤を含む。
In this specification, the term "therapeutic agent" refers broadly to any drug capable of treating a target condition (e.g., a disease such as a malignant tumor). In one embodiment of the present disclosure, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition may be produced, for example, by mixing the active ingredient with the carrier and using any method known in the technical field of pharmaceutical formulations. The therapeutic agent may be in any form as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and any other ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The therapeutic agent for malignant tumors includes drugs used for preventing malignant tumors (prophylactic drugs) or agents for suppressing the proliferation of malignant tumor cells.
本明細書において「予防」とは、ある疾患または障害(例えば、悪性腫瘍)について、そのような状態になる前に、そのような状態にならないようにすることをいう。本開示の薬剤を用いて、診断を行い、必要に応じて本開示の薬剤を用いて例えば、悪性腫瘍等の予防をするか、あるいは予防のための対策を講じることができる。
As used herein, "prevention" refers to preventing a certain disease or disorder (e.g., malignant tumor) from occurring before that state occurs. A diagnosis can be made using the drug disclosed herein, and if necessary, the drug disclosed herein can be used to prevent, for example, malignant tumors, or preventive measures can be taken.
本明細書において「予防薬(剤)」とは、広義には、目的の状態(例えば、悪性腫瘍等の疾患など)を予防できるあらゆる薬剤をいう。
In this specification, the term "prophylactic drug" refers broadly to any drug that can prevent a target condition (e.g., a disease such as a malignant tumor).
本明細書において「相互作用」とは、2つの物質についていうとき、一方の物質と他方の物質との間で力(例えば、分子間力(ファンデルワールス力)、水素結合、疎水性相互作用など)を及ぼしあうこという。通常、相互作用をした2つの物質は、会合または結合している状態にある。本開示の検出、検査および診断は、このような相互作用を利用して実現することができる。
As used herein, "interaction" refers to the mutual exertion of force (e.g., intermolecular forces (van der Waals forces), hydrogen bonds, hydrophobic interactions, etc.) between one substance and the other. Usually, two substances that have interacted are in an associated or bonded state. The detection, testing, and diagnosis disclosed herein can be achieved by utilizing such interactions.
本明細書中で使用される用語「結合」は、2つの物質の間、あるいはそれらの組み合わせの間での、物理的相互作用または化学的相互作用を意味する。結合には、イオン結合、非イオン結合、水素結合、ファンデルワールス結合、疎水性相互作用などが含まれる。物理的相互作用(結合)は、直接的または間接的であり得、間接的なものは、別のタンパク質または化合物の効果を介するかまたは起因する。直接的な結合とは、別のタンパク質または化合物の効果を介してもまたはそれらに起因しても起こらず、他の実質的な化学中間体を伴わない、相互作用をいう。
As used herein, the term "binding" refers to a physical or chemical interaction between two substances or a combination thereof. Binding includes ionic bonds, non-ionic bonds, hydrogen bonds, van der Waals bonds, hydrophobic interactions, and the like. Physical interactions (binding) can be direct or indirect, where indirect is through or due to the effect of another protein or compound. Direct binding refers to an interaction that does not occur through or due to the effect of another protein or compound and does not involve other substantial chemical intermediates.
従って、本明細書においてポリヌクレオチドまたはポリペプチドなどの生物学的因子に対して「特異的に」相互作用する(または結合する)「因子」(または、薬剤、検出剤等)とは、そのポリヌクレオチドまたはそのポリペプチドなどの生物学的因子に対する親和性が、他の無関連の(特に、同一性が30%未満の)ポリヌクレオチドまたはポリペプチドに対する親和性よりも、代表的には同等またはより高いか、好ましくは有意に(例えば、統計学的に有意に)高いものを包含する。そのような親和性は、例えば、ハイブリダイゼーションアッセイ、結合アッセイなどによって測定することができる。
Thus, as used herein, an "agent" (or drug, detection agent, etc.) that "specifically" interacts with (or binds to) a biological agent such as a polynucleotide or polypeptide includes one whose affinity for the biological agent, such as the polynucleotide or polypeptide, is typically equal to or higher than, and preferably significantly (e.g., statistically significantly) higher than, its affinity for other unrelated polynucleotides or polypeptides (e.g., those with less than 30% identity). Such affinity can be measured, for example, by hybridization assays, binding assays, etc.
本明細書において第一の物質または因子が第二の物質または因子に「特異的に」相互作用する(または結合する)とは、第一の物質または因子が、第二の物質または因子に対して、第二の物質または因子以外の物質または因子(特に、第二の物質または因子を含む試料中に存在する他の物質または因子)に対するよりも高い親和性で相互作用する(または結合する)ことをいう。物質または因子について特異的な相互作用(または結合)としては、例えば、核酸におけるハイブリダイゼーション、タンパク質における抗原抗体反応、酵素-基質反応など、核酸およびタンパク質の反応、タンパク質-脂質相互作用、核酸-脂質相互作用などが挙げられるがそれらに限定されない。従って、物質または因子がともに核酸である場合、第一の物質または因子が第二の物質または因子に「特異的に相互作用する」ことには、第一の物質または因子が、第二の物質または因子に対して少なくとも一部に相補性を有することが包含される。また例えば、物質または因子がともにタンパク質である場合、第一の物質または因子が第二の物質または因子に「特異的に」相互作用する(または結合する)こととしては、例えば、抗原抗体反応による相互作用、レセプター-リガンド反応による相互作用、酵素-基質相互作用などが挙げられるがそれらに限定されない。2種類の物質または因子がタンパク質および核酸を含む場合、第一の物質または因子が第二の物質または因子に「特異的に」相互作用する(または結合する)ことには、抗体と、その抗原との間の相互作用(または結合)が包含される。このような特異的な相互作用または結合の反応を利用することにより、試料中の対象物の検出または定量を行うことができる。
As used herein, a first substance or factor "specifically" interacts with (or binds to) a second substance or factor means that the first substance or factor interacts with (or binds to) the second substance or factor with a higher affinity than it does with substances or factors other than the second substance or factor (particularly other substances or factors present in a sample containing the second substance or factor). Examples of specific interactions (or binding) for a substance or factor include, but are not limited to, hybridization in nucleic acids, antigen-antibody reactions in proteins, enzyme-substrate reactions, and other reactions between nucleic acids and proteins, protein-lipid interactions, and nucleic acid-lipid interactions. Thus, when both substances or factors are nucleic acids, "specifically interacting" with a first substance or factor includes the first substance or factor having at least partial complementarity to the second substance or factor. For example, when both substances or factors are proteins, examples of "specific" interaction (or binding) of a first substance or factor with a second substance or factor include, but are not limited to, interactions due to antigen-antibody reactions, interactions due to receptor-ligand reactions, enzyme-substrate interactions, etc. When two types of substances or factors include proteins and nucleic acids, "specific" interaction (or binding) of a first substance or factor with a second substance or factor includes interactions (or binding) between an antibody and its antigen. By utilizing such specific interactions or binding reactions, it is possible to detect or quantify a target substance in a sample.
本明細書においてポリヌクレオチドまたはポリペプチド発現の「検出」または「定量」は、例えば、検出剤、検査剤または診断剤への結合または相互作用を含む、mRNAの測定および免疫学的測定方法を含む適切な方法を用いて達成され得る。分子生物学的測定方法としては、例えば、ノーザンブロット法、ドットブロット法またはPCR法などが例示される。免疫学的測定方法としては、例えば、方法としては、マイクロタイタープレートを用いるELISA法、RIA法、蛍光抗体法、発光イムノアッセイ(LIA)、免疫沈降法(IP)、免疫拡散法(SRID)、免疫比濁法(TIA)、ウェスタンブロット法、免疫組織染色法などが例示される。また、定量方法としては、ELISA法またはRIA法などが例示される。アレイ(例えば、DNAアレイ、プロテインアレイ)を用いた遺伝子解析方法によっても行われ得る。DNAアレイについては、(秀潤社編、細胞工学別冊「DNAマイクロアレイと最新PCR法」)に広く概説されている。プロテインアレイについては、NatGenet.2002 Dec;32 Suppl:526-532に詳述されている。遺伝子発現の分析法としては、上述に加えて、RT-PCR、RACE法、SSCP法、免疫沈降法、two-hybridシステム、invitro翻訳などが挙げられるがそれらに限定されない。そのようなさらなる分析方法は、例えば、ゲノム解析実験法・中村祐輔ラボ・マニュアル、編集・中村祐輔羊土社(2002)などに記載されており、本明細書においてそれらの記載はすべて参考として援用される。
As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression may be accomplished using any suitable method, including, for example, measurement of mRNA and immunological measurement methods, including binding or interaction with a detection agent, test agent or diagnostic agent. Examples of molecular biological measurement methods include, for example, Northern blot, dot blot, or PCR. Examples of immunological measurement methods include, for example, ELISA, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunodiffusion (SRID), immunoturbidimetric (TIA), Western blot, and immunohistochemical staining using microtiter plates. Examples of quantification methods include ELISA or RIA. Genetic analysis may also be performed using arrays (e.g., DNA arrays, protein arrays). DNA arrays are broadly reviewed in "DNA Microarrays and the Latest PCR Methods," a special edition of Cell Engineering, edited by Shujunsha. Protein arrays are reviewed in NatGenet. 2002 Dec;32 Suppl:526-532. In addition to the above, gene expression analysis methods include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like. Such further analysis methods are described, for example, in Genome Analysis Experimental Methods: Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke Yodosha (2002), and all descriptions therein are incorporated by reference.
本明細書において「発現量」とは、目的の細胞、組織などにおいて、ポリペプチドまたはmRNA等が発現される量をいう。そのような発現量としては、本開示の抗体を用いてELISA法、RIA法、蛍光抗体法、ウェスタンブロット法、免疫組織染色法などの免疫学的測定方法を含む任意の適切な方法により評価される本開示ポリペプチドのタンパク質レベルでの発現量、またはノーザンブロット法、ドットブロット法、PCR法などの分子生物学的測定方法を含む任意の適切な方法により評価される本開示において使用されるポリペプチドのmRNAレベルでの発現量が挙げられる。「発現量の変化」とは、上記免疫学的測定方法または分子生物学的測定方法を含む任意の適切な方法により評価される本開示において使用されるポリペプチドのタンパク質レベルまたはmRNAレベルでの発現量が増加あるいは減少することを意味する。あるマーカーの発現量を測定することによって、マーカーに基づく種々の検出または診断を行うことができる。
As used herein, "expression level" refers to the amount of a polypeptide or mRNA, etc., expressed in a cell, tissue, etc. of interest. Examples of such expression level include the expression level at the protein level of the polypeptide of the present disclosure, evaluated by any appropriate method, including immunological measurement methods such as ELISA, RIA, fluorescent antibody technique, Western blotting, and immunohistochemical staining, using an antibody of the present disclosure, or the expression level at the mRNA level of the polypeptide used in the present disclosure, evaluated by any appropriate method, including molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. "Change in expression level" refers to an increase or decrease in the expression level at the protein level or mRNA level of the polypeptide used in the present disclosure, evaluated by any appropriate method, including the immunological measurement method or molecular biological measurement method. By measuring the expression level of a certain marker, various detections or diagnoses based on the marker can be performed.
本明細書において、活性、発現産物(例えば、タンパク質、転写物(RNAなど))の「減少」または「抑制」あるいはその類義語は、特定の活性、転写物またはタンパク質の量、質または効果における減少、または減少させる活性をいう。減少のうち「消失」した場合は、活性、発現産物等が検出限界未満になることをいい、特に「消失」ということがある。本明細書では、「消失」は「減少」または「抑制」に包含される。
As used herein, "reduction" or "suppression" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to a decrease in the quantity, quality, or effect of a particular activity, transcript, or protein, or an activity that causes a decrease. When "elimination" is used as a reduction, it refers to an activity, expression product, etc. becoming below the detection limit, and is sometimes specifically referred to as "elimination." As used herein, "elimination" is encompassed within "reduction" or "suppression."
本明細書において、活性、発現産物(例えば、タンパク質、転写物(RNAなど))の「増加」または「活性化」あるいはその類義語は、特定の活性、転写物またはタンパク質の量、質または効果における増加または増加させる活性をいう。
As used herein, "increase" or "activation" of an activity or expression product (e.g., protein, transcript (RNA, etc.)) or synonyms thereof refers to an increase or increasing activity in the amount, quality or effect of a particular activity, transcript or protein.
本明細書において「標識」とは、目的となる分子または物質を他から識別するための存在(例えば、物質、エネルギー、電磁波など)をいう。そのような標識方法としては、RI(ラジオアイソトープ)法、蛍光法、ビオチン法、化学発光法等を挙げることができる。本開示のマーカーまたはそれを捕捉する因子または手段を複数、蛍光法によって標識する場合には、蛍光発光極大波長が互いに異なる蛍光物質によって標識を行う。蛍光発光極大波長の差は、10nm以上であることが好ましい。リガンドを標識する場合、機能に影響を与えないものならば何れも用いることができるが、蛍光物質としては、AlexaTM Fluorが望ましい。AlexaTMFluorは、クマリン、ローダミン、フルオレセイン、シアニンなどを修飾して得られた水溶性の蛍光色素であり、広範囲の蛍光波長に対応したシリーズであり、他の該当波長の蛍光色素に比べ、非常に安定で、明るく、またpH感受性が低い。蛍光極大波長が10nm以上ある蛍光色素の組み合わせとしては、AlexaTM555とAlexaTM633の組み合わせ、AlexaTM488とAlexaTM555の組み合わせ等を挙げることができる。核酸を標識する場合は、その塩基部分と結合できるものであれば何れも用いることができるが、シアニン色素(例えば、CyDyeTMシリーズのCy3、Cy5等)、ローダミン6G試薬、2-アセチルアミノフルオレン(AAF)、AAIF(AAFのヨウ素誘導体)等を使用することが好ましい。蛍光発光極大波長の差が10nm以上である蛍光物質としては、例えば、Cy5とローダミン6G試薬との組み合わせ、Cy3とフルオレセインとの組み合わせ、ローダミン6G試薬とフルオレセインとの組み合わせ等を挙げることができる。本開示では、このような標識を利用して、使用される検出手段に検出され得るように目的とする対象を改変することができる。そのような改変は、当該分野において公知であり、当業者は標識におよび目的とする対象に応じて適宜そのような方法を実施することができる。
In this specification, the term "label" refers to an entity (e.g., a substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method, etc. When a plurality of markers of the present disclosure or factors or means for capturing them are labeled by a fluorescence method, the labels are labeled with fluorescent substances having mutually different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any substance that does not affect the function can be used, but Alexa TM Fluor is preferable as the fluorescent substance. Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. It is very stable, bright, and has low pH sensitivity compared to other fluorescent dyes of the corresponding wavelengths. Examples of combinations of fluorescent dyes having a maximum fluorescence wavelength of 10 nm or more include a combination of Alexa TM 555 and Alexa TM 633, and a combination of Alexa TM 488 and Alexa TM 555. When labeling a nucleic acid, any dye that can bind to the base moiety can be used, but it is preferable to use cyanine dyes (e.g., Cy3 and Cy5 of the CyDye TM series), rhodamine 6G reagent, 2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), and the like. Examples of fluorescent substances having a difference in maximum fluorescence wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. In the present disclosure, such labels can be used to modify the target object so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the target object.
本明細書において使用される場合、「タグ」とは、受容体-リガンドのような特異的認識機構により分子を選別するための物質、より具体的には、特定の物質を結合するための結合パートナーの役割を果たす物質(例えば、ビオチン-アビジン、ビオチン-ストレプトアビジンのような関係を有する)をいい、「標識」の範疇に含まれうる。よって、例えば、タグが結合した特定の物質は、タグ配列の結合パートナーを結合させた基材を接触させることで、この特定の物質を選別することができる。このようなタグまたは標識は、当該分野で周知である。代表的なタグ配列としては、mycタグ、Hisタグ、HA、Aviタグなどが挙げられるが、これらに限定されない。
As used herein, a "tag" refers to a substance for selecting a molecule by a specific recognition mechanism such as a receptor-ligand, more specifically, a substance that acts as a binding partner for binding a specific substance (e.g., having a relationship such as biotin-avidin or biotin-streptavidin), and may be included in the category of a "label." Thus, for example, a specific substance bound to a tag can be selected by contacting the specific substance with a substrate bound to a binding partner of the tag sequence. Such tags or labels are well known in the art. Representative tag sequences include, but are not limited to, myc tags, His tags, HA, and Avi tags.
本明細書において「インビボ」(in vivo)とは、生体の内部をいう。特定の文脈において、「生体内」は、目的とする物質が配置されるべき位置をいう。
As used herein, "in vivo" refers to the inside of a living organism. In certain contexts, "within the organism" refers to the location where a substance of interest is to be placed.
本明細書において「インビトロ」(in vitro)とは、種々の研究目的のために生体の一部分が「生体外に」(例えば、試験管内に)摘出または遊離されている状態をいう。インビボと対照をなす用語である。
In this specification, "in vitro" refers to a state in which a part of a living organism is removed or isolated "outside of a living organism" (e.g., in a test tube) for various research purposes. This term contrasts with in vivo.
本明細書において「キット」とは、通常2つ以上の区画に分けて、提供されるべき部分(例えば、検査薬、診断薬、治療薬、抗体、標識、説明書など)が提供されるユニットをいう。安定性等のため、混合されて提供されるべきでなく、使用直前に混合して使用することが好ましいような組成物の提供を目的とするときに、このキットの形態は好ましい。そのようなキットは、好ましくは、提供される部分(例えば、検査薬、診断薬、治療薬をどのように使用するか、あるいは、試薬をどのように処理すべきかを記載する指示書または説明書を備えていることが有利である。本明細書においてキットが試薬キットとして使用される場合、キットには、通常、検査薬、診断薬、治療薬、抗体等の使い方などを記載した指示書などが含まれる。
As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit is advantageously provided with instructions or instructions describing how to use the parts to be provided (e.g., test agents, diagnostic agents, therapeutic agents, etc.) or how to handle the reagents. When the kit is used as a reagent kit in this specification, the kit usually includes instructions describing how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.
本明細書において「指示書」は、本開示を使用する方法を医師または他の使用者に対する説明を記載したものである。この指示書は、本開示の検出方法、診断薬の使い方、または医薬などを投与することを指示する文言が記載されている。また、指示書には、投与部位として、経口、食道への投与(例えば、注射などによる)することを指示する文言が記載されていてもよい。この指示書は、本開示が実施される国の監督官庁(例えば、日本であれば厚生労働省、米国であれば食品医薬品局(FDA)など)が規定した様式に従って作成され、その監督官庁により承認を受けた旨が明記される。指示書は、いわゆる添付文書(package insert)であり、通常は紙媒体で提供されるが、それに限定されず、例えば、電子媒体(例えば、インターネットで提供されるホームページ、電子メール)のような形態でも提供され得る。
In this specification, "instructions" refers to instructions for a physician or other user on how to use the present disclosure. The instructions include instructions on how to use the present disclosure's detection method, how to use a diagnostic agent, or how to administer a medicine. The instructions may also include instructions for administration via the mouth or esophagus (e.g., by injection, etc.) as the site of administration. The instructions are prepared in accordance with a format stipulated by the supervisory authority of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and it is clearly stated that the instructions have been approved by the supervisory authority. The instructions are so-called package inserts, and are usually provided in paper form, but are not limited to this, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, e-mail, etc.).
本明細書で使用される「細胞内移行(インターナライズ/インターナライゼーション)」とは、細胞表面上の抗原に結合した物質を媒介してエンドサイトーシスまたは食作用によって細胞が抗原に結合した物質を取り込むことを指す。このような活性を有するLSRに結合する物質(例えば、抗LSR抗体)は、LSRを細胞表面に発現する細胞に対して目的の有効成分を細胞内移行させ、目的の有効成分による所望の効果をLSR発現細胞において生じさせることができる。目的の有効成分としては、細胞傷害性活性を有する薬剤、抗がん剤、造影剤、siRNA、アンチセンス核酸、リボザイムなどが挙げられるが、これらに限定されない。
As used herein, "internalization" refers to a cell taking up an antigen-bound substance on the cell surface via endocytosis or phagocytosis, with the substance being mediated by the antigen. A substance that binds to an LSR having such activity (e.g., an anti-LSR antibody) can cause the intracellular internalization of a target active ingredient in a cell expressing LSR on the cell surface, thereby producing the desired effect of the target active ingredient in the LSR-expressing cell. Examples of target active ingredients include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, etc.
本明細書において「抗体薬物複合体(ADC)」とは、1つまたは複数の目的の有効成分と化学的に連結された抗体これらの抗原結合フラグメントを指す。好ましい実施形態において、ADCは、リンカーを介して作動可能に連結されている。本明細書において「作動可能に連結されている」とは、連結される物質が予測された様式で作動することが可能な関係にあることを指す。ADCに含まれ得る目的の有効成分としては、以下に限定されないが、細胞傷害性活性を有する薬剤、抗がん剤、造影剤、siRNA、アンチセンス核酸、リボザイムなどが挙げられる。リンカーとして使用され得るものは、開裂型リンカーであっても、非開裂型のリンカーであってもよい。開裂型リンカーとしては、タンパク質分解酵素による切断配列を有するリンカー、酸不安定性のリンカー、ジスルフィドリンカーなどが挙げられるが、これらに限定されない。非開裂型リンカーとしては、MCCリンカーなどが挙げられるが、これに限定されない。
As used herein, "antibody drug conjugate (ADC)" refers to an antibody or antigen-binding fragment thereof chemically linked to one or more active ingredients of interest. In a preferred embodiment, the ADC is operably linked via a linker. As used herein, "operably linked" refers to a relationship in which the linked substances are capable of operating in a predicted manner. Active ingredients of interest that may be included in an ADC include, but are not limited to, drugs with cytotoxic activity, anticancer drugs, contrast agents, siRNA, antisense nucleic acids, ribozymes, and the like. The linker may be a cleavable linker or a non-cleavable linker. Examples of cleavable linkers include, but are not limited to, linkers having a sequence cleaved by a protease, acid-labile linkers, disulfide linkers, and the like. Examples of non-cleavable linkers include, but are not limited to, MCC linkers, and the like.
本明細書において「細胞傷害活性」とは、例えば、細胞に病理的な変化をもたらすこと、直接的な外傷にとどまらず、DNAの切断や塩基の二量体の形成、染色体の切断、細胞分裂システムの損傷、各種酵素活性の低下などあらゆる細胞の構造や機能上の損傷により、細胞の機能を直接または間接的に遮断することによって細胞死をもたらすことをいう。したがって、「細胞傷害活性を有する薬剤」としては、例えば、以下に限定されないが、アルキル化剤、腫瘍壊死因子阻害剤、インターカレーター、微小管阻害剤、キナーゼ阻害剤、プロテアソーム阻害剤、及びトポイソメラーゼ阻害剤などが挙げられる。
In this specification, "cytotoxic activity" refers to, for example, causing pathological changes in cells, not only direct trauma, but also any damage to the structure or function of cells, such as DNA breaks, formation of base dimers, chromosome breaks, damage to the cell division system, and reduced activity of various enzymes, thereby directly or indirectly blocking cell function and causing cell death. Therefore, examples of "drugs having cytotoxic activity" include, but are not limited to, alkylating agents, tumor necrosis factor inhibitors, intercalators, microtubule inhibitors, kinase inhibitors, proteasome inhibitors, and topoisomerase inhibitors.
本明細書において「50%阻害濃度(IC50)」とは、50%の細胞が死滅させるために必要な化合物の濃度をいう。本明細書では、実施例7に記載の方法を使用してIC50が測定される。
As used herein, the term "50% inhibitory concentration ( IC50 )" refers to the concentration of a compound required to kill 50% of cells. IC50 is measured herein using the method described in Example 7.
(好ましい実施形態)
以下に本開示の好ましい実施形態を説明する。以下に提供される実施形態は、本開示のよりよい理解のために提供されるものであり、本開示の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本開示の範囲内で適宜改変を行うことができることは明らかである。また、本開示の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。 Preferred Embodiments
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.
以下に本開示の好ましい実施形態を説明する。以下に提供される実施形態は、本開示のよりよい理解のために提供されるものであり、本開示の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本開示の範囲内で適宜改変を行うことができることは明らかである。また、本開示の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。 Preferred Embodiments
Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.
本発明者らは、抗LSR抗体は4T1シンジェニックモデルマウスに抗腫瘍効果を発揮し、この抗腫瘍効果がCD8+T細胞の除去により失われること、抗LSR抗体の投与により腫瘍内に浸潤したCD8+T細胞の数および活性化の割合の上昇を確認した。これらの結果、抗LSR抗体は腫瘍細胞上のLSRと、CD8+T細胞上の未知の受容体との結合を阻害することでCD8+T細胞の活性化を介して抗腫瘍効果を示していることが示唆された。したがって、1つの局面において、本開示は、脂肪分解刺激リポタンパク質受容体(LSR)の調節因子を含む、免疫チェックポイントを調節するための組成物を提供する。
The present inventors confirmed that anti-LSR antibodies exert an anti-tumor effect in 4T1 syngenic model mice, that this anti-tumor effect is lost by the removal of CD8+ T cells, and that administration of anti-LSR antibodies increases the number and activation rate of CD8+ T cells infiltrating into the tumor. These results suggest that anti-LSR antibodies exert their anti-tumor effect through activation of CD8+ T cells by inhibiting the binding of LSR on tumor cells to an unknown receptor on CD8+ T cells. Thus, in one aspect, the present disclosure provides a composition for regulating immune checkpoints, comprising a regulator of lipolysis-stimulating lipoprotein receptor (LSR).
脂肪分解刺激リポタンパク質受容体(LSR)の調節を行うことで、免疫チェックポイントを調節することができることは、本発明者らが本開示において実証したものであり、以下のメカニズムで説明され得る。
The inventors have demonstrated in this disclosure that immune checkpoints can be regulated by modulating the lipolysis-stimulating lipoprotein receptor (LSR), and this can be explained by the following mechanism.
本発明者らは抗LSR抗体がCD8+T細胞依存的に抗腫瘍効果を発揮することを動物モデルにおいて確認した。ここで、理論に束縛されることを望まないが、LSR調節することで免疫チェックポイントが調節しうることについては、以下のように説明され得る。すなわち、腫瘍細胞上に発現しているLSRがCD8+T細胞上に存在する受容体と相互作用する。その結果、CD8+T細胞に対して抑制性の刺激が細胞内に伝達され、腫瘍免疫を抑制する機能をLSRが有する。本明細書では、実際に、LSRを高発現する卵巣癌、胃癌や子宮内膜癌ではLSRを低発現と比較して有意に予後不良であることが確認されており、このことが適切であると合理的に説明され得る。その理由の1つとして、LSRが腫瘍免疫を抑制することで腫瘍増殖の進展が引き起こされていることが考えられる(Hiramatsu, Naka et al., Cancer Res. 2018 Jan 15;78(2):516-527, Sugase, Naka et al, Oncotarget. 2018 Aug 31;9(68):32917-32928, Nagase, Naka et al., BMC Cancer. 2022 Jun 21;22(1):679.)。
The inventors have confirmed in an animal model that anti-LSR antibodies exert an antitumor effect in a CD8+ T cell-dependent manner. Here, without wishing to be bound by theory, the fact that immune checkpoints can be regulated by regulating LSR can be explained as follows. That is, LSR expressed on tumor cells interacts with receptors present on CD8+ T cells. As a result, inhibitory stimuli are transmitted to CD8+ T cells intracellularly, and LSR has the function of suppressing tumor immunity. In this specification, it has actually been confirmed that ovarian cancer, gastric cancer, and endometrial cancer with high LSR expression have a significantly poorer prognosis compared to those with low LSR expression, and this can be reasonably explained as being appropriate. One of the reasons for this is thought to be that LSR suppresses tumor immunity, thereby causing tumor growth to progress (Hiramatsu, Naka et al., Cancer Res. 2018 Jan 15;78(2):516-527, Sugase, Naka et al., Oncotarget. 2018 Aug 31;9(68):32917-32928, Nagase, Naka et al., BMC Cancer. 2022 Jun 21;22(1):679.).
いくつかの実施形態において、LSRの調節因子は、LSRに対する抑制因子であり得る。LSRに対する抑制因子としては、例えば、抗LSR抗体またはその抗原結合フラグメント、アンチセンス核酸またはsiRNA等の核酸、小分子化合物が挙げられるが、これらに限定されない。siRNAは、例えば、配列番号9~14等を含みうる。
In some embodiments, the modulator of LSR may be an inhibitor of LSR. Examples of inhibitors of LSR include, but are not limited to, anti-LSR antibodies or antigen-binding fragments thereof, nucleic acids such as antisense nucleic acids or siRNAs, and small molecule compounds. siRNAs may include, for example, SEQ ID NOs: 9 to 14.
いくつかの実施形態において、LSRまたはそのフラグメントに対する結合因子は、抗LSR抗体またはその抗原結合フラグメントもしくは機能的等価物であり得る。
In some embodiments, the binding agent for LSR or a fragment thereof can be an anti-LSR antibody or an antigen-binding fragment thereof or a functional equivalent.
本開示の抗体は、本開示において別の箇所に記載された具体的な配列であり得る。抗体は、全長配列のCDRを含む任意の配列を含む抗体またはその抗原結合フラグメント、あるいは、以下の配列の可変領域を含む抗体またはその抗原結合フラグメントであって、そのフレームワーク領域において、1個、2個、3個、4個、5個、6個、7個、8個、9個、10個、12個、15個、17個、もしくは、20個、またはそれ以上の置換、付加、もしくは、欠失を含む抗体またはその抗原結合フラグメントであってもよい。抗体の製造等については、本開示の他の箇所に記載された実施形態および/または当該分野で公知の手法を用いることができる。なお、本開示の治療または予防の目的では、このような抗体またはそのフラグメントもしくは機能的等価物は、好ましくは、LSRまたはその情報伝達経路の下流の抑制活性を有することが好ましい。そのような活性は、LSRの発現量またはその活性をみるか、あるいは卵巣明細脂肪癌細胞等の悪性腫瘍細胞株を直接使用して細胞の増殖阻害、またはモデル動物に移植して腫瘍の退縮を観察する等をみることで確認してもよい。これらの手法は、当該分野において周知であり、本開示において使用される手法を用いてもよい。
The antibody of the present disclosure may be a specific sequence described elsewhere in this disclosure. The antibody may be an antibody or antigen-binding fragment thereof that includes any sequence including the CDRs of the full-length sequence, or an antibody or antigen-binding fragment thereof that includes a variable region of the following sequence, and that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, or 20 or more substitutions, additions, or deletions in the framework region. The antibody may be produced, etc., using the embodiments described elsewhere in this disclosure and/or techniques known in the art. For the therapeutic or preventive purposes of the present disclosure, such an antibody or fragment or functional equivalent preferably has an inhibitory activity downstream of the LSR or its signal transduction pathway. Such activity may be confirmed by observing the expression level or activity of the LSR, or by directly using a malignant tumor cell line such as ovarian leiomyoma cells to inhibit cell growth, or by transplanting the cell line into a model animal to observe tumor regression. These techniques are well known in the art and may be used in the present disclosure.
本開示の組成物の投与経路は、治療に際して効果的なものを使用するのが好ましく、例えば、静脈内、皮下、筋肉内、腹腔内、または経口投与等であってもよい。投与形態としては、例えば、注射剤、カプセル剤、錠剤、顆粒剤等であってもよい。抗体またはポリヌクレオチドを投与する場合には、注射剤として用いることが効果的である。注射用の水溶液は、例えば、バイアル、またはステンレス容器で保存してもよい。また注射用の水溶液は、例えば生理食塩水、糖(例えばトレハロース)、NaCl、またはNaOH等を配合してもよい。また治療薬は、例えば、緩衝剤(例えばリン酸塩緩衝液)、安定剤等を配合してもよい。
The administration route of the composition of the present disclosure is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, or granule. When administering an antibody or polynucleotide, it is effective to use it as an injection. The aqueous solution for injection may be stored, for example, in a vial or stainless steel container. The aqueous solution for injection may also be mixed with, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The therapeutic agent may also be mixed with, for example, a buffer (for example, a phosphate buffer), a stabilizer, etc.
一般的に、本開示の組成物、医薬、治療剤、予防剤等は、治療有効量の治療剤または有効成分、および薬学的に許容しうるキャリアもしくは賦形剤を含む。本明細書において「薬学的に許容しうる」は、動物、そしてより詳細にはヒトにおける使用のため、政府の監督官庁に認可されたか、あるいは薬局方または他の一般的に認められる薬局方に列挙されていることを意味する。本開示において使用される「キャリア」は、治療剤を一緒に投与する、希釈剤、アジュバント、賦形剤、またはビヒクルを指す。このようなキャリアは、無菌液体、例えば水および油であることも可能であり、石油、動物、植物または合成起源のものが含まれ、限定されるわけではないが、ピーナツ油、ダイズ油、ミネラルオイル、ゴマ油等が含まれる。医薬を経口投与する場合は、水が好ましいキャリアである。医薬組成物を静脈内投与する場合は、生理食塩水および水性デキストロースが好ましいキャリアである。好ましくは、生理食塩水溶液、並びに水性デキストロースおよびグリセロール溶液が、注射可能溶液の液体キャリアとして使用される。適切な賦形剤には、軽質無水ケイ酸、結晶セルロース、マンニトール、デンプン、グルコース、ラクトース、スクロース、ゼラチン、モルト、米、小麦粉、チョーク、シリカゲル、ステアリン酸ナトリウム、モノステアリン酸グリセロール、タルク、塩化ナトリウム、脱脂粉乳、グリセロール、プロピレン、グリコール、水、エタノール、カルメロースカルシウム、カルメロースナトリウム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルアセタールジエチルアミノアセテート、ポリビニルピロリドン、ゼラチン、中鎖脂肪酸トリグリセライド、ポリオキシエチレン硬化ヒマシ油60、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩等が含まれる。組成物は、望ましい場合、少量の湿潤剤または乳化剤、あるいはpH緩衝剤もまた含有することも可能である。これらの組成物は、溶液、懸濁物、エマルジョン、錠剤、ピル、カプセル、粉末、持続放出配合物等の形を取ることも可能である。伝統的な結合剤およびキャリア、例えばトリグリセリドを用いて、組成物を座薬として配合することも可能である。経口配合物は、医薬等級のマンニトール、ラクトース、デンプン、ステアリン酸マグネシウム、サッカリン・ナトリウム、セルロース、炭酸マグネシウムなどの標準的キャリアを含むことも可能である。適切なキャリアの例は、E.W.Martin, Remington’s Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A)に記載される。このような組成物は、患者に適切に投与する形を提供するように、適切な量のキャリアと一緒に、治療有効量の療法剤、好ましくは精製型のものを含有する。配合物は、投与様式に適していなければならない。これらのほか、例えば、界面活性剤、賦形剤、着色料、着香料、保存料、安定剤、緩衝剤、懸濁剤、等張化剤、結合剤、崩壊剤、滑沢剤、流動性促進剤、矯味剤等を含んでいてもよい。
Generally, the compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present disclosure include a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharma-
ceutically acceptable carrier or excipient. As used herein, "pharma-ceutically acceptable" means approved by a government regulatory agency or listed in a pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. As used in this disclosure, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the medicament is administered orally, water is the preferred carrier. When the pharmaceutical composition is administered intravenously, saline and aqueous dextrose are the preferred carriers. Preferably, saline solutions, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethyl amino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. The composition can also contain a small amount of a wetting or emulsifying agent, or a pH buffer, if desired. These compositions can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. The composition can also be formulated as a suppository, using traditional binders and carriers, such as triglycerides. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable carriers are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide a form that is properly administered to the patient. The formulation should be suitable for the mode of administration. In addition to these, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavorings, etc. may be included.
本開示の抗体、複合体、組成物等を医薬として投与する場合、種々の送達(デリバリー)系が知られ、そしてこのような系を用いて、本開示の治療剤を適切な部位(例えば、食道)に投与することも可能であり、このような系には、例えばリポソーム、微小粒子、および微小カプセル中の被包:治療剤(例えば、ポリペプチド)を発現可能な組換え細胞の使用、受容体が仲介するエンドサイトーシスの使用;レトロウイルスベクターまたは他のベクターの一部としての療法核酸の構築などがある。導入法には、限定されるわけではないが、皮内、筋内、腹腔内、静脈内、皮下、鼻内、硬膜外、および経口経路が含まれる。好適な経路いずれによって、例えば注入によって、ボーラス(bolus)注射によって、上皮または皮膚粘膜裏打ち(例えば口腔、直腸および腸粘膜など)を通じた吸収によって、医薬を投与することも可能であるし、必要に応じてエアロゾル化剤を用いて吸入器または噴霧器を使用しうるし、そして他の生物学的活性剤と一緒に投与することも可能である。投与は全身性または局所であることも可能である。本開示が卵巣領域で使用される場合、さらに、卵巣等の患部に直接注入する等、適切な経路いずれかによって投与されうる。
When the antibodies, conjugates, compositions, etc. of the present disclosure are administered as pharmaceuticals, various delivery systems are known and such systems can be used to administer the therapeutic agents of the present disclosure to the appropriate site (e.g., esophagus), such as encapsulation in liposomes, microparticles, and microcapsules; using recombinant cells capable of expressing the therapeutic agent (e.g., polypeptides); using receptor-mediated endocytosis; constructing the therapeutic nucleic acid as part of a retroviral or other vector. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical agent can be administered by any suitable route, such as by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), using an inhaler or nebulizer with an aerosolizing agent if necessary, and can be administered together with other biologically active agents. Administration can be systemic or local. When the present disclosure is used in the ovarian region, it may be administered by any suitable route, such as by direct injection into the affected area, such as the ovary.
好ましい実施形態において、公知の方法に従って、ヒトへの投与に適応させた医薬組成物として、組成物を配合することができる。このような組成物は注射により投与することができる。代表的には、注射投与のための組成物は、無菌等張水性緩衝剤中の溶液である。必要な場合、組成物はまた、可溶化剤および注射部位での疼痛を和らげるリドカインなどの局所麻酔剤も含むことも可能である。一般的に、成分を別個に供給するか、または単位投薬型中で一緒に混合して供給し、例えば活性剤の量を示すアンプルまたはサシェなどの密封容器中、凍結乾燥粉末または水不含濃縮物として供給することができる。組成物を注入によって投与しようとする場合、無菌薬剤等級の水または生理食塩水を含有する注入ビンを用いて、分配することも可能である。組成物を注射によって投与しようとする場合、投与前に、成分を混合可能であるように、注射用の無菌水または生理食塩水のアンプルを提供することも可能である。
In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for administration to humans, according to known methods. Such a composition can be administered by injection. Typically, a composition for administration by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the composition can also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachette indicating the quantity of active agent. If the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
本開示の組成物、医薬、治療剤、予防剤を中性型または塩型あるいは他のプロドラッグ(例えば、エステル等)で配合することも可能である。薬学的に許容しうる塩には、塩酸、リン酸、酢酸、シュウ酸、酒石酸などに由来する遊離型のカルボキシル基とともに形成されるもの、イソプロピルアミン、トリエチルアミン、2-エチルアミノエタノール、ヒスチジン、プロカインなどに由来するものなどの遊離型のアミン基とともに形成されるもの、並びにナトリウム、カリウム、アンモニウム、カルシウム、および水酸化第二鉄などに由来するものが含まれる。
The compositions, pharmaceuticals, therapeutic agents, and prophylactic agents of the present disclosure may be formulated in neutral or salt form or as other prodrugs (e.g., esters, etc.). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxide, etc.
特定の障害または状態の治療に有効な本開示の治療剤の量は、障害または状態の性質によって変動しうるが、当業者は本明細書の記載に基づき標準的臨床技術によって決定可能である。さらに、場合によって、in vitroアッセイを使用して、最適投薬量範囲を同定するのを補助することも可能である。配合物に使用しようとする正確な用量はまた、投与経路、および疾患または障害の重大性によっても変動しうるため、担当医の判断および各患者の状況に従って、決定すべきである。しかし、投与量は特に限定されないが、例えば、1回あたり0.001、1、5、10、15、100、または1000mg/kg体重であってもよく、それらいずれか2つの値の範囲内であってもよい。投与間隔は特に限定されないが、例えば、1、7、14、21、または28日あたりに1または2回投与してもよく、それらいずれか2つの値の範囲あたりに1または2回投与してもよい。投与量、投与間隔、投与方法は、患者の年齢や体重、症状、対象臓器等により、適宜選択してもよい。また治療薬は、治療有効量、または所望の作用を発揮する有効量の有効成分を含むことが好ましい。悪性腫瘍マーカーが、投与後に有意に減少した場合に、治療効果があったと判断してもよい。
The amount of the therapeutic agent of the present disclosure effective in treating a particular disorder or condition may vary depending on the nature of the disorder or condition, but can be determined by one of skill in the art using standard clinical techniques based on the description herein. In addition, in vitro assays may be used in some cases to help identify optimal dosage ranges. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dosage is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dose, or within any two of these values. The administration interval is not particularly limited, and may be, for example, 1 or 2 doses per 1, 7, 14, 21, or 28 days, or 1 or 2 doses per two of these values. The dosage, administration interval, and administration method may be selected appropriately depending on the age, weight, symptoms, target organ, etc. of the patient. In addition, the therapeutic agent preferably contains an active ingredient in a therapeutically effective amount, or in an effective amount that exerts a desired effect. If the malignant tumor marker is significantly reduced after administration, it may be determined that the therapeutic effect has been achieved.
本開示の一実施形態において「患者」は、ヒト、またはヒトを除く哺乳動物(例えば、マウス、モルモット、ハムスター、ラット、ネズミ、ウサギ、ブタ、ヒツジ、ヤギ、ウシ、ウマ、ネコ、イヌ、マーモセット、サル、またはチンパンジー等の1種以上)を含む。また患者は、LSR陽性悪性腫瘍を発症していると判断または診断された患者であってもよい。このとき、判断または診断は、LSRのタンパク質レベルを検出することにより行われることが好ましい。
In one embodiment of the present disclosure, a "patient" includes a human or a non-human mammal (e.g., one or more of mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, marmoset, monkey, or chimpanzee). The patient may also be a patient who has been determined or diagnosed as having an LSR-positive malignant tumor. In this case, the determination or diagnosis is preferably made by detecting the protein level of LSR.
本開示の医薬組成物または治療剤もしくは予防剤はキットとして提供することができる。
The pharmaceutical composition or therapeutic or prophylactic agent disclosed herein can be provided as a kit.
特定の実施形態では、本開示は、本開示の抗体、複合体、組成物または医薬の1以上の成分が充填された、1以上の容器を含む、薬剤パックまたはキットを提供する。場合によって、このような容器に付随して、医薬または生物学的製品の製造、使用または販売を規制する政府機関によって規定された形で、政府機関による、ヒト投与のための製造、使用または販売の認可を示す情報を示すことも可能である。
In certain embodiments, the disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of an antibody, conjugate, composition, or medicament of the disclosure. Optionally, such containers may bear information indicating approval by a government agency of the manufacture, use, or sale for human administration, in a manner prescribed by the government agency regulating the manufacture, use, or sale of pharmaceutical or biological products.
別の実施形態では、前記抗LSR抗体は、LSRのエピトープに特異的に結合する抗LSR抗体でありうる。より詳細には、抗体は、配列番号7の116~135位および/または216~230位をエピトープとして有するものであってもよい。
In another embodiment, the anti-LSR antibody may be an anti-LSR antibody that specifically binds to an epitope of LSR. More specifically, the antibody may have an epitope at positions 116-135 and/or 216-230 of SEQ ID NO:7.
本開示一実施形態は、(a)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示されるアミノ酸配列を含む抗体、(b)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示されるアミノ酸配列を含む抗体、(c)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、(d)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、(e)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、および(f)重鎖CDR1、2、3、軽鎖CDR1、2、および3が、それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示されるアミノ酸配列を含む抗体、からなる群から選ばれる1種以上の抗体、あるいは該抗体の変異体であって、該変異体において該抗体のフレームワークに1または数個の置換、付加もしくは欠失を含むが、該CDRには変異を含まない、変異体である、抗LSR抗体でありうる。この抗LSR抗体を用いれば、LSR陽性悪性腫瘍細胞の増殖を特に効果的に抑制することができる。また、効率的にLSR陽性悪性腫瘍の診断をすることができる。また本開示の別の実施形態は、上に列挙した重鎖CDR1、2、および3のアミノ酸配列のセットのうち、少なくとも1つのセットを含む抗LSR抗体である。これらの抗体は、モノクローナル抗体、ポリクローナル抗体、キメラ抗体、ヒト化抗体、ヒト抗体、多機能抗体、二重特異性またはオリゴ特異性(oligospecific)抗体、単鎖抗体、scFV、ダイアボディー、sc(Fv)2(single chain(Fv)2)、およびscFv-Fcから選択される抗体であってもよい。
One embodiment of the present disclosure relates to an antibody comprising: (a) a heavy chain CDR1, 2, 3, and a light chain CDR1, 2, and 3, each of which comprises the amino acid sequence set forth in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 230 of SEQ ID NO: 1; and (b) a heavy chain CDR1, 2, 3, and a light chain CDR1, 2, and 3, each of which comprises the amino acid sequence set forth in positions 31 to 35, 50 to 66, 99 to 103, 152 to 165, 182 to 188, and 221 to 230 of SEQ ID NO: 2. (c) an antibody in which heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 comprise the amino acid sequences shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO: 3, respectively; (d) an antibody in which heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 comprise the amino acid sequences shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO: 4, respectively. 5, (e) an antibody in which heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3 comprise the amino acid sequences represented by positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; and (f) an antibody in which heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and The anti-LSR antibody may be an anti-LSR antibody, in which the amino acid sequences of heavy chain CDR1, 2, and 3 are at least one antibody selected from the group consisting of antibodies comprising the amino acid sequences of positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 6, respectively, or a mutant of the antibody, in which the mutant comprises one or several substitutions, additions, or deletions in the framework of the antibody, but does not comprise a mutation in the CDR. The use of this anti-LSR antibody can be particularly effective in suppressing the proliferation of LSR-positive malignant tumor cells. In addition, LSR-positive malignant tumors can be efficiently diagnosed. Another embodiment of the present disclosure is an anti-LSR antibody comprising at least one of the sets of amino acid sequences of heavy chain CDR1, 2, and 3 listed above. These antibodies may be antibodies selected from monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFVs, diabodies, sc(Fv) 2 (single chain (Fv) 2 ), and scFv-Fc.
別の実施形態において、LSRに対する結合性を維持しつつ、抗体の6つのCDRのうちの1つ、2つ、3つ、4つ、5つまたは6つのCDRに1または数個の置換、付加もしくは欠失を含んでもよい。別の実施形態において、3つ以下、好ましくは2つ以下、より好ましくは1つのCDRに2つ以下、好ましくは1つの置換、付加もしくは欠失を含み得る。好ましい実施形態において、置換は保存的置換であり得る。
In another embodiment, the antibody may contain one or several substitutions, additions or deletions in one, two, three, four, five or six of the six CDRs while maintaining binding to the LSR. In another embodiment, the antibody may contain up to three, preferably up to two, more preferably up to two, preferably one substitution, addition or deletion in one CDR. In a preferred embodiment, the substitutions may be conservative substitutions.
本開示の一実施形態に係る抗LSR抗体は、重鎖CDR1、2、および3、ならびに軽鎖CDR1、2および3のアミノ酸配列のセットを含み、さらに、重鎖FR1、2、3、4、軽鎖FR1、2、3、および4のうち少なくとも1つ、好ましくは2つ、3つ、4つ、5つ、6つ、7つ、あるいはすべてのフレームワークが配列番号1~6のいずれかのものと同一または実質的に同一あるいは保存的置換を除き同一であるものであり得る。1種以上の抗体であってもよい。また本開示の別の実施形態は、上に列挙した重鎖FR1、2、3、および4のアミノ酸配列のセットのうち、少なくとも1つのセットを含む抗LSR抗体である。
An anti-LSR antibody according to one embodiment of the present disclosure comprises a set of amino acid sequences of heavy chain CDR1, 2, and 3, and light chain CDR1, 2, and 3, and further, at least one, preferably two, three, four, five, six, seven, or all of the frameworks of heavy chain FR1, 2, 3, and 4, and light chain FR1, 2, 3, and 4 may be identical or substantially identical to any of SEQ ID NOs: 1 to 6, or identical except for conservative substitutions. There may be one or more types of antibodies. Another embodiment of the present disclosure is an anti-LSR antibody comprising at least one of the sets of amino acid sequences of heavy chain FR1, 2, 3, and 4 listed above.
本開示の一実施形態に係る抗LSR抗体は、scFvの形態であってもよく、その場合、重鎖と軽鎖間のリンカーが、配列番号1の116~132位、配列番号2の116~132位、配列番号3の116~132位、配列番号4の116~132位、配列番号5の116~132位、または配列番号6の116~132位で示されるアミノ酸配列を有していてもよい。
The anti-LSR antibody according to one embodiment of the present disclosure may be in the form of an scFv, in which case the linker between the heavy and light chains may have the amino acid sequence shown in positions 116 to 132 of SEQ ID NO:1, positions 116 to 132 of SEQ ID NO:2, positions 116 to 132 of SEQ ID NO:3, positions 116 to 132 of SEQ ID NO:4, positions 116 to 132 of SEQ ID NO:5, or positions 116 to 132 of SEQ ID NO:6.
なお、後述する実施例に記載の#9-7、#16-6、No.26-2、No.27-6、No.1-25、No.1-43のVHは、それぞれ配列番号1の1~115位、配列番号2の1~115位、配列番号3の1~115位、配列番号4の1~115位、配列番号5の1~115位、配列番号6の1~115位である。また、後述する実施例に記載の#9-7、#16-6、No.26-2、No.27-6、No.1-25、およびNo.1-43のVLは、それぞれ配列番号1の133~238位、配列番号2の133~239位、配列番号3の133~238位、配列番号4の133~238位、配列番号5の133~238位、配列番号6の133~238位である。
The VHs of #9-7, #16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in the Examples below are positions 1 to 115 of SEQ ID NO:1, positions 1 to 115 of SEQ ID NO:2, positions 1 to 115 of SEQ ID NO:3, positions 1 to 115 of SEQ ID NO:4, positions 1 to 115 of SEQ ID NO:5, and positions 1 to 115 of SEQ ID NO:6, respectively. The VLs of #9-7, #16-6, No.26-2, No.27-6, No.1-25, and No.1-43 described in the Examples below are positions 133 to 238 of SEQ ID NO:1, positions 133 to 239 of SEQ ID NO:2, positions 133 to 238 of SEQ ID NO:3, positions 133 to 238 of SEQ ID NO:4, positions 133 to 238 of SEQ ID NO:5, and positions 133 to 238 of SEQ ID NO:6, respectively.
上に列挙したアミノ酸配列は、抗LSR抗体が所望の効果を有する限り、(i)上記のアミノ酸配列において、1または数個の塩基配列が欠失、置換、挿入、もしくは付加しているアミノ酸配列、(ii)上記のアミノ酸配列に対して、90%以上(例えば、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、または99%以上)の同一性を有するアミノ酸配列、および(iii)上記のアミノ酸配列をコードする塩基配列に相補的な塩基配列からなるポリヌクレオチドに、ストリンジェントな条件下で特異的にハイブリダイズするポリヌクレオチドがコードするアミノ酸配列、からなる群から選ばれる1つ以上のアミノ酸配列であってもよい。
The amino acid sequences listed above may be one or more amino acid sequences selected from the group consisting of: (i) the above amino acid sequences in which one or more base sequences have been deleted, substituted, inserted, or added; (ii) amino acid sequences having 90% or more identity (e.g., 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the above amino acid sequences; and (iii) amino acid sequences encoded by polynucleotides that specifically hybridize under stringent conditions to polynucleotides consisting of a base sequence complementary to the base sequence encoding the above amino acid sequences, so long as the anti-LSR antibody has the desired effect.
本開示の一実施形態に係る抗LSR抗体をコードするポリヌクレオチドまたはベクターを細胞に導入することによって、形質転換体を作成できる。この形質転換体を用いれば、本開示の実施形態に係る抗LSR抗体を作製できる。形質転換体は、ヒトまたはヒトを除く哺乳動物(例えば、ラット、マウス、モルモット、ウサギ、ウシ、サル等)の細胞であってもよい。哺乳動物細胞としては、例えば、チャイニーズハムスター卵巣細胞(CHO細胞)、サル細胞COS-7などが挙げられる。または、形質転換体はEscherichia属菌、酵母等であってもよい。
A transformant can be created by introducing a polynucleotide or vector encoding an anti-LSR antibody according to an embodiment of the present disclosure into a cell. The transformant can be used to produce an anti-LSR antibody according to an embodiment of the present disclosure. The transformant may be a cell of a human or a mammal other than a human (e.g., rat, mouse, guinea pig, rabbit, cow, monkey, etc.). Examples of mammalian cells include Chinese hamster ovary cells (CHO cells) and monkey cells COS-7. Alternatively, the transformant may be Escherichia bacteria, yeast, etc.
上記のベクターとしては、例えば大腸菌由来のプラスミド(例えばpET-Blue)、枯草菌由来のプラスミド(例えばpUB110)、酵母由来プラスミド(例えばpSH19)、動物細胞発現プラスミド(例えばpA1-11、pcDNA3.1-V5/His-TOPO)、λファージなどのバクテリオファージ、ウイルス由来のベクターなどを用いることができる。これらのベクターは、プロモーター、複製開始点、または抗生物質耐性遺伝子など、タンパク質発現に必要な構成要素を含んでいてもよい。ベクターは発現ベクターであってもよい。
The above vectors may be, for example, plasmids derived from E. coli (e.g., pET-Blue), plasmids derived from Bacillus subtilis (e.g., pUB110), yeast-derived plasmids (e.g., pSH19), animal cell expression plasmids (e.g., pA1-11, pcDNA3.1-V5/His-TOPO), bacteriophages such as λ phage, and vectors derived from viruses. These vectors may contain components necessary for protein expression, such as a promoter, an origin of replication, or an antibiotic resistance gene. The vector may be an expression vector.
上記のポリヌクレオチドまたはベクターの細胞への導入方法としては、例えば、リン酸カルシウム法、リポフェクション法、エレクトロポレーション法、アデノウイルスによる方法、レトロウイルスによる方法、またはマイクロインジェクションなどを使用できる(改訂第4版新 遺伝子工学ハンドブック, 羊土社(2003):152-179.)。抗体の細胞を用いた生産方法としては、例えば、"タンパク質実験ハンドブック,羊土社(2003):128-142."に記載の方法を使用できる。抗体の精製においては、例えば、硫酸アンモニウム、エタノール沈殿、プロテインA、プロテインG、ゲルろ過クロマトグラフィー、陰イオン、陽イオン交換クロマトグラフィー、ホスホセルロースクロマトグラフィー、疎水性相互作用クロマトグラフィー、アフィニティークロマトグラフィー、ヒドロキシルアパタイトクロマトグラフィー、またはレクチンクロマトグラフィーなどを用いることができる(タンパク質実験ハンドブック,羊土社(2003):27-52.)。
The above-mentioned polynucleotides or vectors can be introduced into cells by, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, or microinjection (Revised 4th Edition New Genetic Engineering Handbook, Yodosha (2003): 152-179.). For the production of antibodies using cells, for example, the method described in "Protein Experiment Handbook, Yodosha (2003): 128-142." can be used. For the purification of antibodies, for example, ammonium sulfate, ethanol precipitation, protein A, protein G, gel filtration chromatography, anion and cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, or lectin chromatography can be used (Protein Experiment Handbook, Yodosha (2003): 27-52.).
別の局面において、本開示は、LSRの調節因子を含む、腫瘍浸潤CD8+T細胞の数を増加および/または活性化させるための組成物を提供する。いくつかの実施形態において、CD8+Tは、CD69+であり得る。CD69は、CD8+T細胞の活性化マーカーとして知られている。
In another aspect, the present disclosure provides a composition for increasing the number and/or activating tumor-infiltrating CD8+ T cells, comprising a modulator of an LSR. In some embodiments, the CD8+ T cells can be CD69+. CD69 is known as an activation marker for CD8+ T cells.
いくつかの実施形態において、本開示の組成物等は、悪性腫瘍を治療または予防するためのものであり得る。悪性腫瘍は、LSR陽性であり得る。悪性腫瘍は、乳癌、卵巣癌、子宮内膜癌、膵臓癌、肺癌、胃癌または大腸癌であり得、これらはLSR陽性であり得る。
In some embodiments, the compositions and the like of the present disclosure may be for treating or preventing malignant tumors. The malignant tumor may be LSR positive. The malignant tumor may be breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer, or colon cancer, which may be LSR positive.
さらなる局面において、LSRの検出試薬を含む、本開示の複合体、組成物、医薬等による治療を必要とするかどうかを判断するためのコンパニオン試薬を提供する。
In a further aspect, a companion reagent for determining whether or not treatment with the complex, composition, pharmaceutical, etc. of the present disclosure is required, the companion reagent including a detection reagent for LSR.
さらなる局面において、LSRの検出試薬と、本開示の複合体、組成物、医薬等と、指示書とを含む、悪性腫瘍を治療または予防するためのキットを提供する。
In a further aspect, a kit for treating or preventing malignant tumors is provided, comprising an LSR detection reagent, a complex, composition, medicine, etc. of the present disclosure, and instructions.
本明細書において引用された、科学文献、特許、特許出願などの参考文献は、その全体が、各々具体的に記載されたのと同じ程度に本明細書において参考として援用される。
All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth herein.
以上、本開示を、理解の容易のために好ましい実施形態を示して説明してきた。以下に、実施例に基づいて本開示を説明するが、上述の説明および以下の実施例は、例示の目的のみに提供され、本開示を限定する目的で提供したのではない。従って、本開示の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限定されず、請求の範囲によってのみ限定される。
The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims.
以下、本開示を実施例によりさらに説明するが、本開示はこれらに限定されるものではない。
The present disclosure will be further explained below with reference to examples, but the present disclosure is not limited to these.
実施例1:LSRおよびB7ファミリー分子のV-type Ig領域の配列解析
LSRの配列解析(図1左)
ヒトLSR(Q86X29)とマウスLSR(Q99KG5)のアミノ酸配列の同一性をUniprotのAlimentツールにより解析した。 Example 1: Sequence analysis of LSR and V-type Ig region of B7 family molecules Sequence analysis of LSR (Figure 1, left)
The identity of the amino acid sequences of human LSR (Q86X29) and mouse LSR (Q99KG5) was analyzed using the Aliment tool of Uniprot.
LSRの配列解析(図1左)
ヒトLSR(Q86X29)とマウスLSR(Q99KG5)のアミノ酸配列の同一性をUniprotのAlimentツールにより解析した。 Example 1: Sequence analysis of LSR and V-type Ig region of B7 family molecules Sequence analysis of LSR (Figure 1, left)
The identity of the amino acid sequences of human LSR (Q86X29) and mouse LSR (Q99KG5) was analyzed using the Aliment tool of Uniprot.
LSRとB7ファミリー分子のV-type Ig領域の配列解析(図1右)
ヒトLSR(Q86X29)、ヒトCD80(P33681)、ヒトCD86(P42081)、ヒトICOSL(O75144)、ヒトPDL1(Q9NZQ7)、ヒトPDL2(Q9BQ51)、ヒトCD276(Q5ZPR3)、ヒトVTCN1(B7-H4)(Q7Z7D3)、ヒトVISTA(Q9H7M9)のV-type Ig領域のアミノ酸配列の同一性をUniprotのAlimentツールにより解析した。 Sequence analysis of the V-type Ig region of LSR and B7 family molecules (Figure 1, right)
The amino acid sequence identity of the V-type Ig region of human LSR (Q86X29), human CD80 (P33681), human CD86 (P42081), human ICOSL (O75144), human PDL1 (Q9NZQ7), human PDL2 (Q9BQ51), human CD276 (Q5ZPR3), human VTCN1 (B7-H4) (Q7Z7D3), and human VISTA (Q9H7M9) was analyzed using the Aliment tool of Uniprot.
ヒトLSR(Q86X29)、ヒトCD80(P33681)、ヒトCD86(P42081)、ヒトICOSL(O75144)、ヒトPDL1(Q9NZQ7)、ヒトPDL2(Q9BQ51)、ヒトCD276(Q5ZPR3)、ヒトVTCN1(B7-H4)(Q7Z7D3)、ヒトVISTA(Q9H7M9)のV-type Ig領域のアミノ酸配列の同一性をUniprotのAlimentツールにより解析した。 Sequence analysis of the V-type Ig region of LSR and B7 family molecules (Figure 1, right)
The amino acid sequence identity of the V-type Ig region of human LSR (Q86X29), human CD80 (P33681), human CD86 (P42081), human ICOSL (O75144), human PDL1 (Q9NZQ7), human PDL2 (Q9BQ51), human CD276 (Q5ZPR3), human VTCN1 (B7-H4) (Q7Z7D3), and human VISTA (Q9H7M9) was analyzed using the Aliment tool of Uniprot.
(結果)
ヒトLSR(UniProtKB accession: Q86X29-1)とマウス(LSR UniProtKB accession: Q99KG5-1)のアミノ酸配列を比較した。ヒトLSRのシグナルペプチドは予測ソフトにより判定出来なかったが、マウスLSRは35アミノ酸がシグナルペプチド配列であった。ヒトLSRはlong V-type Igドメイン、stalk領域、および膜貫通ドメインを有していた。ヒトLSRとマウスLSRの全長でのアミノ酸配列同一性は73.2%であったが、V-type Igドメイン間では91.3%のアミノ酸同一性を示した(図1)。 (result)
The amino acid sequences of human LSR (UniProtKB accession: Q86X29-1) and mouse (LSR UniProtKB accession: Q99KG5-1) were compared. The signal peptide of human LSR could not be determined by prediction software, but mouse LSR had a signal peptide sequence of 35 amino acids. Human LSR had a long V-type Ig domain, a stalk region, and a transmembrane domain. The amino acid sequence identity of human LSR and mouse LSR over their entire length was 73.2%, but the V-type Ig domain showed 91.3% amino acid identity (Figure 1).
ヒトLSR(UniProtKB accession: Q86X29-1)とマウス(LSR UniProtKB accession: Q99KG5-1)のアミノ酸配列を比較した。ヒトLSRのシグナルペプチドは予測ソフトにより判定出来なかったが、マウスLSRは35アミノ酸がシグナルペプチド配列であった。ヒトLSRはlong V-type Igドメイン、stalk領域、および膜貫通ドメインを有していた。ヒトLSRとマウスLSRの全長でのアミノ酸配列同一性は73.2%であったが、V-type Igドメイン間では91.3%のアミノ酸同一性を示した(図1)。 (result)
The amino acid sequences of human LSR (UniProtKB accession: Q86X29-1) and mouse (LSR UniProtKB accession: Q99KG5-1) were compared. The signal peptide of human LSR could not be determined by prediction software, but mouse LSR had a signal peptide sequence of 35 amino acids. Human LSR had a long V-type Ig domain, a stalk region, and a transmembrane domain. The amino acid sequence identity of human LSR and mouse LSR over their entire length was 73.2%, but the V-type Ig domain showed 91.3% amino acid identity (Figure 1).
B7ファミリーはIg superfamilyに属しており、V-typeとconstant (C)-type Igドメインを有する1回膜貫通タンパク質である。ヒトLSRおよびマウスLSRと他のB7様タンパク質との間でアミノ酸配列を比較した結果V-type Igドメイン内に存在するDxGxYxCモチーフが保存されていた(図1)。V-type Igドメイン間におけるヒトLSRと他のB7様タンパク質との間でアミノ酸配列同一性は6.77%から16.9%であった(CD80: 8.87%、CD86: 6.77%、ICOSL: 10.24%、PD-L1: 11.2%、PD-L2: 8.94%、B7-H3: 7.67%、B7-H4: 8.43%、VISTA: 16.89%)。ヒトLSRはVISTAと最も高いアミノ酸相同性を示し、両者はC-type Igドメインを欠損していた。DxGxYxCモチーフはIgドメインに保存されている典型的な配列である。Igドメインを有するタンパク質の一部が免疫チェックポイント活性を有し得る。以上から、LSRもB7様タンパク質であることが示唆され、後述の実施例において、LSRが免疫チェックポイント活性を有するかどうかを確認する。
The B7 family belongs to the Ig superfamily and is a single-pass transmembrane protein with V-type and constant (C)-type Ig domains. Comparison of amino acid sequences between human LSR and mouse LSR and other B7-like proteins revealed that the DxGxYxC motif in the V-type Ig domain is conserved (Figure 1). The amino acid sequence identity between human LSR and other B7-like proteins in the V-type Ig domain ranged from 6.77% to 16.9% (CD80: 8.87%, CD86: 6.77%, ICOSL: 10.24%, PD-L1: 11.2%, PD-L2: 8.94%, B7-H3: 7.67%, B7-H4: 8.43%, VISTA: 16.89%). Human LSR showed the highest amino acid sequence identity with VISTA, and both proteins lacked the C-type Ig domain. The DxGxYxC motif is a typical sequence conserved in Ig domains. Some proteins with Ig domains may have immune checkpoint activity. From the above, it is suggested that LSR is also a B7-like protein, and in the Examples below, we will confirm whether LSR has immune checkpoint activity.
実施例2:ELISAによるニワトリマウスキメラ抗LSR抗体(#27-6mF18)のヒト、カニクイザル、ラット、マウスLSRとの交叉反応の解析
(材料および方法)
(バッファー調製)
本実施例で使用されるバッファーの調製は以下のとおり行った。
・コーティングバッファー:50 mM carbonate-bicarbonate buffer, pH 9.6。
・希釈バッファー:希釈溶液は、10%ブロックエース(Block Ace)を用いており、10%ブロックエースはブロックエース粉末4gをMiliQ 100mLに溶解し、PBS(-)にて10倍希釈して調製した。
・ブロッキングバッファー:ブロックエース粉末4gをMiliQ 100mLで溶解した後、PBS(-)で4倍希釈して調製した。ブロックエース粉末4gをMiliQ 100mLにて溶解した溶液を原液(100%溶液)とした。
・洗浄バッファー:PBS-T(Sigma、P3563)1包を超純水1Lに溶解した(0.05% Tween20/PBS)。 Example 2: Analysis of cross-reactivity of chicken mouse chimeric anti-LSR antibody (#27-6mF18) with human, cynomolgus monkey, rat, and mouse LSR by ELISA (Materials and Methods)
Buffer Preparation
The buffers used in this example were prepared as follows.
- Coating buffer: 50 mM carbonate-bicarbonate buffer, pH 9.6.
Dilution buffer: The dilution solution used was 10% Block Ace, which was prepared by dissolving 4 g of Block Ace powder in 100 mL of MiliQ and diluting the solution 10-fold with PBS(-).
Blocking buffer: 4 g of Block Ace powder was dissolved in 100 mL of MiliQ, and then diluted 4-fold with PBS (-) to prepare a blocking buffer. The solution obtained by dissolving 4 g of Block Ace powder in 100 mL of MiliQ was used as the stock solution (100% solution).
Washing buffer: One packet of PBS-T (Sigma, P3563) was dissolved in 1 L of ultrapure water (0.05% Tween 20/PBS).
(材料および方法)
(バッファー調製)
本実施例で使用されるバッファーの調製は以下のとおり行った。
・コーティングバッファー:50 mM carbonate-bicarbonate buffer, pH 9.6。
・希釈バッファー:希釈溶液は、10%ブロックエース(Block Ace)を用いており、10%ブロックエースはブロックエース粉末4gをMiliQ 100mLに溶解し、PBS(-)にて10倍希釈して調製した。
・ブロッキングバッファー:ブロックエース粉末4gをMiliQ 100mLで溶解した後、PBS(-)で4倍希釈して調製した。ブロックエース粉末4gをMiliQ 100mLにて溶解した溶液を原液(100%溶液)とした。
・洗浄バッファー:PBS-T(Sigma、P3563)1包を超純水1Lに溶解した(0.05% Tween20/PBS)。 Example 2: Analysis of cross-reactivity of chicken mouse chimeric anti-LSR antibody (#27-6mF18) with human, cynomolgus monkey, rat, and mouse LSR by ELISA (Materials and Methods)
Buffer Preparation
The buffers used in this example were prepared as follows.
- Coating buffer: 50 mM carbonate-bicarbonate buffer, pH 9.6.
Dilution buffer: The dilution solution used was 10% Block Ace, which was prepared by dissolving 4 g of Block Ace powder in 100 mL of MiliQ and diluting the solution 10-fold with PBS(-).
Blocking buffer: 4 g of Block Ace powder was dissolved in 100 mL of MiliQ, and then diluted 4-fold with PBS (-) to prepare a blocking buffer. The solution obtained by dissolving 4 g of Block Ace powder in 100 mL of MiliQ was used as the stock solution (100% solution).
Washing buffer: One packet of PBS-T (Sigma, P3563) was dissolved in 1 L of ultrapure water (0.05% Tween 20/PBS).
(1次抗体調製)
本実施例で使用される1次抗体を調製した。ニワトリマウスキメラ抗LSR抗体(#27-6mF18)を希釈バッファーで1000、100、10、1、0.1、0.01、0.001nMに段階的に希釈した。抗原は、組み換えヒトLSR-Fc、カニクイザルLSR-Fc、ラットLSR-Fc、マウスLSR-Fcを使用した。 (Primary antibody preparation)
The primary antibody used in this example was prepared. Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was serially diluted with dilution buffer to 1000, 100, 10, 1, 0.1, 0.01, and 0.001 nM. The antigens used were recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc.
本実施例で使用される1次抗体を調製した。ニワトリマウスキメラ抗LSR抗体(#27-6mF18)を希釈バッファーで1000、100、10、1、0.1、0.01、0.001nMに段階的に希釈した。抗原は、組み換えヒトLSR-Fc、カニクイザルLSR-Fc、ラットLSR-Fc、マウスLSR-Fcを使用した。 (Primary antibody preparation)
The primary antibody used in this example was prepared. Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was serially diluted with dilution buffer to 1000, 100, 10, 1, 0.1, 0.01, and 0.001 nM. The antigens used were recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc.
(手法)
組み換えヒトLSR-Fc、カニクイザルLSR-Fc、ラットLSR-Fc、マウスLSR-Fcを50mM carbonate-bicarbonate buffer, pH 9.6で2.5μg/mLに希釈し、50μL/wellでImmuno 96well MicroWell Solid Plate(Maxisorp, Nunc)に添加し、プレートにシールをした。プレートを揺らして、液が全体に行き渡ることを確認した。次いで、以下の手順に従った。
・静置(4℃、一晩)
・洗浄(200 μL/well x3)
・ブロッキングバッファーを200μL/wellずつ96wellマイクロプレートに添加
・室温で1時間反応
・洗浄(200μL/well、3回)
・ニワトリマウスキメラ抗LSR抗体(#27-6mF18)を希釈バッファーで100nMから10倍希釈系列で希釈し、100μL/wellで96wellマイクロプレートに添加
・室温で1時間反応
・洗浄(200μL/well、3回)
・Goat Anti-Chicken IgY H&L (HRP) (abcam社, cat.No.: ab6877)を希釈バッファー(10% BlockAce)で2μg/mlに希釈し(5000倍希釈を用時調製)、100μL/wellずつ96wellマイクロプレートに添加
・室温で1時間反応
・洗浄(200μL/well、3回)
・TMB(SurModics、TMBW-1000-01)を使用前に常温に戻し、100μL/wellずつ96wellマイクロプレートに添加
・撹拌(遮光下、室温)
・1N-硫酸50μL/wellで停止
・450nmで吸光度測定
・GraphPad Prism 6.04でデータ解析。 (Method)
Recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc were diluted to 2.5 μg/mL with 50 mM carbonate-bicarbonate buffer, pH 9.6, and added to an Immuno 96-well MicroWell Solid Plate (Maxisorp, Nunc) at 50 μL/well, and the plate was sealed. The plate was rocked to ensure that the liquid was distributed throughout. The following procedure was then followed.
- Leave it still (4℃, overnight)
Washing (200 μL/well x 3)
Add 200 μL of blocking buffer per well to a 96-well microplate. Incubate at room temperature for 1 hour. Wash (200 μL per well, 3 times).
・Dilute chicken mouse chimeric anti-LSR antibody (#27-6mF18) in dilution buffer in a 10-fold serial dilution starting from 100 nM, add 100 μL/well to a 96-well microplate, react at room temperature for 1 hour, and wash (200 μL/well, 3 times).
・Goat Anti-Chicken IgY H&L (HRP) (Abcam, cat. No.: ab6877) was diluted to 2 μg/ml with dilution buffer (10% BlockAce) (prepared 5000-fold dilution just before use), and added to a 96-well microplate at 100 μL/well. Incubated at room temperature for 1 hour and washed (200 μL/well, 3 times).
・Bring TMB (SurModics, TMBW-1000-01) to room temperature before use, add 100 μL/well to a 96-well microplate, and mix (in the dark, at room temperature).
- Stop with 50 μL/well of 1N sulfuric acid - Measure absorbance at 450 nm - Data analysis with GraphPad Prism 6.04.
組み換えヒトLSR-Fc、カニクイザルLSR-Fc、ラットLSR-Fc、マウスLSR-Fcを50mM carbonate-bicarbonate buffer, pH 9.6で2.5μg/mLに希釈し、50μL/wellでImmuno 96well MicroWell Solid Plate(Maxisorp, Nunc)に添加し、プレートにシールをした。プレートを揺らして、液が全体に行き渡ることを確認した。次いで、以下の手順に従った。
・静置(4℃、一晩)
・洗浄(200 μL/well x3)
・ブロッキングバッファーを200μL/wellずつ96wellマイクロプレートに添加
・室温で1時間反応
・洗浄(200μL/well、3回)
・ニワトリマウスキメラ抗LSR抗体(#27-6mF18)を希釈バッファーで100nMから10倍希釈系列で希釈し、100μL/wellで96wellマイクロプレートに添加
・室温で1時間反応
・洗浄(200μL/well、3回)
・Goat Anti-Chicken IgY H&L (HRP) (abcam社, cat.No.: ab6877)を希釈バッファー(10% BlockAce)で2μg/mlに希釈し(5000倍希釈を用時調製)、100μL/wellずつ96wellマイクロプレートに添加
・室温で1時間反応
・洗浄(200μL/well、3回)
・TMB(SurModics、TMBW-1000-01)を使用前に常温に戻し、100μL/wellずつ96wellマイクロプレートに添加
・撹拌(遮光下、室温)
・1N-硫酸50μL/wellで停止
・450nmで吸光度測定
・GraphPad Prism 6.04でデータ解析。 (Method)
Recombinant human LSR-Fc, cynomolgus monkey LSR-Fc, rat LSR-Fc, and mouse LSR-Fc were diluted to 2.5 μg/mL with 50 mM carbonate-bicarbonate buffer, pH 9.6, and added to an Immuno 96-well MicroWell Solid Plate (Maxisorp, Nunc) at 50 μL/well, and the plate was sealed. The plate was rocked to ensure that the liquid was distributed throughout. The following procedure was then followed.
- Leave it still (4℃, overnight)
Washing (200 μL/well x 3)
Add 200 μL of blocking buffer per well to a 96-well microplate. Incubate at room temperature for 1 hour. Wash (200 μL per well, 3 times).
・Dilute chicken mouse chimeric anti-LSR antibody (#27-6mF18) in dilution buffer in a 10-fold serial dilution starting from 100 nM, add 100 μL/well to a 96-well microplate, react at room temperature for 1 hour, and wash (200 μL/well, 3 times).
・Goat Anti-Chicken IgY H&L (HRP) (Abcam, cat. No.: ab6877) was diluted to 2 μg/ml with dilution buffer (10% BlockAce) (prepared 5000-fold dilution just before use), and added to a 96-well microplate at 100 μL/well. Incubated at room temperature for 1 hour and washed (200 μL/well, 3 times).
・Bring TMB (SurModics, TMBW-1000-01) to room temperature before use, add 100 μL/well to a 96-well microplate, and mix (in the dark, at room temperature).
- Stop with 50 μL/well of 1N sulfuric acid - Measure absorbance at 450 nm - Data analysis with GraphPad Prism 6.04.
(結果)
発明者等が作成した抗LSRモノクローナル抗体#27-6はマウスLSRにも交叉反応を示す(WO/2015/098113)。抗LSRモノクローナル抗体#27-6のFc領域のアミノ酸配列を置換することで、ADCC活性とCDC活性を示さない抗LSRモノクローナル抗体#27-6 mF-18を作成した。これは、以下の実施例において示される抗腫瘍効果が、ADCC活性によるものでもCDC活性によるものでもないことを示すためのものである。抗LSRモノクローナル抗体#27-6 mF-18はヒトLSRおよびマウスLSRに反応することをELISA解析により明らかにした(図2)。 (result)
The anti-LSR monoclonal antibody #27-6 created by the inventors also cross-reacts with mouse LSR (WO/2015/098113). By substituting the amino acid sequence of the Fc region of the anti-LSR monoclonal antibody #27-6, the anti-LSR monoclonal antibody #27-6 mF-18, which does not exhibit ADCC activity or CDC activity, was created. This is to demonstrate that the antitumor effect shown in the following examples is not due to ADCC activity or CDC activity. ELISA analysis revealed that the anti-LSR monoclonal antibody #27-6 mF-18 reacts with human LSR and mouse LSR (Figure 2).
発明者等が作成した抗LSRモノクローナル抗体#27-6はマウスLSRにも交叉反応を示す(WO/2015/098113)。抗LSRモノクローナル抗体#27-6のFc領域のアミノ酸配列を置換することで、ADCC活性とCDC活性を示さない抗LSRモノクローナル抗体#27-6 mF-18を作成した。これは、以下の実施例において示される抗腫瘍効果が、ADCC活性によるものでもCDC活性によるものでもないことを示すためのものである。抗LSRモノクローナル抗体#27-6 mF-18はヒトLSRおよびマウスLSRに反応することをELISA解析により明らかにした(図2)。 (result)
The anti-LSR monoclonal antibody #27-6 created by the inventors also cross-reacts with mouse LSR (WO/2015/098113). By substituting the amino acid sequence of the Fc region of the anti-LSR monoclonal antibody #27-6, the anti-LSR monoclonal antibody #27-6 mF-18, which does not exhibit ADCC activity or CDC activity, was created. This is to demonstrate that the antitumor effect shown in the following examples is not due to ADCC activity or CDC activity. ELISA analysis revealed that the anti-LSR monoclonal antibody #27-6 mF-18 reacts with human LSR and mouse LSR (Figure 2).
実施例3:FACSおよびウェスタンブロットによるLSRの発現解析
FACSによるLSRの発現解析(図3左)
4T1(マウス乳癌細胞株)、HM-1(OV2944-HM-1、マウス卵巣癌細胞株)、B16F10(マウス悪性黒色腫細胞株)、CT26(マウス大腸癌細胞株)、MC-38(マウス大腸癌)、およびMC-38-mLSR-13(mLSR発現マウス大腸癌)について、FACS解析によりLSRの発現を解析した。ニワトリマウスキメラ抗LSR抗体(#27-6mF18)を1次抗体として用い、2次抗体としてはFITC標識GoatAnti-Mouse IgG (H+L chain specific)(southern biothech社)を用い、FACS CantoII (BD社)を用いて測定し、測定データは、FlowJo(商標)ソフトウェア(Tree star社)を用いて解析した。 Example 3: LSR expression analysis by FACS and Western blot LSR expression analysis by FACS (Figure 3, left)
The expression of LSR was analyzed by FACS analysis for 4T1 (mouse breast cancer cell line), HM-1 (OV2944-HM-1, mouse ovarian cancer cell line), B16F10 (mouse malignant melanoma cell line), CT26 (mouse colon cancer cell line), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mLSR-expressing mouse colon cancer). Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was used as the primary antibody, and FITC-labeled Goat Anti-Mouse IgG (H+L chain specific) (southern biotech) was used as the secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using FlowJo (trademark) software (Tree star).
FACSによるLSRの発現解析(図3左)
4T1(マウス乳癌細胞株)、HM-1(OV2944-HM-1、マウス卵巣癌細胞株)、B16F10(マウス悪性黒色腫細胞株)、CT26(マウス大腸癌細胞株)、MC-38(マウス大腸癌)、およびMC-38-mLSR-13(mLSR発現マウス大腸癌)について、FACS解析によりLSRの発現を解析した。ニワトリマウスキメラ抗LSR抗体(#27-6mF18)を1次抗体として用い、2次抗体としてはFITC標識GoatAnti-Mouse IgG (H+L chain specific)(southern biothech社)を用い、FACS CantoII (BD社)を用いて測定し、測定データは、FlowJo(商標)ソフトウェア(Tree star社)を用いて解析した。 Example 3: LSR expression analysis by FACS and Western blot LSR expression analysis by FACS (Figure 3, left)
The expression of LSR was analyzed by FACS analysis for 4T1 (mouse breast cancer cell line), HM-1 (OV2944-HM-1, mouse ovarian cancer cell line), B16F10 (mouse malignant melanoma cell line), CT26 (mouse colon cancer cell line), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mLSR-expressing mouse colon cancer). Chicken mouse chimeric anti-LSR antibody (#27-6mF18) was used as the primary antibody, and FITC-labeled Goat Anti-Mouse IgG (H+L chain specific) (southern biotech) was used as the secondary antibody. Measurements were performed using FACS CantoII (BD), and the measurement data was analyzed using FlowJo (trademark) software (Tree star).
ウェスタンブロットによるLSRの発現解析(図3右)
10cm plateに撒いた細胞株が90%コンフルエント程度に達した後、培養上清を捨て、氷冷PBSで洗浄後、セルスクレイパーで細胞を剥がして回収し、RIPA buffer (10 mM Tris-HCl,pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1×phosphatase inhibitor cocktail (Nacalai Tesque) および 1×protease inhibitor cocktail (Nacalai Tesque))を用いてタンパク質抽出を行い、ウェスタンブロット法によりタンパク質発現差の解析を行った。一次抗体は以下のものを用いた。抗LSR抗体(CST #14804)、抗beta-actin抗体(sc-69879) (Santa Cruz Biotechnology (Santa Cruz, CA))。 Expression analysis of LSR by Western blot (Figure 3, right)
After the cell lines seeded on a 10 cm plate reached approximately 90% confluence, the culture supernatant was discarded, the cells were washed with ice-cold PBS, and then detached and collected with a cell scraper. Proteins were extracted using RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1× phosphatase inhibitor cocktail (Nacalai Tesque) and 1× protease inhibitor cocktail (Nacalai Tesque)), and differential protein expression was analyzed by Western blotting. The primary antibodies used were anti-LSR antibody (CST #14804) and anti-beta-actin antibody (sc-69879) (Santa Cruz Biotechnology, Santa Cruz, CA).
10cm plateに撒いた細胞株が90%コンフルエント程度に達した後、培養上清を捨て、氷冷PBSで洗浄後、セルスクレイパーで細胞を剥がして回収し、RIPA buffer (10 mM Tris-HCl,pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1×phosphatase inhibitor cocktail (Nacalai Tesque) および 1×protease inhibitor cocktail (Nacalai Tesque))を用いてタンパク質抽出を行い、ウェスタンブロット法によりタンパク質発現差の解析を行った。一次抗体は以下のものを用いた。抗LSR抗体(CST #14804)、抗beta-actin抗体(sc-69879) (Santa Cruz Biotechnology (Santa Cruz, CA))。 Expression analysis of LSR by Western blot (Figure 3, right)
After the cell lines seeded on a 10 cm plate reached approximately 90% confluence, the culture supernatant was discarded, the cells were washed with ice-cold PBS, and then detached and collected with a cell scraper. Proteins were extracted using RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 1× phosphatase inhibitor cocktail (Nacalai Tesque) and 1× protease inhibitor cocktail (Nacalai Tesque)), and differential protein expression was analyzed by Western blotting. The primary antibodies used were anti-LSR antibody (CST #14804) and anti-beta-actin antibody (sc-69879) (Santa Cruz Biotechnology, Santa Cruz, CA).
(結果)
4T1(マウス乳癌)、HM-1(OV2944-HM-1、マウス卵巣癌)、B16F10(マウスメラノーマ) 、CT-26(マウス大腸癌)、MC-38(マウス大腸癌)およびMC-38-mLSR-13(マウス大腸癌)細胞株を用いてマウスLSRの発現を確認したところ、FACSおよびウェスタンブロット法のいずれにおいても4T1およびMC-38-mLSR-13においてマウスLSRの発現が認められた(図3)。 (result)
Expression of mouse LSR was confirmed using 4T1 (mouse breast cancer), HM-1 (OV2944-HM-1, mouse ovarian cancer), B16F10 (mouse melanoma), CT-26 (mouse colon cancer), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mouse colon cancer) cell lines. Expression of mouse LSR was confirmed in 4T1 and MC-38-mLSR-13 by both FACS and Western blotting (Figure 3).
4T1(マウス乳癌)、HM-1(OV2944-HM-1、マウス卵巣癌)、B16F10(マウスメラノーマ) 、CT-26(マウス大腸癌)、MC-38(マウス大腸癌)およびMC-38-mLSR-13(マウス大腸癌)細胞株を用いてマウスLSRの発現を確認したところ、FACSおよびウェスタンブロット法のいずれにおいても4T1およびMC-38-mLSR-13においてマウスLSRの発現が認められた(図3)。 (result)
Expression of mouse LSR was confirmed using 4T1 (mouse breast cancer), HM-1 (OV2944-HM-1, mouse ovarian cancer), B16F10 (mouse melanoma), CT-26 (mouse colon cancer), MC-38 (mouse colon cancer), and MC-38-mLSR-13 (mouse colon cancer) cell lines. Expression of mouse LSR was confirmed in 4T1 and MC-38-mLSR-13 by both FACS and Western blotting (Figure 3).
実施例4:4T1シンジェニックマウスモデル(皮下移植)を用いた抗LSR抗体(#27-6mF18)のin vivo薬効試験
パラフィン包埋組織の薄切を脱パラフィン後、アルコールによる脱水を行った。LSRに対する免疫組織化学染色法は抗LSR抗体(CST #14804)とChemMate Envision kit HRP 500T(Dako社:K5007)を用いて行った。 Example 4: In vivo efficacy test of anti-LSR antibody (#27-6mF18) using 4T1 syngenic mouse model (subcutaneous transplantation) Paraffin-embedded tissue was sliced, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for LSR was performed using anti-LSR antibody (CST #14804) and ChemMate Envision kit HRP 500T (Dako: K5007).
パラフィン包埋組織の薄切を脱パラフィン後、アルコールによる脱水を行った。LSRに対する免疫組織化学染色法は抗LSR抗体(CST #14804)とChemMate Envision kit HRP 500T(Dako社:K5007)を用いて行った。 Example 4: In vivo efficacy test of anti-LSR antibody (#27-6mF18) using 4T1 syngenic mouse model (subcutaneous transplantation) Paraffin-embedded tissue was sliced, deparaffinized, and dehydrated with alcohol. Immunohistochemical staining for LSR was performed using anti-LSR antibody (CST #14804) and ChemMate Envision kit HRP 500T (Dako: K5007).
本実施例における4T1シンジェニックマウスモデル(皮下移植)を用いた抗LSR抗体(#27-6mF18)のin vivo薬効試験の模式図を図4に示す。具体的には、8週齢のBalb/c雌性マウスに4T1細胞株を2.0×105個皮下移植し、移植した翌日に4群に群分けし、PBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および抗LSR抗体(#27-6mF18)(20mg/kg)の投与を開始した。図4の実験概要に記載の時点で薬剤の腹腔内投与、腫瘍体積計測、体重測定を実施した。
A schematic diagram of the in vivo efficacy test of anti-LSR antibody (#27-6mF18) using the 4T1 syngenic mouse model (subcutaneous transplantation) in this example is shown in Figure 4. Specifically, 2.0 x 10 5 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and the day after transplantation, the mice were divided into 4 groups, and administration of PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) was started. Intraperitoneal administration of drugs, measurement of tumor volume, and body weight were performed at the time points described in the experimental outline in Figure 4.
(結果)
4T1シンジェニックマウスモデルを作成し、抗LSRモノクローナル抗体#27-6 mF-18の薬効を図4の実験スケジュールで評価した。PBS投与群、コントロール抗体投与群(20mg/kg)と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群は5mg/kgおよび20mg/kgのいずれの投与量においても有意な抗腫瘍効果が認められた(図5)。 (result)
A 4T1 syngenic mouse model was created, and the efficacy of anti-LSR monoclonal antibody #27-6 mF-18 was evaluated according to the experimental schedule in Figure 4. Compared to the PBS-administered group and the control antibody-administered group (20 mg/kg), the anti-LSR monoclonal antibody #27-6 mF-18-administered group showed significant antitumor effects at both doses of 5 mg/kg and 20 mg/kg (Figure 5).
4T1シンジェニックマウスモデルを作成し、抗LSRモノクローナル抗体#27-6 mF-18の薬効を図4の実験スケジュールで評価した。PBS投与群、コントロール抗体投与群(20mg/kg)と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群は5mg/kgおよび20mg/kgのいずれの投与量においても有意な抗腫瘍効果が認められた(図5)。 (result)
A 4T1 syngenic mouse model was created, and the efficacy of anti-LSR monoclonal antibody #27-6 mF-18 was evaluated according to the experimental schedule in Figure 4. Compared to the PBS-administered group and the control antibody-administered group (20 mg/kg), the anti-LSR monoclonal antibody #27-6 mF-18-administered group showed significant antitumor effects at both doses of 5 mg/kg and 20 mg/kg (Figure 5).
実施例5:抗LSR抗体(#27-6mF18)の薬効におけるCD8+T細胞の影響
本実施例における、抗LSR抗体(#27-6mF18)の薬効におけるCD8+T細胞の影響を検証するin vivo薬効試験の模式図を図6に示す。 Example 5: Effect of CD8 + T cells on efficacy of anti-LSR antibody (#27-6mF18) A schematic diagram of the in vivo efficacy test in this example to verify the effect of CD8 + T cells on the efficacy of anti-LSR antibody (#27-6mF18) is shown in FIG.
本実施例における、抗LSR抗体(#27-6mF18)の薬効におけるCD8+T細胞の影響を検証するin vivo薬効試験の模式図を図6に示す。 Example 5: Effect of CD8 + T cells on efficacy of anti-LSR antibody (#27-6mF18) A schematic diagram of the in vivo efficacy test in this example to verify the effect of CD8 + T cells on the efficacy of anti-LSR antibody (#27-6mF18) is shown in FIG.
具体的には、8週齢のBalb/c雌性マウスに4T1細胞株を2.0×105個皮下移植し、移植した翌日に4群に群分けし、isotype control抗体(5mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、抗CD8抗体(0.2mg/body)(BioXCell)および抗LSR抗体(#27-6mF18)(5mg/kg)+抗CD8抗体(0.2mg/body)の投与を開始した。図6の実験概要に記載の時点で薬剤の腹腔内投与、腫瘍体積計測、体重測定を実施した。
Specifically, 2.0x105 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and the day after transplantation, the mice were divided into 4 groups and administered isotype control antibody (5mg/kg), anti-LSR antibody (#27-6mF18) (5mg/kg), anti-CD8 antibody (0.2mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5mg/kg) + anti-CD8 antibody (0.2mg/body). Drugs were intraperitoneally administered, tumor volume was measured, and body weight was measured at the time points described in the experimental outline in Figure 6.
(結果)
4T1シンジェニックマウスモデルに対する抗LSRモノクローナル抗体#27-6 mF-18の抗腫瘍効果に腫瘍免疫が関与しているかどうか検証するために、抗CD8抗体を投与することでCD8+T細胞を欠失させた条件での抗LSRモノクローナル抗体#27-6 mF-18による薬効試験を図6に示す実験スケジュールで実施した。抗CD8抗体と抗LSRモノクローナル抗体#27-6 mF-18を組み合わせた条件では、抗LSRモノクローナル抗体#27-6 mF-18により発揮された抗腫瘍効果が打ち消されることが確認された(図7)。このことから、抗LSR抗体による抗腫瘍効果は、CD8+T細胞に依存するものであることが示された。 (result)
To verify whether tumor immunity is involved in the antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 on the 4T1 syngenic mouse model, a drug efficacy test using anti-LSR monoclonal antibody #27-6 mF-18 under conditions in which CD8+ T cells were deleted by administration of anti-CD8 antibody was performed according to the experimental schedule shown in Figure 6. It was confirmed that the antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 was negated under conditions in which anti-CD8 antibody and anti-LSR monoclonal antibody #27-6 mF-18 were combined (Figure 7). This indicates that the antitumor effect of anti-LSR antibody is dependent on CD8+ T cells.
4T1シンジェニックマウスモデルに対する抗LSRモノクローナル抗体#27-6 mF-18の抗腫瘍効果に腫瘍免疫が関与しているかどうか検証するために、抗CD8抗体を投与することでCD8+T細胞を欠失させた条件での抗LSRモノクローナル抗体#27-6 mF-18による薬効試験を図6に示す実験スケジュールで実施した。抗CD8抗体と抗LSRモノクローナル抗体#27-6 mF-18を組み合わせた条件では、抗LSRモノクローナル抗体#27-6 mF-18により発揮された抗腫瘍効果が打ち消されることが確認された(図7)。このことから、抗LSR抗体による抗腫瘍効果は、CD8+T細胞に依存するものであることが示された。 (result)
To verify whether tumor immunity is involved in the antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 on the 4T1 syngenic mouse model, a drug efficacy test using anti-LSR monoclonal antibody #27-6 mF-18 under conditions in which CD8+ T cells were deleted by administration of anti-CD8 antibody was performed according to the experimental schedule shown in Figure 6. It was confirmed that the antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 was negated under conditions in which anti-CD8 antibody and anti-LSR monoclonal antibody #27-6 mF-18 were combined (Figure 7). This indicates that the antitumor effect of anti-LSR antibody is dependent on CD8+ T cells.
実施例6:抗CD8抗体投与によるCD8+T細胞depletionの確認実験
抗CD8抗体を投与することでマウス体内のCD8+T細胞が除去出来ていることを確認する実験を行った。8週齢のBalb/c雌性マウスに4T1細胞株を2.0×105個皮下移植し、移植した当日と2日後の2回、抗CD8抗体(0.2mg/body)を腹腔内投与した。4日目に脾臓から細胞を単離し、anti-CD45 (30-F11; catalog No.103116), anti-CD8 (53-6.7; catalog No.100734), anti-CD4 (RM4-5; catalog No. 100516)の抗体で表面抗原を4度で30分間染色した。染色した細胞を洗浄後、FACS CantoII (BD社)を用いて測定し、BD FACS Diva software (BD Biosciences)を用いてデータを解析した。 Example 6: Experiment to confirm CD8+ T cell depletion by administration of anti-CD8 antibody An experiment was conducted to confirm that CD8+ T cells in mice were removed by administration of anti-CD8 antibody. 2.0×10 5 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and anti-CD8 antibody (0.2 mg/body) was intraperitoneally administered twice, on the day of transplantation and two days later. On the fourth day, cells were isolated from the spleen, and surface antigens were stained with anti-CD45 (30-F11; catalog No. 103116), anti-CD8 (53-6.7; catalog No. 100734), and anti-CD4 (RM4-5; catalog No. 100516) antibodies at 4°C for 30 minutes. After washing the stained cells, they were measured using FACS CantoII (BD), and the data was analyzed using BD FACS Diva software (BD Biosciences).
抗CD8抗体を投与することでマウス体内のCD8+T細胞が除去出来ていることを確認する実験を行った。8週齢のBalb/c雌性マウスに4T1細胞株を2.0×105個皮下移植し、移植した当日と2日後の2回、抗CD8抗体(0.2mg/body)を腹腔内投与した。4日目に脾臓から細胞を単離し、anti-CD45 (30-F11; catalog No.103116), anti-CD8 (53-6.7; catalog No.100734), anti-CD4 (RM4-5; catalog No. 100516)の抗体で表面抗原を4度で30分間染色した。染色した細胞を洗浄後、FACS CantoII (BD社)を用いて測定し、BD FACS Diva software (BD Biosciences)を用いてデータを解析した。 Example 6: Experiment to confirm CD8+ T cell depletion by administration of anti-CD8 antibody An experiment was conducted to confirm that CD8+ T cells in mice were removed by administration of anti-CD8 antibody. 2.0×10 5 4T1 cell lines were subcutaneously transplanted into 8-week-old Balb/c female mice, and anti-CD8 antibody (0.2 mg/body) was intraperitoneally administered twice, on the day of transplantation and two days later. On the fourth day, cells were isolated from the spleen, and surface antigens were stained with anti-CD45 (30-F11; catalog No. 103116), anti-CD8 (53-6.7; catalog No. 100734), and anti-CD4 (RM4-5; catalog No. 100516) antibodies at 4°C for 30 minutes. After washing the stained cells, they were measured using FACS CantoII (BD), and the data was analyzed using BD FACS Diva software (BD Biosciences).
(結果)
抗CD8抗体投与によるマウスからCD8+T細胞の除去については、4T1シンジェニックマウスモデルに抗CD8抗体を投与した後、脾臓中のT細胞をFACS解析することで、CD8+T細胞が消失していることを確認した(図8)。 (result)
Regarding the removal of CD8+ T cells from mice by administration of anti-CD8 antibodies, we administered anti-CD8 antibodies to a 4T1 syngenic mouse model, and then performed FACS analysis of T cells in the spleen to confirm the disappearance of CD8+ T cells (Figure 8).
抗CD8抗体投与によるマウスからCD8+T細胞の除去については、4T1シンジェニックマウスモデルに抗CD8抗体を投与した後、脾臓中のT細胞をFACS解析することで、CD8+T細胞が消失していることを確認した(図8)。 (result)
Regarding the removal of CD8+ T cells from mice by administration of anti-CD8 antibodies, we administered anti-CD8 antibodies to a 4T1 syngenic mouse model, and then performed FACS analysis of T cells in the spleen to confirm the disappearance of CD8+ T cells (Figure 8).
実施例7:腫瘍内浸潤CD8+T細胞の解析
抗LSR抗体投与により腫瘍内浸潤CD8+T細胞の数と、活性化の割合を評価するために、実施例5のisotype control抗体(5mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)の2群について、最終日(19日目)の腫瘍を摘出した。摘出した腫瘍組織はgentleMACS Octo Dissociator (Miltenyi Biotec)とmouse Tumor Dissociation Kit cocktail (Miltenyi Biotec; cat#130-096-730)を用いてシングルセル懸濁液を作成した。細胞懸濁液を100μm pre-separation filters (Miltenyi Biotec; cat#130-098-463, cat#130-098-458)を通し、フィルターを通過した細胞懸濁液を回収した。細胞懸濁液をmouse FcR Blocking Reagent (Miltenyi Biotec; catalog No. 130-092-575)で反応させた後、anti-CD3 (145-2C11; catalog No. 553062, BD Biosciences), anti-CD69 (H1.2F3; catalog No.104508, BioLegend), anti-CD45 (30-F11; catalog No.103116, BioLegend), anti-CD8 (53-6.7; catalog No.100734, BioLegend)を用いて表面抗原の染色を4度で30分間染色した。染色した細胞を洗浄後、FACS CantoII (BD社)を用いて測定し、FlowJo software (Tree Star)を用いてデータを解析した。 Example 7: Analysis of tumor-infiltrating CD8+ T cells To evaluate the number of tumor-infiltrating CD8+ T cells and the rate of activation by anti-LSR antibody administration, tumors were excised on the final day (day 19) for the two groups of isotype control antibody (5 mg/kg) and anti-LSR antibody (#27-6mF18) (5 mg/kg) in Example 5. Single cell suspensions were prepared from the excised tumor tissues using gentleMACS Octo Dissociator (Miltenyi Biotec) and mouse Tumor Dissociation Kit cocktail (Miltenyi Biotec; cat#130-096-730). The cell suspension was passed through 100 μm pre-separation filters (Miltenyi Biotec; cat#130-098-463, cat#130-098-458), and the cell suspension that passed through the filters was collected. The cell suspension was reacted with mouse FcR Blocking Reagent (Miltenyi Biotec; catalog No. 130-092-575), and then surface antigens were stained with anti-CD3 (145-2C11; catalog No. 553062, BD Biosciences), anti-CD69 (H1.2F3; catalog No.104508, BioLegend), anti-CD45 (30-F11; catalog No.103116, BioLegend), and anti-CD8 (53-6.7; catalog No.100734, BioLegend) at 4°C for 30 minutes. After washing, the stained cells were measured using a FACS CantoII (BD) and the data were analyzed using FlowJo software (Tree Star).
抗LSR抗体投与により腫瘍内浸潤CD8+T細胞の数と、活性化の割合を評価するために、実施例5のisotype control抗体(5mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)の2群について、最終日(19日目)の腫瘍を摘出した。摘出した腫瘍組織はgentleMACS Octo Dissociator (Miltenyi Biotec)とmouse Tumor Dissociation Kit cocktail (Miltenyi Biotec; cat#130-096-730)を用いてシングルセル懸濁液を作成した。細胞懸濁液を100μm pre-separation filters (Miltenyi Biotec; cat#130-098-463, cat#130-098-458)を通し、フィルターを通過した細胞懸濁液を回収した。細胞懸濁液をmouse FcR Blocking Reagent (Miltenyi Biotec; catalog No. 130-092-575)で反応させた後、anti-CD3 (145-2C11; catalog No. 553062, BD Biosciences), anti-CD69 (H1.2F3; catalog No.104508, BioLegend), anti-CD45 (30-F11; catalog No.103116, BioLegend), anti-CD8 (53-6.7; catalog No.100734, BioLegend)を用いて表面抗原の染色を4度で30分間染色した。染色した細胞を洗浄後、FACS CantoII (BD社)を用いて測定し、FlowJo software (Tree Star)を用いてデータを解析した。 Example 7: Analysis of tumor-infiltrating CD8+ T cells To evaluate the number of tumor-infiltrating CD8+ T cells and the rate of activation by anti-LSR antibody administration, tumors were excised on the final day (day 19) for the two groups of isotype control antibody (5 mg/kg) and anti-LSR antibody (#27-6mF18) (5 mg/kg) in Example 5. Single cell suspensions were prepared from the excised tumor tissues using gentleMACS Octo Dissociator (Miltenyi Biotec) and mouse Tumor Dissociation Kit cocktail (Miltenyi Biotec; cat#130-096-730). The cell suspension was passed through 100 μm pre-separation filters (Miltenyi Biotec; cat#130-098-463, cat#130-098-458), and the cell suspension that passed through the filters was collected. The cell suspension was reacted with mouse FcR Blocking Reagent (Miltenyi Biotec; catalog No. 130-092-575), and then surface antigens were stained with anti-CD3 (145-2C11; catalog No. 553062, BD Biosciences), anti-CD69 (H1.2F3; catalog No.104508, BioLegend), anti-CD45 (30-F11; catalog No.103116, BioLegend), and anti-CD8 (53-6.7; catalog No.100734, BioLegend) at 4°C for 30 minutes. After washing, the stained cells were measured using a FACS CantoII (BD) and the data were analyzed using FlowJo software (Tree Star).
(結果)
抗LSRモノクローナル抗体#27-6 mF-18投与群の腫瘍内に浸潤したCD8+T細胞をFACSで解析した。その結果、コントロール抗体投与群と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群では腫瘍内に浸潤したCD8+T細胞の数と、活性化(CD69)の割合の有意な上昇が認められた(図9)。したがって、LSRを阻害することにより免疫チェックポイント刺激が阻害され、腫瘍内へのCD8+T細胞の遊走と、活性化が促進されると考えられる。 (result)
CD8+ T cells infiltrating into the tumors of the anti-LSR monoclonal antibody #27-6 mF-18 treated group were analyzed by FACS. As a result, the number of CD8+ T cells infiltrating into the tumors and the proportion of activated (CD69) cells were significantly increased in the anti-LSR monoclonal antibody #27-6 mF-18 treated group compared to the control antibody treated group (Figure 9). Therefore, it is believed that inhibition of LSR inhibits immune checkpoint stimulation and promotes migration and activation of CD8+ T cells into the tumors.
抗LSRモノクローナル抗体#27-6 mF-18投与群の腫瘍内に浸潤したCD8+T細胞をFACSで解析した。その結果、コントロール抗体投与群と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群では腫瘍内に浸潤したCD8+T細胞の数と、活性化(CD69)の割合の有意な上昇が認められた(図9)。したがって、LSRを阻害することにより免疫チェックポイント刺激が阻害され、腫瘍内へのCD8+T細胞の遊走と、活性化が促進されると考えられる。 (result)
CD8+ T cells infiltrating into the tumors of the anti-LSR monoclonal antibody #27-6 mF-18 treated group were analyzed by FACS. As a result, the number of CD8+ T cells infiltrating into the tumors and the proportion of activated (CD69) cells were significantly increased in the anti-LSR monoclonal antibody #27-6 mF-18 treated group compared to the control antibody treated group (Figure 9). Therefore, it is believed that inhibition of LSR inhibits immune checkpoint stimulation and promotes migration and activation of CD8+ T cells into the tumors.
実施例8:4T1腫瘍組織中のケモカイン発現解析
8週齢のBalb/c雌性マウスに4T1細胞株を2.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれPBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および、抗LSR抗体(#27-6mF18)(20mg/kg)を週2回の頻度で合計6回、腹腔内に投与した。細胞移植後19日目に、各群のマウスから腫瘍を摘出した。摘出した4T1腫瘍組織に対して、1 mgの前記腫瘍組織あたり2μlのPBSを添加し、次いで、前記腫瘍組織を37℃で4時間インキュベートした。その後、前記腫瘍組織を遠心分離に供し(14,000 rpm, 4°C , 10 min)、次いで、上清を回収した。前記上清中のケモカイン(CXCL9、CXCL10)の濃度は、BioLegend Legendplex(商標), mouse proinflammatory chemokine panelを用いて定量した。 Example 8: Analysis of chemokine expression in 4T1 tumor tissue 2.0 x 10 5 cells of the 4T1 cell line were subcutaneously transplanted into 8-week-old Balb/c female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. On the 19th day after cell transplantation, tumors were excised from the mice in each group. 2 μl of PBS was added per 1 mg of the tumor tissue to the excised 4T1 tumor tissue, and then the tumor tissue was incubated at 37° C. for 4 hours. The tumor tissue was then centrifuged (14,000 rpm, 4° C., 10 min), and the supernatant was then collected. The concentrations of chemokines (CXCL9, CXCL10) in the supernatants were quantified using BioLegend Legendplex™, a mouse proinflammatory chemokine panel.
8週齢のBalb/c雌性マウスに4T1細胞株を2.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれPBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および、抗LSR抗体(#27-6mF18)(20mg/kg)を週2回の頻度で合計6回、腹腔内に投与した。細胞移植後19日目に、各群のマウスから腫瘍を摘出した。摘出した4T1腫瘍組織に対して、1 mgの前記腫瘍組織あたり2μlのPBSを添加し、次いで、前記腫瘍組織を37℃で4時間インキュベートした。その後、前記腫瘍組織を遠心分離に供し(14,000 rpm, 4°C , 10 min)、次いで、上清を回収した。前記上清中のケモカイン(CXCL9、CXCL10)の濃度は、BioLegend Legendplex(商標), mouse proinflammatory chemokine panelを用いて定量した。 Example 8: Analysis of chemokine expression in 4T1 tumor tissue 2.0 x 10 5 cells of the 4T1 cell line were subcutaneously transplanted into 8-week-old Balb/c female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. On the 19th day after cell transplantation, tumors were excised from the mice in each group. 2 μl of PBS was added per 1 mg of the tumor tissue to the excised 4T1 tumor tissue, and then the tumor tissue was incubated at 37° C. for 4 hours. The tumor tissue was then centrifuged (14,000 rpm, 4° C., 10 min), and the supernatant was then collected. The concentrations of chemokines (CXCL9, CXCL10) in the supernatants were quantified using BioLegend Legendplex™, a mouse proinflammatory chemokine panel.
(結果)
CXCL9とCXCL10とは、CD8+T細胞を誘引するケモカインとして知られていることから、前記腫瘍組織中のケモカイン(CXCL9、CXCL10)を定量した。その結果、前記腫瘍組織中では、コントロール抗体投与群と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群において、CXCL9とCXCL10との濃度が有意に高いことを確認した(図10)。したがって、抗LSRモノクローナル抗体#27-6 mF-18投与群では、腫瘍内のCXCL9とCXCL10との濃度の上昇によりCD8+T細胞の腫瘍内浸潤が促進されると考えられる。 (result)
CXCL9 and CXCL10 are known to be chemokines that attract CD8+ T cells, so the chemokines (CXCL9, CXCL10) in the tumor tissue were quantified. As a result, it was confirmed that the concentrations of CXCL9 and CXCL10 in the tumor tissue were significantly higher in the anti-LSR monoclonal antibody #27-6 mF-18 administration group than in the control antibody administration group (Figure 10). Therefore, it is considered that the increase in the concentrations of CXCL9 and CXCL10 in the tumor promotes the infiltration of CD8+ T cells into the tumor in the anti-LSR monoclonal antibody #27-6 mF-18 administration group.
CXCL9とCXCL10とは、CD8+T細胞を誘引するケモカインとして知られていることから、前記腫瘍組織中のケモカイン(CXCL9、CXCL10)を定量した。その結果、前記腫瘍組織中では、コントロール抗体投与群と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群において、CXCL9とCXCL10との濃度が有意に高いことを確認した(図10)。したがって、抗LSRモノクローナル抗体#27-6 mF-18投与群では、腫瘍内のCXCL9とCXCL10との濃度の上昇によりCD8+T細胞の腫瘍内浸潤が促進されると考えられる。 (result)
CXCL9 and CXCL10 are known to be chemokines that attract CD8+ T cells, so the chemokines (CXCL9, CXCL10) in the tumor tissue were quantified. As a result, it was confirmed that the concentrations of CXCL9 and CXCL10 in the tumor tissue were significantly higher in the anti-LSR monoclonal antibody #27-6 mF-18 administration group than in the control antibody administration group (Figure 10). Therefore, it is considered that the increase in the concentrations of CXCL9 and CXCL10 in the tumor promotes the infiltration of CD8+ T cells into the tumor in the anti-LSR monoclonal antibody #27-6 mF-18 administration group.
実施例9:免疫不全マウスを用いた抗LSR抗体(#27-6mF18)の薬効解析
7週齢のBalb/c nu/nu雌性マウスに4T1細胞株を2.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれPBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および、抗LSR抗体(#27-6mF18)(20mg/kg)を週2回の頻度で合計6回、腹腔内に投与した。図11に記載の時点で、腫瘍体積計測、体重測定および腫瘍重量の測定を実施した。 Example 9: Drug efficacy analysis of anti-LSR antibody (#27-6mF18) using immunodeficient mice 2.0 x 10 5 cells of the 4T1 cell line were subcutaneously transplanted into 7-week-old Balb/c nu/nu female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. At the time points shown in FIG. 11, tumor volume, body weight, and tumor weight were measured.
7週齢のBalb/c nu/nu雌性マウスに4T1細胞株を2.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれPBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および、抗LSR抗体(#27-6mF18)(20mg/kg)を週2回の頻度で合計6回、腹腔内に投与した。図11に記載の時点で、腫瘍体積計測、体重測定および腫瘍重量の測定を実施した。 Example 9: Drug efficacy analysis of anti-LSR antibody (#27-6mF18) using immunodeficient mice 2.0 x 10 5 cells of the 4T1 cell line were subcutaneously transplanted into 7-week-old Balb/c nu/nu female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. At the time points shown in FIG. 11, tumor volume, body weight, and tumor weight were measured.
(結果)
T細胞機能が欠如しているBalb/c nu/nuマウスを用いて4T1を皮下移植した担癌マウスを作成し、抗LSRモノクローナル抗体#27-6 mF-18のin vivoでの薬効を調べた。その結果、抗腫瘍効果が認められなかった。したがって、4T1担癌マウスに対する抗LSRモノクローナル抗体#27-6 mF-18の薬効は、T細胞依存的であることが確認された(図11)。 (result)
Balb/c nu/nu mice lacking T cell function were used to create tumor-bearing mice subcutaneously implanted with 4T1, and the in vivo efficacy of anti-LSR monoclonal antibody #27-6 mF-18 was examined. As a result, no antitumor effect was observed. Therefore, it was confirmed that the efficacy of anti-LSR monoclonal antibody #27-6 mF-18 in 4T1 tumor-bearing mice is T cell-dependent (Figure 11).
T細胞機能が欠如しているBalb/c nu/nuマウスを用いて4T1を皮下移植した担癌マウスを作成し、抗LSRモノクローナル抗体#27-6 mF-18のin vivoでの薬効を調べた。その結果、抗腫瘍効果が認められなかった。したがって、4T1担癌マウスに対する抗LSRモノクローナル抗体#27-6 mF-18の薬効は、T細胞依存的であることが確認された(図11)。 (result)
Balb/c nu/nu mice lacking T cell function were used to create tumor-bearing mice subcutaneously implanted with 4T1, and the in vivo efficacy of anti-LSR monoclonal antibody #27-6 mF-18 was examined. As a result, no antitumor effect was observed. Therefore, it was confirmed that the efficacy of anti-LSR monoclonal antibody #27-6 mF-18 in 4T1 tumor-bearing mice is T cell-dependent (Figure 11).
実施例10:MC38-mLSR細胞をC57BL/6マウスに移植して作成したシンジェニックモデルマウスを用いた抗LSR抗体(#27-6mF18)の薬効解析
8週齢のC57BL/6雌性マウスにMC38-mLSR細胞株を5.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれPBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および、抗LSR抗体(#27-6mF18)(20mg/kg)を週2回の頻度で合計6回、腹腔内に投与した。図12に記載の時点で腫瘍体積計測、体重測定および腫瘍重量の測定を実施した。 Example 10: Analysis of the efficacy of anti-LSR antibody (#27-6mF18) using syngenic model mice created by transplanting MC38-mLSR cells into C57BL/6 mice 5.0 x 10 5 MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. Measurements of tumor volume, body weight, and tumor weight were performed at the time points shown in FIG. 12.
8週齢のC57BL/6雌性マウスにMC38-mLSR細胞株を5.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれPBS、isotype control抗体(20mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、および、抗LSR抗体(#27-6mF18)(20mg/kg)を週2回の頻度で合計6回、腹腔内に投与した。図12に記載の時点で腫瘍体積計測、体重測定および腫瘍重量の測定を実施した。 Example 10: Analysis of the efficacy of anti-LSR antibody (#27-6mF18) using syngenic model mice created by transplanting MC38-mLSR cells into C57BL/6 mice 5.0 x 10 5 MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and PBS, isotype control antibody (20 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), and anti-LSR antibody (#27-6mF18) (20 mg/kg) were intraperitoneally administered to the mice in each group twice a week for a total of 6 times. Measurements of tumor volume, body weight, and tumor weight were performed at the time points shown in FIG. 12.
(結果)
実施例3では、マウス大腸癌細胞株MC-38においてはLSRの発現が陰性であるため、マウスLSR遺伝子を安定発現させたMC38-mLSR-13を樹立した(図3)。MC38-mLSR-13を用いてin vivoでの抗LSRモノクローナル抗体#27-6 mF-18の抗腫瘍効果を調べることにより、4T1を用いた場合(図5)と同様に抗腫瘍効果が認められるかどうか調べた。その結果、PBS投与群、コントロール抗体投与群(20 mg/kg)と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群は、5 mg/kgおよび20 mg/kgのいずれの投与量においても有意な抗腫瘍効果が認められた(図12)。 (result)
In Example 3, since the expression of LSR is negative in the mouse colon cancer cell line MC-38, MC38-mLSR-13, which stably expresses the mouse LSR gene, was established (Figure 3). By examining the in vivo antitumor effect of the anti-LSR monoclonal antibody #27-6 mF-18 using MC38-mLSR-13, it was examined whether the antitumor effect was observed as in the case of using 4T1 (Figure 5). As a result, the anti-LSR monoclonal antibody #27-6 mF-18 administration group showed a significant antitumor effect at both doses of 5 mg/kg and 20 mg/kg, compared with the PBS administration group and the control antibody administration group (20 mg/kg) (Figure 12).
実施例3では、マウス大腸癌細胞株MC-38においてはLSRの発現が陰性であるため、マウスLSR遺伝子を安定発現させたMC38-mLSR-13を樹立した(図3)。MC38-mLSR-13を用いてin vivoでの抗LSRモノクローナル抗体#27-6 mF-18の抗腫瘍効果を調べることにより、4T1を用いた場合(図5)と同様に抗腫瘍効果が認められるかどうか調べた。その結果、PBS投与群、コントロール抗体投与群(20 mg/kg)と比較して、抗LSRモノクローナル抗体#27-6 mF-18投与群は、5 mg/kgおよび20 mg/kgのいずれの投与量においても有意な抗腫瘍効果が認められた(図12)。 (result)
In Example 3, since the expression of LSR is negative in the mouse colon cancer cell line MC-38, MC38-mLSR-13, which stably expresses the mouse LSR gene, was established (Figure 3). By examining the in vivo antitumor effect of the anti-LSR monoclonal antibody #27-6 mF-18 using MC38-mLSR-13, it was examined whether the antitumor effect was observed as in the case of using 4T1 (Figure 5). As a result, the anti-LSR monoclonal antibody #27-6 mF-18 administration group showed a significant antitumor effect at both doses of 5 mg/kg and 20 mg/kg, compared with the PBS administration group and the control antibody administration group (20 mg/kg) (Figure 12).
実施例11:MC38-mLSRシンジェニックモデルマウスにおける抗LSR抗体(#27-6mF18)の薬効におけるCD8+T細胞の影響を検証するin vivo薬効試験
8週齢のC57BL/6雌性マウスにMC38-mLSR細胞株を5.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれisotype control抗体(5mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、抗CD8抗体(0.2mg/body)(BioXCell)および抗LSR抗体(#27-6mF18)(5mg/kg)+抗CD8抗体(0.2mg/body)の投与を開始した。図13に記載の時点で腫瘍体積計測、体重測定および腫瘍重量の測定を実施した。 Example 11: In vivo efficacy test to verify the influence of CD8 + T cells on the efficacy of anti-LSR antibody (#27-6mF18) in MC38-mLSR syngenic model mice 5.0 × 10 5 MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and each group was administered isotype control antibody (5 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), anti-CD8 antibody (0.2 mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5 mg/kg) + anti-CD8 antibody (0.2 mg/body). Tumor volume, body weight, and tumor weight were measured at the time points shown in Figure 13.
8週齢のC57BL/6雌性マウスにMC38-mLSR細胞株を5.0×105個皮下移植した。移植した翌日に前記マウスを4群に群分けし、各群のマウスに対して、それぞれisotype control抗体(5mg/kg)、抗LSR抗体(#27-6mF18)(5mg/kg)、抗CD8抗体(0.2mg/body)(BioXCell)および抗LSR抗体(#27-6mF18)(5mg/kg)+抗CD8抗体(0.2mg/body)の投与を開始した。図13に記載の時点で腫瘍体積計測、体重測定および腫瘍重量の測定を実施した。 Example 11: In vivo efficacy test to verify the influence of CD8 + T cells on the efficacy of anti-LSR antibody (#27-6mF18) in MC38-mLSR syngenic model mice 5.0 × 10 5 MC38-mLSR cell lines were subcutaneously transplanted into 8-week-old C57BL/6 female mice. The day after transplantation, the mice were divided into 4 groups, and each group was administered isotype control antibody (5 mg/kg), anti-LSR antibody (#27-6mF18) (5 mg/kg), anti-CD8 antibody (0.2 mg/body) (BioXCell), and anti-LSR antibody (#27-6mF18) (5 mg/kg) + anti-CD8 antibody (0.2 mg/body). Tumor volume, body weight, and tumor weight were measured at the time points shown in Figure 13.
(結果)
MC38-mLSR-13移植マウスに対して抗CD8抗体と抗LSRモノクローナル抗体#27-6 mF-18を投与することによって、CD8+T細胞を除去した場合の抗LSRモノクローナル抗体#27-6 mF-18による抗腫瘍効果を調べた。その結果、抗LSRモノクローナル抗体#27-6 mF-18により発揮された抗腫瘍効果が打ち消されることが確認された(図13)。LSRモノクローナル抗体#27-6 mF-18が免疫チェックポイント阻害活性により抗腫瘍効果を発揮することについて、4T1だけでなくMC38-mLSR-13においても確認することが出来た。 (result)
The antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 was examined when CD8+ T cells were removed by administering anti-CD8 antibody and anti-LSR monoclonal antibody #27-6 mF-18 to MC38-mLSR-13-implanted mice. As a result, it was confirmed that the antitumor effect exerted by anti-LSR monoclonal antibody #27-6 mF-18 was negated (Figure 13). It was confirmed that LSR monoclonal antibody #27-6 mF-18 exerts an antitumor effect by immune checkpoint inhibitory activity not only in 4T1 but also in MC38-mLSR-13.
MC38-mLSR-13移植マウスに対して抗CD8抗体と抗LSRモノクローナル抗体#27-6 mF-18を投与することによって、CD8+T細胞を除去した場合の抗LSRモノクローナル抗体#27-6 mF-18による抗腫瘍効果を調べた。その結果、抗LSRモノクローナル抗体#27-6 mF-18により発揮された抗腫瘍効果が打ち消されることが確認された(図13)。LSRモノクローナル抗体#27-6 mF-18が免疫チェックポイント阻害活性により抗腫瘍効果を発揮することについて、4T1だけでなくMC38-mLSR-13においても確認することが出来た。 (result)
The antitumor effect of anti-LSR monoclonal antibody #27-6 mF-18 was examined when CD8+ T cells were removed by administering anti-CD8 antibody and anti-LSR monoclonal antibody #27-6 mF-18 to MC38-mLSR-13-implanted mice. As a result, it was confirmed that the antitumor effect exerted by anti-LSR monoclonal antibody #27-6 mF-18 was negated (Figure 13). It was confirmed that LSR monoclonal antibody #27-6 mF-18 exerts an antitumor effect by immune checkpoint inhibitory activity not only in 4T1 but also in MC38-mLSR-13.
これらの結果から、抗LSR抗体は腫瘍細胞上のLSRとT細胞上の未知の受容体の相互作用を阻害することで腫瘍免疫を活性化し、CD8+T細胞の活性化を介して抗腫瘍効果を示すことが示唆された。
These results suggest that anti-LSR antibodies activate tumor immunity by inhibiting the interaction between LSR on tumor cells and an unknown receptor on T cells, and exert an antitumor effect through activation of CD8+ T cells.
実施例12:その他のLSRの抑制因子
本実施例では、抗LSR抗体#9-7、#16-6、#26-2、#1-25、#1-43、siRNA(配列番号15または16)、または小分子化合物のLSR阻害剤を使用して、実施例4~11と同様の試験を行う。 Example 12: Other LSR inhibitors In this example, tests similar to those in Examples 4 to 11 are performed using anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, and #1-43, siRNA (SEQ ID NO: 15 or 16), or small molecule compounds as LSR inhibitors.
本実施例では、抗LSR抗体#9-7、#16-6、#26-2、#1-25、#1-43、siRNA(配列番号15または16)、または小分子化合物のLSR阻害剤を使用して、実施例4~11と同様の試験を行う。 Example 12: Other LSR inhibitors In this example, tests similar to those in Examples 4 to 11 are performed using anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, and #1-43, siRNA (SEQ ID NO: 15 or 16), or small molecule compounds as LSR inhibitors.
具体的には、実施例4と同様に、抗LSR抗体#9-7、#16-6、#26-2、#1-25、#1-43、siRNA(配列番号15または16)、または小分子化合物のLSR阻害剤を使用して、4T1シンジェニックマウスモデル(皮下移植)におけるin vivo薬効試験を確認する。また、実施例7と同様に、抗LSR抗体#9-7、#16-6、#26-2、#1-25、#1-43、siRNA(配列番号15または16)、または小分子化合物のLSR阻害剤を使用して、腫瘍内浸潤CD8+T細胞の増加および活性化を確認する。
Specifically, as in Example 4, anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, #1-43, siRNA (SEQ ID NO: 15 or 16), or a small molecule LSR inhibitor will be used to confirm in vivo efficacy tests in a 4T1 syngenic mouse model (subcutaneous implantation). Also, as in Example 7, anti-LSR antibodies #9-7, #16-6, #26-2, #1-25, #1-43, siRNA (SEQ ID NO: 15 or 16), or a small molecule LSR inhibitor will be used to confirm the increase and activation of tumor-infiltrating CD8+ T cells.
これらの結果から、LSRを調節することで、その調節因子の種類にかかわりなく、免疫チェックポイントの調節ができることが理解される。
These results suggest that regulating LSR can regulate immune checkpoints, regardless of the type of regulator.
以上、本開示を実施例に基づいて説明した。この実施例はあくまで例示であり、種々の変形例が可能なこと、またそうした変形例も本開示の範囲にあることは当業者に理解されるところである。
The present disclosure has been described above based on examples. These examples are merely illustrative, and those skilled in the art will understand that various modifications are possible and that such modifications are also within the scope of the present disclosure.
以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本開示は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。
As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted only in terms of the claims. It is understood that the patents, patent applications, and literature cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth in this specification.
この出願は、2022年11月9日に出願された日本出願特願2022-179712を基礎とする優先権を主張し、その開示のすべてをここに取り込む。
This application claims priority based on Japanese Patent Application No. 2022-179712, filed on November 9, 2022, the disclosure of which is incorporated herein in its entirety.
悪性腫瘍制御技術が提供され、悪性腫瘍の治療および予防に関連する技術に関与する産業(試薬、製薬等)において利用可能な技術が提供される。
Malignant tumor control technology is provided, and technology that can be used in industries involved in technologies related to the treatment and prevention of malignant tumors (reagents, pharmaceuticals, etc.) is provided.
配列番号1:抗LSR抗体9-7配列
配列番号2:抗LSR抗体16-6配列
配列番号3:抗LSR抗体26-2配列
配列番号4:抗LSR抗体27-6配列
配列番号5:抗LSR抗体1-25配列
配列番号6:抗LSR抗体1-43配列
配列番号7:ヒトLSRタンパク質配列(NP_991403.1)
配列番号8:ヒトLSR核酸配列(NM_205834.3)
配列番号9:LSR siRNA 1のコア配列(ガイド配列)
配列番号10:LSR siRNA 2コア配列(ガイド配列)
配列番号11:LSR siRNA 1のコア配列(ガイド配列)のアンチセンス配列
配列番号12:LSR siRNA 2コア配列(ガイド配列)のアンチセンス配列
配列番号13:LSR siRNA 1のセンス全長配列
配列番号14:LSR siRNA 2のセンス全長配列
配列番号15:LSR siRNA 1のアンチセンス全長配列
配列番号16:LSR siRNA 2のアンチセンス全長配列 SEQ ID NO:1: Anti-LSR antibody 9-7 sequence SEQ ID NO:2: Anti-LSR antibody 16-6 sequence SEQ ID NO:3: Anti-LSR antibody 26-2 sequence SEQ ID NO:4: Anti-LSR antibody 27-6 sequence SEQ ID NO:5: Anti-LSR antibody 1-25 sequence SEQ ID NO:6: Anti-LSR antibody 1-43 sequence SEQ ID NO:7: Human LSR protein sequence (NP_991403.1)
SEQ ID NO: 8: Human LSR nucleic acid sequence (NM_205834.3)
SEQ ID NO: 9: Core sequence (guide sequence) of LSR siRNA 1
SEQ ID NO: 10: LSR siRNA 2 core sequence (guide sequence)
SEQ ID NO: 11: Antisense sequence of LSR siRNA 1 core sequence (guide sequence) SEQ ID NO: 12: Antisense sequence of LSR siRNA 2 core sequence (guide sequence) SEQ ID NO: 13: Full-length sense sequence of LSR siRNA 1 SEQ ID NO: 14: Full-length sense sequence of LSR siRNA 2 SEQ ID NO: 15: Full-length antisense sequence of LSR siRNA 1 SEQ ID NO: 16: Full-length antisense sequence of LSR siRNA 2
配列番号2:抗LSR抗体16-6配列
配列番号3:抗LSR抗体26-2配列
配列番号4:抗LSR抗体27-6配列
配列番号5:抗LSR抗体1-25配列
配列番号6:抗LSR抗体1-43配列
配列番号7:ヒトLSRタンパク質配列(NP_991403.1)
配列番号8:ヒトLSR核酸配列(NM_205834.3)
配列番号9:LSR siRNA 1のコア配列(ガイド配列)
配列番号10:LSR siRNA 2コア配列(ガイド配列)
配列番号11:LSR siRNA 1のコア配列(ガイド配列)のアンチセンス配列
配列番号12:LSR siRNA 2コア配列(ガイド配列)のアンチセンス配列
配列番号13:LSR siRNA 1のセンス全長配列
配列番号14:LSR siRNA 2のセンス全長配列
配列番号15:LSR siRNA 1のアンチセンス全長配列
配列番号16:LSR siRNA 2のアンチセンス全長配列 SEQ ID NO:1: Anti-LSR antibody 9-7 sequence SEQ ID NO:2: Anti-LSR antibody 16-6 sequence SEQ ID NO:3: Anti-LSR antibody 26-2 sequence SEQ ID NO:4: Anti-LSR antibody 27-6 sequence SEQ ID NO:5: Anti-LSR antibody 1-25 sequence SEQ ID NO:6: Anti-LSR antibody 1-43 sequence SEQ ID NO:7: Human LSR protein sequence (NP_991403.1)
SEQ ID NO: 8: Human LSR nucleic acid sequence (NM_205834.3)
SEQ ID NO: 9: Core sequence (guide sequence) of LSR siRNA 1
SEQ ID NO: 10: LSR siRNA 2 core sequence (guide sequence)
SEQ ID NO: 11: Antisense sequence of LSR siRNA 1 core sequence (guide sequence) SEQ ID NO: 12: Antisense sequence of LSR siRNA 2 core sequence (guide sequence) SEQ ID NO: 13: Full-length sense sequence of LSR siRNA 1 SEQ ID NO: 14: Full-length sense sequence of LSR siRNA 2 SEQ ID NO: 15: Full-length antisense sequence of LSR siRNA 1 SEQ ID NO: 16: Full-length antisense sequence of LSR siRNA 2
Claims (27)
- 脂肪分解刺激リポタンパク質受容体(LSR)の調節因子を含む、免疫チェックポイントを調節するための組成物。 A composition for regulating immune checkpoints, comprising a regulator of the lipolysis-stimulating lipoprotein receptor (LSR).
- 前記LSRの調節因子が、LSRに対する抑制因子である、請求項1に記載の組成物。 The composition according to claim 1, wherein the LSR regulator is an inhibitor of LSR.
- 前記LSRに対する抑制因子が、抗LSR抗体またはその抗原結合フラグメントである、請求項2に記載の組成物。 The composition according to claim 2, wherein the inhibitor against LSR is an anti-LSR antibody or an antigen-binding fragment thereof.
- 前記抗体のエピトープが、配列番号7の116~135位および/または216~230位を含む、請求項3に記載の組成物。 The composition according to claim 3, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
- 前記抗LSR抗体またはその抗原結合フラグメントが、以下:
(a)それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(b)それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(c)それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(d)それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(e)それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、または
(f)それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
請求項3または4に記載の組成物。 The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
5. The composition according to claim 3 or 4. - LSRの調節因子を含む、腫瘍浸潤CD8+T細胞の数を増加および/または活性化させるための組成物。 A composition for increasing and/or activating tumor-infiltrating CD8+ T cells, comprising an LSR regulator.
- 前記腫瘍浸潤CD8+T細胞が、CD69陽性である、請求項6に記載の組成物。 The composition of claim 6, wherein the tumor-infiltrating CD8+ T cells are CD69 positive.
- 前記LSRの調節因子が、LSRに対する抑制因子である、請求項6または7に記載の組成物。 The composition according to claim 6 or 7, wherein the LSR regulator is an inhibitor of LSR.
- 前記LSRの抑制因子が、抗LSR抗体またはその抗原結合フラグメントである、請求項8に記載の組成物。 The composition of claim 8, wherein the LSR inhibitor is an anti-LSR antibody or an antigen-binding fragment thereof.
- 前記抗体のエピトープが、配列番号7の116~135位および/または216~230位を含む、請求項9に記載の組成物。 The composition according to claim 9, wherein the epitope of the antibody comprises positions 116-135 and/or 216-230 of SEQ ID NO:7.
- 前記抗LSR抗体またはその抗原結合フラグメントが、以下:
(a)それぞれ配列番号1の31~35位、50~66位、99~104位、153~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(b)それぞれ配列番号2の31~35位、50~66位、99~103位、152~165位、182~188位、および221~230位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(c)それぞれ配列番号3の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(d)それぞれ配列番号4の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
(e)それぞれ配列番号5の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、または
(f)それぞれ配列番号6の31~35位、50~66位、99~104位、153~165位、182~188位、および221~229位で示される重鎖CDR1、2、3、軽鎖CDR1、2、および3のアミノ酸配列を含む、
からなる群から選択される、請求項9または10に記載の組成物。 The anti-LSR antibody or antigen-binding fragment thereof is
(a) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-230 of SEQ ID NO:1, respectively;
(b) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-103, 152-165, 182-188, and 221-230 of SEQ ID NO:2, respectively;
(c) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO: 3, respectively;
(d) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188, and 221-229 of SEQ ID NO:4, respectively;
(e) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:5, respectively; or (f) comprising the amino acid sequences of heavy chain CDR1, 2, 3 and light chain CDR1, 2, and 3 shown in positions 31 to 35, 50 to 66, 99 to 104, 153 to 165, 182 to 188, and 221 to 229 of SEQ ID NO:6, respectively;
The composition according to claim 9 or 10, selected from the group consisting of: - 前記組成物が、悪性腫瘍を治療または予防するためのものである、請求項1~11のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 11, wherein the composition is for treating or preventing a malignant tumor.
- 前記悪性腫瘍が、LSR陽性悪性腫瘍である、請求項12に記載の組成物。 The composition according to claim 12, wherein the malignant tumor is an LSR-positive malignant tumor.
- 前記悪性腫瘍が、乳癌、卵巣癌、子宮内膜癌、膵臓癌、肺癌、胃癌または大腸癌である、請求項12または13に記載の組成物。 The composition according to claim 12 or 13, wherein the malignant tumor is breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, lung cancer, gastric cancer or colon cancer.
- 請求項1~5のいずれか一項に記載の組成物を使用する、免疫チェックポイントの調節方法。 A method for regulating immune checkpoints using a composition according to any one of claims 1 to 5.
- in vitroまたはin vivoで使用する、請求項15に記載の方法。 The method of claim 15, for use in vitro or in vivo.
- 患者に、治療有効量の組成物を投与する工程を含む、請求項15または16に記載の方法。 The method of claim 15 or 16, comprising administering to a patient a therapeutically effective amount of the composition.
- 前記免疫チェックポイントの調節は、免疫チェックポイントの阻害である、請求項15~17のいずれか一項に記載の方法。 The method according to any one of claims 15 to 17, wherein the modulation of an immune checkpoint is inhibition of an immune checkpoint.
- 請求項6~11のいずれか一項に記載の組成物を使用する、腫瘍浸潤CD8+T細胞の数を増加および/または活性化方法。 A method for increasing and/or activating tumor-infiltrating CD8+ T cells using a composition according to any one of claims 6 to 11.
- in vitroまたはin vivoで使用する、請求項19に記載の方法。 The method of claim 19, for use in vitro or in vivo.
- 患者に、治療有効量の組成物を投与する工程を含む、請求項19または20に記載の方法。 The method of claim 19 or 20, comprising administering to a patient a therapeutically effective amount of the composition.
- 請求項1~15のいずれか一項に記載の組成物を使用する、悪性腫瘍の治療または予防方法。 A method for treating or preventing malignant tumors using a composition according to any one of claims 1 to 15.
- in vitroまたはin vivoで使用する、請求項22に記載の方法。 The method of claim 22, for use in vitro or in vivo.
- 患者に、治療有効量の組成物を投与する工程を含む、請求項22または23に記載の方法。 The method of claim 22 or 23, comprising administering to a patient a therapeutically effective amount of the composition.
- 免疫チェックポイントの調節に用いるための、請求項1~5のいずれか一項に記載の組成物。 A composition according to any one of claims 1 to 5 for use in regulating immune checkpoints.
- 腫瘍浸潤CD8+T細胞の数を増加および/または活性化に用いるための、請求項6~11のいずれか一項に記載の組成物。 The composition according to any one of claims 6 to 11, for use in increasing the number and/or activating tumor-infiltrating CD8+ T cells.
- 悪性腫瘍の治療または予防に用いるための、請求項1~15のいずれか一項に記載の組成物。
A composition according to any one of claims 1 to 15 for use in the treatment or prevention of malignant tumors.
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