Anti-SIRP alpha antibody

Abstract

To provide an anti-SIRPα antibody usable as an anti-tumor agent, and anti-tumor agents containing the antibody as an effective ingredient.SOLUTION: The invention includes an antibody that specifically binds to human SIRP α and inhibits binding of human SIRP α to CD47, the antibody comprising: (a) a light chain CDRL1 comprising an amino acid sequence of SEQ ID NO: 1; (b) a light chain CDRL2 comprising an amino acid sequence of SEQ ID NO:2; (c) a light chain CDRL3 comprising an amino acid sequence of SEQ ID NO:3; (d) a heavy chain CDRH1 comprising an amino acid sequence of SEQ ID NO:4; (e) a heavy chain CDRH2 comprising an amino acid sequence of SEQ ID NO:5; (f) a heavy chain CDRH3 comprising an amino acid sequence of SEQ ID NO:6, where a heavy chain constant region is that of human IgG4, and phenylalanine at position 234 is substituted with alanine, leucine at position 235 with alanine and serine at position 228 with proline as numbered according to an EU index as in Kabat et al.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an anti-SIRPα antibody useful for treating tumors, and an antitumor agent containing said antibody. . [Background technology]

[0002] SIRPα (SHPS-1) regulates myeloid cells such as macrophages, dendritic cells, and neutrophils. It is a single-pass transmembrane molecule of the Ig superfamily present in cells and glial cells (non- Patent Document 1). The extracellular region consists of one IgV domain and two IgC domains. There are 10 variants in the IgV domain, which is the binding site for D47, in humans. It has been reported (Non-Patent Document 2). On the other hand, the intracellular domain is an immunoreceptor. Contains tyrosine-based inhibition motifs (ITIMs) By binding to CD47, the tyrosine phosphatase SHP-1 and SHP-2 Binding to β-terminal β-terminal is induced, transmitting an inhibitory signal.

[0003] As a physiological phenomenon resulting from SIRPα-CD47 interaction, SIRPα on macrophages CD47 on red blood cells binds to α and transmits the "Don't eat me" signal. It has been shown that it prevents unnecessary phagocytosis of red blood cells (Non-Patent Document 3). Even in a small environment, SIRPα on macrophages and dendritic cells is highly expressed in tumor cells It has been suggested that CD47 binding suppresses the phagocytic activity of tumor cells. Suppression of phagocytosis subsequently suppresses tumor antigen presentation to T cells and further suppresses tumor immune responses. Therefore, the immune phenomenon of phagocytosis of tumor cells is thought to be related to the uptake of tumor antigens. This is considered to be a checkpoint for entry.

[0004] To date, we have reported that SIRPα-CD47 antibodies, which are the ligand for SIRPα, 7 It has been reported that inhibition of this interaction enhances the phagocytic activity of tumor cells. This is because, even when anti-SIRPα antibodies are used, tumor cells are not isolated from immune cells (Non-Patent Document 4). A similar phenomenon was observed when anti-cancer antibodies with effector activity that attracts In addition, in a mouse allogeneic tumor model using anti-CD47 antibody, It has been suggested that it not only has an antitumor effect but also induces tumor immunity (Non-Patent Document 7). A similar effect can be expected for anti-SIRPα antibodies when used in combination with anti-cancer antibodies.

[0005] On the other hand, immune checkpoint inhibitors, such as PD-1 / PD-L1, are used to treat immune deficiencies on T cells. Several antibodies against immune-suppressing molecules have been developed, and their effectiveness has been demonstrated in clinical trials ( Non-patent documents 8 and 9). SIRPα-CD47 is the only phagocytosis-suppressing molecule currently proven. Blocking antibodies against this molecule could serve as a new checkpoint for targets other than T cells. It is expected that this drug will be a promising candidate for the treatment of patients who are resistant to conventional immune checkpoint inhibitors. It also has the potential to be widely effective against a range of conditions.

[0006] To date, we have performed a study to detect human Burkitt's syndrome using anti-mouse SIRPα antibody (MY-1). In a study using a lymphoma subcutaneous transplant model, the combination of rituximab and In addition, in a mouse colon cancer model, antitumor effects were observed when combined with PD-1 antibodies. In addition, anti-mSIRPα antibodies from different clones ( In a study using a mouse liver cancer model, anti-PD-L1 antibody and anti-4-1BB antibody Antitumor and life-prolonging effects have been observed in mice that showed a life-prolonging effect. When the same tumor cells were re-implanted, further antitumor effects and survival benefits were observed. This suggests that inhibiting certain immune checkpoints may be able to induce a strong tumor immune response. These results indicate that the effector activity of Combination with anti-cancer antibodies as well as immune checkpoint inhibitors targeting T cells This is an example of a combination effect that was also observed in anti-human SIRPα antibodies. It's promising. In recent years, various companies have been reporting patents related to anti-SIRPα antibodies (Patent Documents 1 and 2). For example, OSE-172 is an IgG4Pro type antibody that inhibits V1 of SIRPα. It binds to SIRPα type V2 and SIRPγ, but not to SIRPβ1. KWAR23 is an IgG1N279A type antibody that inhibits 10 types of SIRPs. It binds to the α variant as well as SIRPβ1 and SIRPγ. It is a gG2 type antibody that exhibits binding to 10 types of SIRPα variants and SIRPγ. It is not clear which antibody is most suitable as a medicine, and efforts to obtain superior antibodies are ongoing. It is being done.

[0007] Furthermore, in a study using anti-CD47 antibodies, in addition to the antibody drugs mentioned above, conventional SOC Chemotherapy agents widely used as standard of care It has also been reported that it exhibits sufficient antitumor and life-prolonging effects even when used in combination with radiation therapy. In particular, in cases where chemotherapy was used in combination, chemotherapy was administered first, followed by administration of anti-CD47 antibody. This administration resulted in stronger antitumor effects and life extension than the simultaneous administration of chemotherapy and anti-CD47 antibodies. Since this effect is observed (Non-Patent Document 7), it is considered that the pre-administration of a chemotherapy agent facilitates the uptake of tumor antigens. By creating such an environment, we could block antigen uptake by inhibiting SIRPα-CD47 interaction. This suggests the possibility of enhancing the immune-activating effect of the antibody.

[0008] Based on the above, it is possible that anti-SIRPα antibodies, when used in combination with various antitumor agents, may provide stronger tumor immunity. It can be speculated that this is a drug that can induce an immune response. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2017 / 178653 [Patent Document 2] International Publication No. 2018 / 026600 [Patent Document 3] International Publication No. 2018 / 190719 [Non-patent literature]

[0010] [Non-Patent Document 1] Matozaki et al. Trends in cell biol. 2009(19) 2, 72-80 [Non-Patent Document 2] Takenaka et al. Nat Immunol. 2007(8)12, 1313-1323 [Non-Patent Document 3] Matozaki et al. Trends in cell biol. 2009(19) 2, 72-80 [Non-Patent Document 4] Liu et al. Plos One, 2015 (10) 9 [Non-Patent Document 5] Yanagita et al. JCI Insight, 2017 (2) 1, 1-15 [Non-Patent Document 6] Ring et al. PNAS, 2017 (114) 49, E10578-E10585 [Non-Patent Document 7] Liu et al, Nat Med. 2015 (21) 10, 1209-1215 [Non-Patent Document 8] Lee et al. The Oncologist, 2017(22)11, 1392-1399 [Non-Patent Document 9] Weinstock et al. Clin Can Res. 2017(23)16, 4534-4539 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention relates to an anti-SIRPα antibody that can be used as an antitumor agent, and a method for effectively treating the same. The present invention aims to provide an antitumor agent containing the compound as a component. [Means for solving the problem]

[0012] The present inventors have identified SIRPα expressed in phagocytes with phagocytic activity and SIRPα expressed in tumor cells. Anti-SIRPα antibody inhibits the interaction with CD47, which is a marker of proliferation and secretion from tumor cells into phagocytes. It inhibits the transmission of the “Don't-eat-me” signal and inhibits the phagocyte-mediated elimination of tumor cells. The present inventors investigated a method for enhancing the phagocytosis of cells. We attempted to generate an antibody with higher affinity and greater inhibitory effect on the interaction between SIRPα and CD47. In addition, anti-SIRPα antibodies have effector functions such as ADCC or ADCP. Considering the possibility that the effector may attack the immune cells of the patient's own body, We also investigated the generation of anti-SIRPα antibodies that lack effector functions. To reduce the effector function, the antibody subclass is changed to IgG4. The Fc region of the antibody was modified to suppress the interaction between SIRPα and CD47. To generate anti-SIRPα antibodies that strongly inhibit SIRPα but have reduced effector functions. This antibody exerts effector function without binding to the Fc receptor of effector cells. Therefore, in addition to having effector functions, When used in combination with other antibody drugs that have immune checkpoint inhibitory effects, It was confirmed that the compound exhibited a favorable antitumor effect, and thus the present invention was completed.

[0013] That is, the present invention is as follows. [1] (a) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO:1; (b) a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO:2; (c) a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:3; (d) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO:4; (e) a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO:5; and (f) heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO:6 Specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. antibody. [2] The heavy chain constant region is a human IgG4 heavy chain constant region, and the heavy chain constant region is a human IgG4 heavy chain constant region. An antibody of [1] that contains a mutation that results in reduced activity. [3] The heavy chain constant region is that of human IgG4, and is the EU-interacting constant region of Kabat et al. The phenylalanine at position 234 was replaced by alanine as indicated by the index, and A leucine is replaced by an alanine, and serine at position 228 is replaced by a proline. Antibody of [1] or [2]. [4] The amino acid sequence of the heavy chain constant region is selected from the group consisting of amino acid residues 140 to 466 of SEQ ID NO: 25. An antibody having an amino acid sequence consisting of [3] groups.

[0014] [5] (ai) an amino acid sequence consisting of amino acid residues 21 to 126 of SEQ ID NO: 23 or (aii) an amino acid sequence consisting of amino acid residues 21 to 126 of SEQ ID NO: 23 that is 95% or more identical to and a light chain having binding activity to human SIRPα. Chain variable regions, and (bi) a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 139 of SEQ ID NO: 25 Variable region or (bii) an amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 25, the amino acid sequence consisting of the 20th to 139th amino acid residues, or and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. Chain variable region wherein the heavy chain constant region is a heavy chain constant region of human IgG4, and specifically binds to human SIRPα, which contains a mutation that results in reduced P activity, and and an antibody that inhibits CD47 binding. [6] The heavy chain constant region is that of human IgG4, and the EU-Immuninhibitor The phenylalanine at position 234 was replaced by alanine as indicated by the index, and A leucine is replaced by an alanine, and serine at position 228 is replaced by a proline. [5] Antibody. [7] The amino acid sequence of the heavy chain constant region is selected from the group consisting of amino acid residues 140 to 466 of SEQ ID NO: 25. An antibody having an amino acid sequence consisting of [6].

[0015] [8] (a) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO:7; (b) a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO:8; (c) a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:9; (d) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 10; (e) a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 11, and (f) heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 12 Specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. antibody. [9] The heavy chain constant region is a human IgG4 heavy chain constant region, and [8] antibodies that contain mutations that result in reduced activity.

[10] The heavy chain constant region is that of human IgG4, and the EU-I The phenylalanine at position 234, indicated by the index, was replaced by alanine, and the The leucine at position 1 is replaced by alanine, and the serine at position 228 is replaced by proline. [8] or [9] specifically binds to human SIRPα, and inhibits the binding of human SIRPα to CD47. Inhibitory antibodies.

[11] The amino acid sequence of the heavy chain constant region is the amino acids 139 to 465 of SEQ ID NO: 29. An antibody of

[10] , which is an amino acid sequence consisting of residues.

[0016]

[12] (ai) an amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO: 27 or a light chain variable region comprising (aii) an amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 27, the 21st to 127th amino acid residues of which are 95% or more identical and a light chain having binding activity to human SIRPα. Chain variable regions, and (bi) a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 138 of SEQ ID NO: 29 Variable region or (bii) an amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 29, the amino acid sequence consisting of the 20th to 138th amino acid residues, or and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. Chain variable region wherein the heavy chain constant region is a heavy chain constant region of human IgG4, and specifically binds to human SIRPα, which contains a mutation that results in reduced P activity, and and an antibody that inhibits CD47 binding.

[13] The heavy chain constant region is that of human IgG4, and the EU-I The phenylalanine at position 234, indicated by the index, was replaced by alanine, and the The leucine at position 1 is replaced by alanine, and the serine at position 228 is replaced by proline.

[12] specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. Antibody.

[14] The amino acid sequence of the heavy chain constant region is the amino acids 139 to 465 of SEQ ID NO: 29. An antibody of

[13] , which is an amino acid sequence consisting of residues.

[0017]

[15] (a) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 13; (b) a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 14; (c) a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 15; (d) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 16; (e) a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 17, and (f) heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 18 Specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. antibody.

[16] The heavy chain constant region is a human IgG4 heavy chain constant region, and

[15] antibodies that contain mutations that result in reduced P activity.

[17] The heavy chain constant region is that of human IgG4, and the EU-I The phenylalanine at position 234, indicated by the index, was replaced by alanine, and the The leucine at position 1 is replaced by alanine, and the serine at position 228 is replaced by proline. The antibody specifically binds to human SIRPα,

[15] or

[16] , and inhibits the binding of human SIRPα to CD47. Antibody that inhibits binding.

[18] The amino acid sequence of the heavy chain constant region is the amino acids 144 to 470 of SEQ ID NO: 33. An antibody of

[17] , which is an amino acid sequence consisting of residues.

[0018]

[19] (ai) an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 or a light chain variable region comprising (aii) an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 which is 95% or more identical to and a light chain having binding activity to human SIRPα. Chain variable regions, and (bi) a heavy chain consisting of an amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33 Variable region or (bii) an amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33 which is at least 95% identical to and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. Chain variable region wherein the heavy chain constant region is a heavy chain constant region of human IgG4, and specifically binds to human SIRPα, which contains a mutation that results in reduced P activity, and and an antibody that inhibits CD47 binding.

[20] The heavy chain constant region is that of human IgG4, and the EU-I The phenylalanine at position 234, indicated by the index, was replaced by alanine, and the The leucine at position 1 is replaced by alanine, and the serine at position 228 is replaced by proline.

[19] specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. Antibody.

[21] The amino acid sequence of the heavy chain constant region is the amino acids 144 to 470 of SEQ ID NO: 33. An antibody, which is an amino acid sequence consisting of residues,

[20] .

[0019]

[22] Any of the following antibodies (1) to (8) and (1) to (4): (1) A complex consisting of an amino acid sequence consisting of amino acid residues 20 to 466 of SEQ ID NO: 41 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:37. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. Antibodies; (2) An amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 41, the amino acid sequence consisting of the 20th to 466th amino acid residues, or more than 95% identical to that of SEQ ID NO: 41 and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. and has a sequence identity of 95% or more with the amino acid residues 21 to 234 of SEQ ID NO: 37. and a human SIR having a light chain with binding activity to human SIRPα. An antibody that specifically binds to Pα and inhibits the binding of human SIRPα to CD47; (3) a polymerase consisting of an amino acid sequence consisting of amino acid residues 20 to 466 of SEQ ID NO: 41 and a light chain having an amino acid sequence consisting of the 21st to 234th amino acid residues of SEQ ID NO:39. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. Antibodies; (4) An amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 41, the amino acid sequence consisting of the 20th to 466th amino acid residues, or more than 95% identical to that of SEQ ID NO: 41. and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. and an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 39, which has a repeat sequence of 95% or more. and a light chain having binding activity to human SIRPα. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. Antibodies; (5) A complex consisting of an amino acid sequence consisting of amino acid residues 20 to 466 of SEQ ID NO: 43. and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:35. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. Antibodies; (6) An amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 43, the amino acid sequence consisting of amino acid residues 20 to 466, and having an amino acid sequence identical to that of SEQ ID NO: 43, the amino acid sequence ... and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. and an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 35, which has a repeat sequence of 95% or more. and a light chain having binding activity to human SIRPα. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. Antibodies; (7) A polymerase consisting of an amino acid sequence consisting of amino acid residues 20 to 466 of SEQ ID NO: 43 and a light chain consisting of an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO:37. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. and (8) An amino acid sequence having an amino acid sequence identical to that of SEQ ID NO: 43, the amino acid sequence consisting of the 20th to 466th amino acid residues, or more than 95% identical to that of SEQ ID NO: 43. and a polypeptide having binding activity to human SIRPα, the polypeptide comprising the amino acid sequence having the above sequence identity. and an amino acid sequence consisting of amino acid residues 21 to 234 of SEQ ID NO: 37, which has a repeat sequence of 95% or more. and a light chain having binding activity to human SIRPα. It specifically binds to human SIRPα, which consists of two chains, and inhibits the binding of human SIRPα to CD47. Antibody.

[23] The antibody of

[22] , having reduced ADCC and / or ADCP activity.

[0020]

[24] The amino acid sequence of human SIRPα represented by SEQ ID NO:57, namely, Gln at position 82 and Lys at position 83 , binds to an epitope containing Glu at position 84 and Gly at position 85 of human SIRPα An antibody that specifically binds and inhibits the binding of human SIRPα to CD47.

[25] The heavy chain constant region is a human IgG4 heavy chain constant region, and

[24] antibodies that contain mutations that result in reduced P activity.

[26] The heavy chain constant region is that of human IgG4, and the EU-I The phenylalanine at position 234, indicated by the index, was replaced by alanine, and the The leucine at position 1 is replaced by alanine, and the serine at position 228 is replaced by proline. ,

[24] or

[25] .

[27] The amino acid sequence of the heavy chain constant region is the amino acids 140 to 466 of SEQ ID NO: 25. An antibody,

[26] , which is an amino acid sequence consisting of residues.

[28] Any of the antibodies [1]-

[27] that enhance the phagocytosis of macrophages.

[29] The antibody according to any one of [1] to

[28] , wherein the heavy chain carboxyl-terminal lysine residue is Missing antibodies.

[0021]

[30] An antigen-binding fragment of any of the antibodies [1] to

[29] .

[31] An antigen-binding fragment of the antibody of

[0030] selected from the group consisting of Fab, F(ab')2, Fab' and scFv.

[32] An antibody of any one of [1] to

[29] or an antigen-binding fragment of the antibody of

[30] or

[31] A pharmaceutical composition comprising as an active ingredient.

[33] The pharmaceutical composition of

[32] , which is an antitumor agent.

[34] The active ingredients of the antitumor agent may further include immune checkpoint inhibitors and / or cancer A pharmaceutical composition according to

[0033] , comprising an antibody drug that specifically reacts with an antigen and has ADCC and / or ADCP activity.

[35] An antigen-binding fragment of any one of the antibodies [1] to

[29] or the antibody

[30] or

[31] as an active ingredient, specifically reacts to immune checkpoint inhibitors and / or cancer antigens and a pharmaceutical composition to be used in combination with an antibody drug having ADCC and / or ADCP activity.

[36] Immune checkpoint inhibitors are inhibitors of the binding of PD-L1 and PD-1, or The pharmaceutical composition of

[34] or

[35] , which is a TLA4 inhibitor.

[37] Antibody drugs that specifically react with cancer antigens and have ADCC and / or ADCP activity The pharmaceutical composition of

[0034] or

[35] , wherein the antibody is selected from the group consisting of an anti-CD20 antibody, an anti-HER2 antibody, and an anti-EGFR antibody.

[38] The tumor is a carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, or carcinoma. one or more selected from the group consisting of nephroblastoma, neuroblastoma, retinoblastoma, and nephroblastoma The pharmaceutical composition according to any one of

[33] to

[37] , wherein the pharmaceutical composition is a tumor.

[39] Tumors include renal cell carcinoma, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival carcinoma, and oral cancer. Laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, and duodenal cancer small intestine cancer, colon cancer, rectal cancer, appendix cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer , pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, Chondrosarcoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant Peripheral nerve sheath tumor, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, sarcoma-like tumor Epithelial sarcoma, B-cell lymphoma, T-NK cell lymphoma, Hodgkin's lymphoma, myeloid leukemia, Lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, testicular cancer, and ovarian cancer

[38] The tumor is one or more types selected from the group consisting of glioma, meningioma, and glioma. A pharmaceutical composition comprising:

[40] A nucleic acid encoding the amino acid sequence of the heavy and light chains of any one of the antibodies [1] to

[29] . A polynucleotide consisting of a nucleic acid sequence.

[41] A vector containing the polynucleotide of

[40] .

[42] A host cell containing the polynucleotide of

[40] or the vector of

[41] .

[43] A method for producing an antibody of any of [1] to

[0029] , comprising culturing a host cell of

[42] and purifying the antibody from the culture.

[44] Antibodies produced by the method of

[43] .

[0022] This specification incorporates the disclosures of Japanese Patent Application No. 2018-131116, which is the priority basis of this application. Includes. Effect of the Invention

[0023] The anti-SIRPα antibody of the present invention binds SIRPα expressed in phagocytes and SIRPα expressed in tumor cells. This strongly inhibits the interaction with CD47, which is a target of tumor cells, and inhibits the “Don’t eat” It inhibits the transmission of t-me signaling but has no effector function. It is safe because it does not attack one's own immune cells.

[0024] The anti-SIRPα antibody of the present invention is effective in treating other antibody drugs and immune checkpoints that have effector functions. When used in combination with other antibody drugs that have point-blocking effects, it exerts an excellent antitumor effect. It is possible. [Brief description of the drawings]

[0025] [Figure 1A] FIG. 1 shows (A) the structures of SIRPA constructs used in epitope analysis of anti-SIRPA antibodies, and (B) the reactivity of rat anti-human SIRPA antibodies to each construct. [Figure 1B] Figures (i)-(iv) showing the reactivity of humanized anti-human SIRPA antibody to the hmSIRPA construct used in epitope analysis of anti-SIRPA antibody, and (v) showing the amino acid sequence of the SIRPA construct used in epitope analysis of anti-SIRPA antibody. [Diagram 2] FIG. 13 shows a ribbon model of the entire complex of an anti-SIRPA antibody Fab fragment and SIRPA_V2_IgV. [Diagram 3] FIG. 1 shows the interaction of the regions before beta5 (A) and after beta5 (B) of human SIRPA with an antibody, SIRPA antibody (antibody D13). [Figure 4]FIG. 1 shows a comparison of the amino acid sequences of each variant of the beta5-6 loop portion of human SIRPA. [Diagram 5] FIG. 1 shows the ADCP activity against gastric cancer cell lines when an anti-SIRPA antibody was used alone (A) and when an anti-SIRPA antibody was used in combination with Trastuzumab (B). [Figure 6] FIG. 1 shows the results of evaluating the binding affinity of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) to human SIRPA. [Figure 7] FIG. 1 shows the results of evaluating the binding affinity of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) to monkey SIRPA. [Figure 8A] FIG. 1 shows the results of evaluating the activity of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) to inhibit the binding of human or monkey SIRPA to CD47 ((i): SIRPA_V1, (ii): SIRPA_V2, (iii): monkey SIRPA). [Figure 8B] FIG. 1 shows the results of evaluating the activity of human chimeric anti-SIRPA antibodies (each isotype of cF44) to inhibit the binding of human or monkey SIRPA to CD47 ((i): SIRPA_V1, (ii): SIRPA_V2, (iii): monkey SIRPA). [Figure 9] FIG. 1 shows the ADCP activity against Burkitt's lymphoma cell lines when an anti-SIRPA antibody was used alone (A) and when an anti-SIRPA antibody was used in combination with Rituximab (B). [Figure 10] 1 shows the results of toxicity evaluation of human chimeric anti-human SIRPA antibodies against PBMCs and macrophages, in which A shows the ADCP activity of cD13, cF44, and cF63 against PBMCs, B shows the ADCP activity of the cF44 antibody with a different constant region, and C shows the abundance ratio of macrophages. [Figure 11] FIG. 1 shows a comparison of the amino acid sequences of the heavy chain variable region of antibody D13, the variable region of humanized antibody heavy chain hH1, and the variable region of humanized antibody heavy chain hH2. [Figure 12]FIG. 1 shows a comparison of the amino acid sequences of the light chain variable region of antibody D13, the variable region of humanized antibody light chain hL2, the variable region of humanized antibody heavy chain hL3, and the variable region of humanized antibody light chain hL4. [Figure 13A] FIG. 1 shows the binding activity of humanized anti-SIRPA antibodies to human SIRPA variants ((i): V1, (ii): V2, (iii): V3, and (iv): V4). [Figure 13B] FIG. 1 shows the binding activity of humanized anti-SIRPA antibodies to human SIRPA variants ((i): V5, (ii): V6, (iii): V7, and (iv): V8). [Figure 13C] FIG. 1 shows the binding activity of humanized anti-SIRPA antibodies to human SIRPA variants ((i): V9, (ii): V10, (iii): monkey SIRPA, and (iv): Mock). [Figure 14A] FIG. 1 shows the binding activity of humanized anti-SIRPA antibodies to mouse SIRPA ((i): C57BL / 6, (ii): Balb / c, (iii): 129sv). [Figure 14B] FIG. 1 shows the binding activity of humanized anti-SIRPA antibodies to mouse SIRPA ((i): NOD, (ii): Mock). [Figure 15] FIG. 1 shows the results of evaluating the inhibitory activity of humanized anti-SIRPA antibodies against the binding of human (A and B) or monkey SIRPA (C) to CD47. [Figure 16] FIG. 1 shows the ADCP activity against cancer cell lines (A and B: Raji line, C and D: Ramos line) when a humanized anti-human SIRPA antibody was used alone (A and C) and when a humanized anti-human SIRPA antibody was used in combination with Rituximab (B and D). [Figure 17] FIG. 1 shows the nucleotide sequence encoding the cD13 light chain and the amino acid sequence of the cD13 light chain. [Figure 18] FIG. 1 shows the nucleotide sequence encoding the cD13 heavy chain and the amino acid sequence of the cD13 heavy chain. [Figure 19] FIG. 1 shows the nucleotide sequence encoding the cF44 light chain and the amino acid sequence of the cF44 light chain. [Figure 20] FIG. 1 shows the nucleotide sequence encoding the cF44 heavy chain and the amino acid sequence of the cF44 heavy chain. [Figure 21] FIG. 1 shows the nucleotide sequence encoding the cF63 light chain and the amino acid sequence of the cF63 light chain. [Figure 22] FIG. 1 shows the nucleotide sequence encoding the cF63 heavy chain and the amino acid sequence of the cF63 heavy chain. [Figure 23] FIG. 1 shows the nucleotide sequence encoding hL2 and the amino acid sequence of hL2. [Figure 24] FIG. 1 shows the nucleotide sequence encoding hL3 and the amino acid sequence of hL3. [Diagram 25] FIG. 1 shows the nucleotide sequence encoding hL4 and the amino acid sequence of hL4. [Figure 26] FIG. 1 shows the nucleotide sequence encoding hH1 and the amino acid sequence of hH1. [Figure 27] FIG. 1 shows the nucleotide sequence encoding hH2 and the amino acid sequence of hH2. [Figure 28] FIG. 1 shows the CDR sequences of antibody D13. [Figure 29] FIG. 1 shows the CDR sequences of antibody F44. [Diagram 30] FIG. 1 shows the CDR sequences of antibody F63. [Diagram 31] FIG. 1 shows the results of evaluating the human SIRPA_V1 / CD47 binding inhibitory activity of various anti-human SIRPA antibodies (A), the results of evaluating the human SIRPA_V2 / CD47 binding inhibitory activity of various anti-human SIRPA antibodies (B), and the IC50 values ​​of various antibodies for inhibition of human SIRPA_V1 / CD47 or SIRPA_V2 / CD47 binding activity by human SIRPA antibodies. [Diagram 32] FIG. 1 shows the results of binding evaluation of various anti-human SIRPA antibodies to human SIRPB (A) and human SIRPG (B), as well as the results of negative control tests for A and B (C). [Diagram 33]ADCP activity against Burkitt's lymphoma cell line (Raji) when Rituximab was used in combination with various anti-human SIRPA antibodies: A comparison of reactivity for each donor at 10μg / mL, with a reaction time of 2 hours (A), a reaction time of 16 hours (B), and a comparison of concentration dependency at a reaction time of 2 hours (C), and a graph showing the self-ADCP activity (D) of macrophages phagocytosing each other by various anti-human SIRPA antibodies. In each figure, "Ab-" indicates a negative control with no antibody added, and "+ Rmab" indicates simultaneous addition of Rituximab. [Diagram 34] FIG. 1 shows the amino acid sequences of the OSE-172 antibody heavy chain (OSE-172_hG4Pro) and light chain (OSE-172_hK). [Diagram 35] FIG. 1 shows the amino acid sequences of the KWAR23 antibody heavy chain (KWAR23_hG4Pro) and light chain (KWAR23_hK). [Diagram 36] FIG. 1 shows the amino acid sequences of the ADU-1805 antibody heavy chain (ADU-1805_hG2) and light chain (ADU-1805_hK). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention will be described in detail below.

[0027] Characteristics of anti-SIRPα antibodies The present invention provides an anti-SIRPα antibody that recognizes and binds to the extracellular IgV domain of the SIRPα protein. It is a body.

[0028] SIRPα (signal regulatory protein α) is a macro Ig subunits present in phages, dendritic cells, myeloid cells such as neutrophils, and glial cells It is a single-pass transmembrane molecule of the parenchymal family. The extracellular domain contains one IgV domain and two The IgC domain is composed of the IgV domain, which is the binding site for CD47. Ten variants, V1 to V10, have been reported. The IgV domain is one of three extracellular Ig-like domains that make up the SIRPα protein. There are two IgV domains. Of these, V1 and V2 are the major variants. The anti-SIRPα antibody of the present invention inhibits all variants including the major variants V1 and V2. In the present invention, "SIRPα" may be referred to as "SIRPA." be.

[0029] The amino acid sequence of human SIRPα protein is available from GenBank Accession No. .:NP_001035111.

[0030] The monoclonal antibodies used in the present invention are derived from mice, rats, rabbits, hamsters, guinea pigs, and the like. Mammals such as rats, horses, monkeys, dogs, pigs, cows, goats, and sheep were treated with SIRPα or its Immunize using the fragment as an immunogen, fuse spleen cells and myelomas to produce hybridomas. The antibody can be obtained as an antibody produced and secreted by the hybridoma. It can be prepared by the method described above.

[0031] SIRPα as an immunogen can be chemically synthesized based on the sequence information, or it can be expressed as a protein. Based on the DNA sequence information encoding the protein, the protein is synthesized as a recombinant protein by a known method. It can also be obtained.

[0032] The antibody screening can be carried out by any method, but preferably, the antibody is selected from the group consisting of SIRPα and SIRPα. Cell-ELISA using animal cells transfected with DNA encoding Screening is possible. The amino acid sequence of the V1 protein of human SIRPα is shown in the sequence table. Number 56 shows the amino acid sequence of the V2 protein of human SIRPα as SEQ ID NO:57 in the sequence listing. .

[0033] The anti-SIRPα antibody of the present invention inhibits the binding of SIRPα to CD47. Tumor cells highly express CD47, and SIR is expressed on phagocytes with phagocytic activity. The binding and interaction of Pα with CD47 induces the phagocyte to express the "Don't eat e” signal and escape phagocytosis by phagocytes. Anti-SIRPα antibody inhibits SIRPα By blocking the binding of RPα to CD47, tumor cells can transmit the “Don’t It inhibits the transmission of the "eat-me" signal, inhibiting the phagocytosis of tumor cells by phagocytes. As a result, it can exert an antitumor effect. , macrophages such as M2 macrophages, and dendritic cells such as imDCs (immature dendritic cells). Examples include cells.

[0034] In this case, anti-SIRPα antibodies have effector functions and act on phagocytes such as macrophages and Fc receptors such as Fcγ receptors on effector cells such as natural killer cells and T cells When bound to a target, it induces ADCC (Antibody Dependent Cellular r Cytotoxicity: Antibody-dependent cytotoxicity) and ADCP (Antidoby cytotoxicity) Dependent Cellular Phagocytosis: Antibody-dependent cellular phagocytosis Phagocytosis (phagocytosis) induces autologous effector cells such as PBMCs (peripheral blood mononuclear cells) and macrophages. I will attack.

[0035] To avoid attacking self-cells, the anti-SIRPα antibody of the present invention has an effector function As a result, the anti-SIRPα antibody of the present invention is capable of inhibiting the binding of SIRPα to CD47 alone. It only has the effect of inhibiting binding, and does not bind to the Fc receptor of effector cells. The vector function is not available.

[0036] The anti-SIRPα antibody of the present invention does not attack the patient's own immune cells, and therefore has no side effects. It can be safely used as a medicine.

[0037] However, since the anti-SIRPα antibody of the present invention has a reduced effector function, Therefore, as described below, it is necessary to combine it with other antitumor agents. Use.

[0038] To reduce effector function, the Fc portion of the anti-SIRPα antibody binds to macrophages. It is necessary that the anti-SI of the present invention does not bind to the Fc receptor of the antigen or T cell. The subclass of the RPα antibody has been replaced with that of IgG4. Among the classes, IgG4 has effects such as ADCC activity, CDC activity, and / or ADCP activity. It is known that this subclass has low kinetic activity (Bruggemann et al. , J. Exp. Med., 1351-1361, 1987). Avoiding toxicity due to effector function-mediated cytotoxicity when targeting molecules that express It is used as one of the IgG formats for the treatment of IgG (such as Opdivo). Although the effector functions of the four subclasses are low, they are not completely nonexistent. The anti-SIRPα antibody of the present invention has a heavy chain constant region modified to further reduce effector function. Mutations, i.e., substitutions of one or more amino acids that result in reduced ADCC and / or ADCP activity The EU index ( Kabat et.al., Sequences of proteins of im munological interest,1991 Fifth edition) The substitution of phenylalanine at position 234 with alanine (F234A) and Substitution of leucine at position 5 with alanine (L235A) is one example (Parekh et al. al., mAbs, 310-318, 2012). Such antibody mutations are called FALA mutations. The phenylalanine at position 234 in the EU index by Kabat et al. It is sometimes called phenylalanine, EU numbering 234.

[0039] In addition, since the formation of SS bonds between antibody heavy chains is not stable in IgG4, To achieve this, a mutation that promotes the formation of SS bonds between antibody heavy chains is introduced. and a proline at position 228 of the serine as indicated by the EU index by Kabat et al. The substitution (S228P) is one example (ANGAL et.al., Molecular (Immunology, 105-108, 1993). This antibody mutation is called a PRO mutation. Oink.

[0040] The constant region of the antibody of the present invention has the above-mentioned FALA mutation and PRO mutation simultaneously introduced therein. (Vafa et.al., Methods, 65, 114-126, 201 4) IgG4 heavy chains with both FALA and Pro mutations are called “IgG4pr "oFALA" type heavy chain, "IgG4PFALA" type heavy chain or "IgG4pf" type Also called the heavy chain.

[0041] The antibody heavy chain constant region is composed of CH1, hinge, CH2 and CH3 regions. CH1 is Index 118 to 215, Hinge EU Index 216 to 230, CH2 EU index 231 to 340, CH3 is defined as EU index 341 to 446 The phenylalanine at position 234 in the EU index by Kabat et al. The alanine substituted from amino acid 25 of SEQ ID NO:25 shows the heavy chain amino acid sequence of antibody D13. the 3rd alanine, the 252nd alanine, and the 3rd alanine, respectively, of SEQ ID NO:29 showing the heavy chain amino acid sequence of antibody F44. Alanine and the 257th alanine of SEQ ID NO: 33 showing the heavy chain amino acid sequence of antibody F63 The alanine substituted for leucine at position 235 corresponds to position 254 of SEQ ID NO:25. alanine at position 253 of SEQ ID NO:29 and alanine at position 258 of SEQ ID NO:33 It corresponds to alanine. It is also 228, as indicated by the EU index by Kabat et al. The proline substituted for the serine at position 247 of SEQ ID NO: 25 is the proline at position 247 of SEQ ID NO: This corresponds to proline at position 246 of SEQ ID NO:29 and proline at position 251 of SEQ ID NO:33.

[0042] The amino acid sequence of the constant region of the "IgG4proFALA" type heavy chain is SEQ ID NO: 25 The amino acid sequence consisting of amino acid residues 140 to 466 of SEQ ID NO: 29 The amino acid sequence consisting of the 5th amino acid residue and the 144th to 470th amino acids of SEQ ID NO: 33 It is an amino acid sequence consisting of amino acid residues.

[0043] Human IgG1 is one of the human IgG subclasses that has the highest CDC activity via complement fixation and antibody The effector functions, such as cell-dependent cytotoxicity, are very strong (Bruggemann et al., J. Exp. Med., 1351-1361, 1987), as a therapeutic antibody When targeting molecules highly expressed in cancer, the effector function of these molecules is mediated by cytotoxicity in cancer cells. It is used as an IgG format that shows therapeutic effects by inducing cell death in cells (tr As the isotype of the antibody of the present invention, When using gG1, the effect can be improved by substituting some of the amino acid residues in the constant region. It is possible to adjust the function of the actuator (WO88 / 007089, W094 / 2802 7, W094 / 29351). For the IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA (IgG1-L235A, G237A), etc. It is also possible to use an IgG1 heavy chain constant region into which these mutations have been introduced.

[0044] Human IgG2 is one of the human IgG subclasses that has the highest CDC activity via complement fixation and antibody The effector functions, such as cell-dependent cytotoxicity, are very weak (Bruggemann et al., J. Exp. Med., 1351-1361, 1987), as a therapeutic antibody Toxic effects due to cytotoxicity via effector functions when targeting molecules expressed in normal organs It is used as one of the IgG formats to avoid side effects (denosumab, The constant region of the antibody of the present invention is It is also possible to use the IgG2 heavy chain constant region. Regarding species cross-reactivity, the anti-SIRPα antibody of the present invention is capable of detecting human SIRPα and monkey (Cyn It binds to mouse SIRPα (omolgus monkey) but not to mouse SIRPα.

[0045] Human Chimeric and Humanized Antibodies The anti-SIRPα antibody of the present invention is a human antibody modified to reduce heterologous antigenicity to humans. These include chimeric antibodies and humanized antibodies. Humanized antibodies are also called CDR-grafted antibodies.

[0046] Human Chimeric Antibodies A human chimeric antibody is a chimeric antibody that combines the light and heavy chain variable regions of a non-human animal antibody with a human antibody. A human chimeric antibody is an antibody that consists of the light chain constant region and the heavy chain constant region of the human antibody. cDNA encoding the light chain variable region and cDNA encoding the heavy chain variable region were obtained from a hybridoma producing RPα antibody. The cDNA encoding the variable region is extracted and encoded by the light chain constant region and the heavy chain constant region of the human antibody. The human chimeric antibody expression vector was constructed by inserting the cDNA encoding the antibody into an expression vector. The gene can be produced by introducing the gene into a host cell and expressing it therein.

[0047] The heavy chain constant region consists of three domains, C H 1. C H 2 and C H This is composed of three parts. In particular, as described above, the human heavy chain constant region of the chimeric antibody is an IgG4 subclass antibody. A heavy chain constant region IgG4pro having a Pro mutation and a FALA mutation The light chain constant region may be any of those belonging to human Ig, and may be either a κ or λ constant region. It is an area.

[0048] As an example of the human chimeric anti-SIRPα antibody of the present invention, a rat anti-human SIRPα antibody is Human chimeric antibody having the variable regions of monoclonal antibodies D13, F44 and F63, These three antibodies are antibodies cD13, cF44 and cF63. An antibody that has high binding affinity to RPα and high inhibitory activity against the binding of SIRPα to CD47. Among these, the antibodies cD13 and cF63, which have high activity, are preferable.

[0049] antibody cD13 The nucleotide sequence of the cDNA encoding the light chain variable region of antibody cD13 is shown in the sequence listing. The nucleotide sequence is from nucleotide 61 to nucleotide 378 of sequence number 22 (Figure 17 The amino acid sequence of the light chain variable region of antibody cD13 is 21 to 12 of SEQ ID NO: 23 in the sequence listing. This is the amino acid sequence consisting of the sixth amino acid residue (Figure 17).

[0050] The nucleotide sequence of the cDNA encoding the heavy chain variable region of antibody cD13 is It is a nucleotide sequence consisting of nucleotides 58 to 417 of SEQ ID NO: 24 in the table ( FIG. 18) The amino acid sequence of the heavy chain variable region of antibody cD13 is 20 of SEQ ID NO: 25 in the sequence listing. The amino acid sequence consists of amino acid residues 1 to 139 (Figure 18).

[0051] That is, the anti-SIRPα antibody of the present invention comprises the amino acid sequence of 21 to 126 of SEQ ID NO:23. a light chain variable region consisting of an amino acid sequence consisting of residues 20 to 139 of SEQ ID NO: 25; Contains a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues that bind to human SIRPα It is an anti-human SIRPα antibody.

[0052] In addition, the nucleotide sequence consisting of nucleotides 61 to 378 of SEQ ID NO: 22 is or a nucleotide sequence consisting of nucleotides 58 to 417 of SEQ ID NO: 24 and CLUS TAL W (Alignment Tool), etc. (e.g., default or initial setting parameters) When calculated using the formula, it is at least 85% or more, preferably 90% or more, and more preferably Preferably, the sequence identity is 95% or more, more preferably 97% or more, 98% or more, or even 99% or more. A DNA having a nucleotide sequence having the same identity as the light chain variable region or heavy chain variable region of an antibody. The protein encoding the variable region of the SIRPα gene has the activity of binding to human SIRPα. The DNA encoding the light chain variable region or heavy chain variable region of the antibody of the present invention is also included in the DNA encoding the light chain variable region or heavy chain variable region of the antibody of the present invention. .

[0053] In addition, the nucleotide sequence consisting of nucleotides 61 to 378 of SEQ ID NO: 22 is or a nucleotide sequence complementary to the nucleotide sequence consisting of nucleotides 58 to 417 of SEQ ID NO:24 can hybridize under stringent conditions to DNA consisting of a sequence similar to that of the The antibody is a DNA having an activity of the light chain variable region or the heavy chain variable region, i.e., to human SIRPα. The DNA encoding a protein having the binding activity of the light chain variable region or heavy chain variable region of the present invention is also The region is contained in the DNA coding for the

[0054] The light chain variable region or the heavy chain variable region is selected from the group consisting of the 21st to 126th addresses of SEQ ID NO: 23. or an amino acid sequence consisting of amino acid residues 20 to 139 of SEQ ID NO:25 Not only a light chain variable region or a heavy chain variable region having an amino acid sequence comprising the amino acid sequence In the above, one or several, for example, 1 to 10, preferably 1 to 5, more preferably or one or two, more preferably one, amino acid deletion, substitution or addition The antibody heavy chain variable region or light chain variable region is composed of a sequence, and the activity of the antibody heavy chain variable region or light chain variable region, i.e., the activity of the antibody heavy chain variable region or light chain variable region, The present invention also includes a light chain variable region or a heavy chain variable region consisting of a protein having the binding activity of

[0055] Such an amino acid sequence consisting of amino acid residues 21 to 126 of SEQ ID NO: 23, or 1 or more amino acid residues in the amino acid sequence consisting of the 20th to 139th amino acid residues of SEQ ID NO:25 is an amino acid sequence in which several amino acids are deleted, substituted or added, as shown in SEQ ID NO: 23. An amino acid sequence consisting of amino acid residues 21 to 126 of SEQ ID NO: 25 or amino acid residues 20 to 139 of SEQ ID NO: 25 The amino acid sequence consisting of the amino acid residue and CLUSTAL W (alignment tool) etc. (e.g. For example, when calculated using the default or initial setting parameters, 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more. % or more, 98% or more, or 99% or more sequence identity.

[0056] Such an amino acid sequence consisting of amino acid residues 21 to 126 of SEQ ID NO: 23, or 1 or more amino acid residues in the amino acid sequence consisting of the 20th to 139th amino acid residues of SEQ ID NO:25 Proteins with amino acid sequences in which several amino acids are deleted, substituted, or added are An amino acid sequence consisting of amino acid residues 21 to 126 of sequence number 23 or SEQ ID NO: 25 A protein having substantially the same amino acid sequence as the 20th to 139th amino acid residues. It is one.

[0057] Furthermore, antibody cD13 has, as a CDR (complementarity determining region) of the light chain variable region, the following sequence: CDRL1 consisting of the amino acid sequence (GASKSVRTYMH) represented by SEQ ID NO:2 CDRL2 consisting of the amino acid sequence (SASNLEA) represented by SEQ ID NO:3 and a heavy chain variable region comprising a CDRL3 having the amino acid sequence (QQSNEPPYT). The CDR of the present invention is an amino acid sequence (GFTFSDYGMI) represented by SEQ ID NO: 4. CDRH1, CDRH2 consisting of the amino acid sequence (SISSSSSYIY) represented by SEQ ID NO:5 RH2, CDR consisting of the amino acid sequence (RYYGFNYPFDY) represented by SEQ ID NO:6 Contains H3 (Figure 28).

[0058] That is, the anti-SIRPα antibody of the present invention has the amino acid sequence represented by SEQ ID NO: 1. CDRL1, consisting of the amino acid sequence represented by SEQ ID NO:2; CDRL2, consisting of the amino acid sequence represented by SEQ ID NO:3 and further comprising, as CDR of the heavy chain variable region, a CDRL3 having an amino acid sequence represented by the following sequence: CDRH1 consisting of the amino acid sequence represented by sequence number 4, and the amino acid sequence represented by sequence number 5. and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO:6. It is a body.

[0059] Each of the above CDRs preferably has one or several amino acid residues in the amino acid sequence represented by each CDR. or an amino acid sequence in which one or two, more preferably one, amino acid is deleted, substituted or added. The CDR also includes a CDR consisting of an amino acid sequence consisting of an amino acid sequence.

[0060] The chimeric or humanized D13 antibody has an SIR consisting of the amino acid sequence represented by SEQ ID NO:73. SI having the amino acid sequence represented by SEQ ID NO: 74 or 75, which binds to the Pα mutant. It does not bind to RPα mutants. NQKEG sequence in the amino acid sequence represented by SEQ ID NO: 73 (SEQ ID NO: 76) is the NQKEE sequence (SEQ ID NO: 77) in SEQ ID NO: 74, 5 is replaced with the SFTEG sequence (SEQ ID NO: 80), and the chimeric or humanized D13 antibody It was revealed that the NQKEG sequence (SEQ ID NO: 76) is necessary for the binding of SIRPα to X-ray crystal structure analysis revealed that antibody cD13 is identical to the human SIR shown in SEQ ID NO:57. Amino acid residues Gln82, Lys83, Glu84, Gly85, and H in Pα variant 2 is86, Phe87 (the position of each amino acid residue corresponds to SEQ ID NO: 57 in the sequence listing) ) and binds to SIRPα via Gln82, Lys83, and Gl The sequence consisting of u84 and Gly85 corresponds to the QKEG portion in the above NQKEG sequence. Therefore, the NQKEG sequence is an essential epitope for the binding of D13 antibody to human SIRPα. An antibody that specifically recognizes the NQKEG sequence (SEQ ID NO: 76), i.e., the NQKEG sequence ( SIRPα mutant having the amino acid sequence represented by SEQ ID NO: 73) having SEQ ID NO: 76 The amino acid sequence represented by SEQ ID NO: 74 or 75 binds to but does not have said sequence. By selecting an antibody that does not bind to SIRPα mutants, we were able to confirm that the antibody has the same epitope as the D13 antibody. It is possible to select an antibody having a specific target gene.

[0061] Antibody cF44 The nucleotide sequence of the cDNA encoding the light chain variable region of the antibody cF44 is shown in the sequence listing. The nucleotide sequence is the nucleotide sequence consisting of nucleotides 61 to 381 of sequence number 26 (Figure 19 The amino acid sequence of the light chain variable region of the antibody cF44 is 21 to 12 of SEQ ID NO: 27 in the sequence listing. This is the amino acid sequence consisting of the seventh amino acid residue (Figure 19).

[0062] The nucleotide sequence of the cDNA encoding the heavy chain variable region of the antibody cF44 is A nucleotide sequence consisting of nucleotides 58 to 414 of SEQ ID NO: 28 in the table ( FIG. 20) The amino acid sequence of the heavy chain variable region of antibody cF44 is 20 of SEQ ID NO: 29 in the sequence listing. The amino acid sequence consists of amino acid residues 1 to 138 (Figure 20).

[0063] That is, the anti-SIRPα antibody of the present invention comprises the amino acid sequence of 21 to 127 of SEQ ID NO:27. a light chain variable region consisting of an amino acid sequence consisting of residues 20 to 138 of SEQ ID NO:29; Contains a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues that bind to human SIRPα It is an anti-human SIRPα antibody.

[0064] In addition, the nucleotide sequence consisting of nucleotides 61 to 381 of SEQ ID NO: 26 is or a nucleotide sequence consisting of nucleotides 58 to 414 of SEQ ID NO: 28 and CLUS TAL W (Alignment Tool), etc. (e.g., default or initial setting parameters) When calculated using the formula, it is at least 85% or more, preferably 90% or more, and more preferably Preferably, the sequence identity is 95% or more, more preferably 97% or more, 98% or more, or even 99% or more. A DNA having a nucleotide sequence having the same identity as the light chain variable region or heavy chain variable region of an antibody. The protein encoding the variable region of the SIRPα gene has the activity of binding to human SIRPα. The DNA encoding the light chain variable region or heavy chain variable region of the antibody of the present invention is also included in the DNA encoding the light chain variable region or heavy chain variable region of the antibody of the present invention. .

[0065] In addition, the nucleotide sequence consisting of nucleotides 61 to 381 of SEQ ID NO: 26 is or a nucleotide sequence complementary to the nucleotide sequence consisting of nucleotides 58 to 414 of SEQ ID NO:28 can hybridize under stringent conditions to DNA consisting of a sequence similar to that of the The antibody is a DNA having an activity of the light chain variable region or the heavy chain variable region, i.e., to human SIRPα. The DNA encoding a protein having the binding activity of the light chain variable region or heavy chain variable region of the present invention is also The region is contained in the DNA coding for the

[0066] The light chain variable region or the heavy chain variable region is selected from the group consisting of the 21st to 127th addresses of SEQ ID NO:27. or an amino acid sequence consisting of amino acid residues 20 to 138 of SEQ ID NO:29 Not only a light chain variable region or a heavy chain variable region having an amino acid sequence comprising the amino acid sequence In the above, one or several, for example, 1 to 10, preferably 1 to 5, more preferably or one or two, more preferably one, amino acid deletion, substitution or addition The antibody heavy chain variable region or light chain variable region is composed of a sequence, and the activity of the antibody heavy chain variable region or light chain variable region, i.e., the activity of the antibody heavy chain variable region or light chain variable region, The present invention also includes a light chain variable region or a heavy chain variable region consisting of a protein having the binding activity of

[0067] Such an amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO: 27, or In the amino acid sequence consisting of the 20th to 138th amino acid residues of SEQ ID NO:29, is an amino acid sequence in which several amino acids are deleted, substituted or added, as shown in SEQ ID NO: 27. An amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO:29 or amino acid residues 20 to 138 of SEQ ID NO:29 The amino acid sequence consisting of the amino acid residue and CLUSTAL W (alignment tool) etc. (e.g. For example, when calculated using the default or initial setting parameters, 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more. % or more, 98% or more, or 99% or more sequence identity.

[0068] Such an amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO: 27, or In the amino acid sequence consisting of the 20th to 138th amino acid residues of SEQ ID NO:29, Proteins with amino acid sequences in which several amino acids are deleted, substituted, or added are The amino acid sequence consisting of the 21st to 127th amino acid residues of SEQ ID NO:27 or the amino acid sequence of SEQ ID NO:29 A protein having substantially the same amino acid sequence as the 20th to 138th amino acid residues. It is one.

[0069] Furthermore, antibody cF44 has, as a CDR (complementarity determining region) of the light chain variable region, the following sequence: CDRL1 consisting of the amino acid sequence (KASKSISKYLA) represented by SEQ ID NO: 8 CDRL2 consisting of the amino acid sequence (SGSTLQS) represented by SEQ ID NO:9 and a heavy chain variable region comprising a CDRL3 having the amino acid sequence (QQHNEYPPT). The CDR of the present invention is composed of the amino acid sequence (GFTFSNYYMA) represented by SEQ ID NO: 10. CDRH1, which consists of the amino acid sequence (YITTGGGSTY) represented by SEQ ID NO:11 CDRH2, a C consisting of the amino acid sequence (ANYGGSYFDY) represented by SEQ ID NO: 12 Includes DRH3 (Figure 29).

[0070] That is, the anti-SIRPα antibody of the present invention has the amino acid sequence represented by SEQ ID NO:7. CDRL1, consisting of the amino acid sequence represented by SEQ ID NO:8; CDRL2, consisting of the amino acid sequence represented by SEQ ID NO:9 and further comprising, as CDR of the heavy chain variable region, a CDRL3 having an amino acid sequence represented by the following sequence: CDRH1 consisting of the amino acid sequence represented by sequence number 10; CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 12, and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 13. It is an antibody comprising

[0071] Each of the above CDRs preferably has one or several amino acid residues in the amino acid sequence represented by each CDR. or an amino acid sequence in which one or two, more preferably one, amino acid is deleted, substituted or added. The CDR also includes a CDR consisting of an amino acid sequence consisting of an amino acid sequence.

[0072] Antibody cF63 The nucleotide sequence of the cDNA encoding the light chain variable region of the antibody cF63 is shown in the sequence listing. The nucleotide sequence is from nucleotide 61 to nucleotide 390 of sequence number 30 (Figure 21 The amino acid sequence of the light chain variable region of antibody F63 is 21 to 130 of SEQ ID NO: 31 in the sequence listing. The amino acid sequence consists of the amino acid residues (Figure 21).

[0073] The nucleotide sequence of the cDNA encoding the heavy chain variable region of the antibody cF63 is A nucleotide sequence consisting of nucleotides 58 to 429 of SEQ ID NO: 32 in the table ( FIG. 22) The amino acid sequence of the heavy chain variable region of antibody cF63 is 20 of SEQ ID NO: 33 in the sequence listing. The amino acid sequence consists of amino acid residues 1 to 143 (Figure 22).

[0074] That is, the anti-SIRPα antibody of the present invention comprises the amino acid sequence of 21 to 130 of SEQ ID NO: 31. a light chain variable region consisting of an amino acid sequence consisting of residues 20 to 143 of SEQ ID NO: 33; Contains a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues that bind to human SIRPα It is an anti-human SIRPα antibody.

[0075] In addition, the nucleotide sequence consisting of nucleotides 61 to 390 of SEQ ID NO: 30 or a nucleotide sequence consisting of nucleotides 58 to 429 of SEQ ID NO: 32 and CLUS TAL W (Alignment Tool), etc. (e.g., default or initial setting parameters) When calculated using the formula, it is at least 85% or more, preferably 90% or more, and more preferably Preferably, the sequence identity is 95% or more, more preferably 97% or more, 98% or more, or even 99% or more. A DNA having a nucleotide sequence having the same identity as the light chain variable region or heavy chain variable region of an antibody. The protein encoding the variable region of the SIRPα gene has the activity of binding to human SIRPα. The DNA encoding the light chain variable region or heavy chain variable region of the antibody of the present invention is also included in the DNA encoding the light chain variable region or heavy chain variable region of the antibody of the present invention. .

[0076] In addition, the nucleotide sequence consisting of nucleotides 61 to 390 of SEQ ID NO: 30 or a nucleotide sequence complementary to the nucleotide sequence consisting of nucleotides 58 to 429 of SEQ ID NO:32 can hybridize under stringent conditions to DNA consisting of a sequence similar to that of the The antibody is a DNA having an activity of the light chain variable region or the heavy chain variable region, i.e., to human SIRPα. The DNA encoding a protein having the binding activity of the light chain variable region or heavy chain variable region of the present invention is also The region is contained in the DNA coding for the

[0077] The light chain variable region or the heavy chain variable region is selected from the group consisting of the 21st to 130th addresses of SEQ ID NO: 31. or an amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33 Not only a light chain variable region or a heavy chain variable region having an amino acid sequence comprising the amino acid sequence In the above, one or several, for example, 1 to 10, preferably 1 to 5, more preferably or one or two, more preferably one, amino acid deletion, substitution or addition The antibody heavy chain variable region or light chain variable region is composed of a sequence, and the activity of the antibody heavy chain variable region or light chain variable region, i.e., the activity of the antibody heavy chain variable region or light chain variable region, The present invention also includes a light chain variable region or a heavy chain variable region consisting of a protein having the binding activity of

[0078] Such an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31, or In the amino acid sequence consisting of the 20th to 143rd amino acid residues of SEQ ID NO: 33, is an amino acid sequence in which several amino acids are deleted, substituted or added, as shown in SEQ ID NO: 31. An amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 33 or amino acid residues 20 to 143 of SEQ ID NO: 33 The amino acid sequence consisting of the amino acid residue and CLUSTAL W (alignment tool) etc. (e.g. For example, when calculated using the default or initial setting parameters, 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 97% or more. % or more, 98% or more, or 99% or more sequence identity.

[0079] Such an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31, or In the amino acid sequence consisting of the 20th to 143rd amino acid residues of SEQ ID NO: 33, Proteins with amino acid sequences in which several amino acids are deleted, substituted, or added are An amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 or SEQ ID NO: 33 A protein having substantially the same amino acid sequence as the 20th to 143rd amino acid residues. It is one.

[0080] Furthermore, antibody cF63 contains, as the CDR (complementarity determining region) of the light chain variable region, the following sequence: CDRL1, consisting of the amino acid sequence (ERSSGDIGDSYVS) represented by 13, CDRL2 consisting of the amino acid sequence (ADDQRPS) represented by number 14, SEQ ID NO: 15 and further comprising a CDRL3 consisting of the amino acid sequence (QSYDSKIDI) represented by the following formula: The CDR of the chain variable region is the amino acid sequence represented by SEQ ID NO: 16 (GFSLASYSL S), and the amino acid sequence represented by SEQ ID NO: 17 (RMYYDGDTA) CDRH2 consisting of the amino acid sequence represented by SEQ ID NO: 18 (DRSMFGTDYPHW YFDF) (Figure 30).

[0081] That is, the anti-SIRPα antibody of the present invention comprises the amino acid sequence represented by SEQ ID NO:13. CDRL1 having the amino acid sequence represented by SEQ ID NO:14; CDRL2 having the amino acid sequence represented by SEQ ID NO:15; and further comprising a heavy chain variable region CDR comprising a CDRL3 having an amino acid sequence represented by the following formula: CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 16; CDRH2 consisting of the amino acid sequence, CDR consisting of the amino acid sequence represented by SEQ ID NO:18 It is an antibody that contains H3.

[0082] Each of the above CDRs preferably has one or several amino acid residues in the amino acid sequence represented by each CDR. or an amino acid sequence in which one or two, more preferably one, amino acid is deleted, substituted or added. The CDR also includes a CDR consisting of an amino acid sequence consisting of an amino acid sequence.

[0083] Humanized antibodies Humanized antibodies (CDR-grafted antibodies) are antibodies that have been synthesized by merging the light and heavy chain variable regions of non-human animals. The amino acid sequence of the CDR of the region is inserted into the appropriate positions of the light chain variable region and the heavy chain variable region of the human antibody. This refers to the transplanted antibody.

[0084] The humanized anti-SIRPα antibody of the present invention binds to human SIRPα and is capable of binding to SIRPα and C A monoclonal antibody that enhances the phagocytic activity of macrophages by inhibiting the binding of D47 A light chain variable region of an antibody of an animal other than a human produced from an antibody-producing hybridoma, and The amino acid sequence of the CDR of the heavy chain variable region is The variable region is grafted into the framework region (FR) of A cDNA was constructed to have genes encoding the light chain constant region and the heavy chain constant region of a human antibody. The humanized antibody expression vector is constructed by inserting the humanized antibody into an expression vector for animal cells. The gene can be produced by introducing the gene into cells and expressing it therein.

[0085] Specifically, the CDR of antibody D13, antibody F44, or antibody F63 and the framework of a human antibody are A DNA sequence designed to link the CDR to the target region can be synthesized. The framework region of the human antibody to be used is designed so that the CDRs form a good antigen-binding site. Also, if necessary, the CDRs of the humanized antibody are selected to form an appropriate antigen-binding site. In this manner, amino acids in the framework regions of the antibody variable region may be substituted. The preparation of a humanized antibody grafted with R can be carried out by known CDR grafting techniques. Cut.

[0086] By the above method, the CDR of the heavy chain variable region of antibody D13 (amino acids shown in SEQ ID NOs: 1 to 6) was The heavy chain of a humanized antibody having six CDRs each comprising a framework region of a variable region As a heavy chain in which some amino acids in the region are replaced, humanized antibody heavy chain hH1 and humanized antibody heavy chain hH2. Also, a light chain variable region of a humanized antibody having CDRs of the light chain variable region of antibody D13 is A light chain in which some amino acids in the framework region of the variable region are replaced, These include the humanized antibody light chain hL2, the humanized antibody light chain hL3 and the humanized antibody light chain hL4.

[0087] The full length nucleotide sequence of the humanized antibody heavy chain hH1 is shown in SEQ ID NO: 40, and the amino acid sequence is shown in SEQ ID NO: The full-length nucleotide sequence of the humanized antibody heavy chain hH2 is shown in SEQ ID NO: 42. The amino acid sequence is shown in SEQ ID NO: 43. The nucleotide sequence consists of a signal sequence, nucleotides 58 to 417 of the The nucleotide sequence of the variable region is The nucleotide sequences of SEQ ID NO: 41 and SEQ ID NO: 42 each encode a constant region. In 43, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, 20 The amino acid sequence consisting of amino acid residues 140 to 466 is the variable region. The amino acid sequence of the constant region is shown in FIG. The heavy chain variable region of the humanized antibody heavy chain hH1 and the variable region of the humanized antibody heavy chain hH2 A comparison of amino acid sequences (including signal sequences) is shown.

[0088] The anti-SIRPα antibody of the present invention is a nucleic acid sequence encoding the 20th to 139th amino acid residues of SEQ ID NO: 41 or 43. The heavy chain variable region is composed of 1, 2, 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, The antibody includes an antibody that

[0089] The full length nucleotide sequence of the humanized antibody light chain hL2 is shown in SEQ ID NO:34, and the amino acid sequence is shown in SEQ ID NO:35. The full-length nucleotide sequence of the humanized antibody light chain hL3 is shown in SEQ ID NO:36. The amino acid sequence is shown in SEQ ID NO: 37. Furthermore, the full length nucleotide sequence of the humanized antibody light chain hL4 is The code sequence is shown in SEQ ID NO: 38, and the amino acid sequence is shown in SEQ ID NO: 39. In 38, the nucleotide sequence consisting of nucleotides 1 to 60 corresponds to a signal sequence. The nucleotide sequence from 61 to 381 defines the variable region, and the nucleotide sequence from 382 to 7 The nucleotide sequence consisting of nucleotide 02 encodes the constant region, In SEQ ID NOs: 35, 37 and 39, the amino acid sequence consisting of the 1st to 20th amino acid residues is The amino acid sequence is a signal sequence, and the amino acid sequence consisting of amino acid residues 21 to 127 is a variable region. The amino acid sequence consisting of amino acid residues 128 to 234 is the amino acid sequence of the constant region. FIG. 12 shows the light chain variable region of antibody D13 and the variable region of the humanized antibody light chain hL2 and the humanized antibody light chain hL3. Amino acid sequences of the variable region of the antibody heavy chain hL3 and the variable region of the humanized antibody light chain hL4 (signal A comparison of the sequences is shown below.

[0090] The anti-SIRPα antibody of the present invention is an antibody having an amino acid sequence similar to that of SEQ ID NOs: 35, 37, and 39, the amino acid sequence of which is 21 to 127. The variable region consists of 128 to 234 amino acid residues, and the light chain constant region consists of 128 to 234 amino acid residues. The present invention includes antibodies having the above structure.

[0091] The heavy chain constant region of the humanized antibody is a heavy chain constant region of the IgG4 subclass having a Pro mutation. and the heavy chain constant region IgG4proFALA carrying the FALA mutation.

[0092] This antibody has high binding affinity to human SIRPα and inhibits the binding of SIRPα to CD47. As an antibody with high affinity, an antibody (hD 13_H1L3 antibody), an antibody consisting of a humanized antibody heavy chain hH1 and a humanized antibody light chain hL4 ( hD13_H1L4 antibody), an antibody consisting of a humanized antibody heavy chain hH2 and a humanized antibody light chain hL2 hD13_H2L2 antibody and humanized antibody heavy chain hH2 and humanized antibody light chain hL3 An example of such an antibody is hD13_H2L3 antibody.

[0093] The hD13_H1L3 antibody consists of amino acid residues 20 to 466 of SEQ ID NO:41. An antibody having a heavy chain and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:37. do.

[0094] The hD13_H1L4 antibody consists of amino acid residues 20 to 466 of SEQ ID NO:41. An antibody having a heavy chain and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:39. do.

[0095] The hD13_H2L2 antibody consists of amino acid residues 20 to 466 of SEQ ID NO:43. An antibody having a heavy chain and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:35. do.

[0096] The hD13_H2L3 antibody consists of amino acid residues 20 to 466 of SEQ ID NO:43. An antibody having a heavy chain and a light chain consisting of amino acid residues 21 to 234 of SEQ ID NO:37. do.

[0097] In addition, the heavy chain of the antibody produced in cultured mammalian cells lacks a lysine residue at the carboxyl terminus. It is known that (Tsubaki et al., Int. J. Biol. Ma cromol, 139-147, 2013). However, the deletion of this heavy chain sequence reduces the antibody's anti- It has no effect on the binding ability and effector functions (such as complement activation and antibody-dependent cellular cytotoxicity). Therefore, the present invention also includes antibodies lacking the lysine residue at the carboxyl terminus of the heavy chain. Included.

[0098] Other antibodies The antibody of the present invention is an antigen-binding fragment of an antibody having an antigen-binding portion of the antibody, or a modified fragment thereof. The antibody may be treated with a protease such as papain or pepsin, or By modifying the gene by genetic engineering techniques and expressing it in suitable cultured cells, Among such antibody fragments, those having the same structure as the full-length antibody molecule can be obtained by the above-mentioned method. Fragments that retain all or part of the antibody's function can be referred to as antigen-binding fragments of an antibody. The functions of an antibody are generally classified into antigen binding activity, neutralizing activity of the antigen, and suppressing activity of the antigen. activity, antibody-dependent cytotoxicity activity, complement-dependent cytotoxicity activity and complement-dependent cell Examples of the cytotoxic activity possessed by the antigen-binding fragment of the antibody of the present invention include The function is binding activity to SIRPα.

[0099] For example, antibody fragments include Fab, F(ab')2, variable regions (Fv), or heavy chains. and single-chain Fv (scFv) in which the light chain Fv is linked with an appropriate linker. Polyspecific antibodies formed from abody (diabodies), linear antibodies, and antibody fragments. In addition, the variable domain of an antibody obtained by treating F(ab')2 under reducing conditions can be used. Fab', which is a monovalent fragment of the region, is also included in antibody fragments.

[0100] Furthermore, the antibody of the present invention is a multispecific antibody having specificity for at least two different antigens. Such molecules usually bind to two different antigens (i.e. That is, bispecific antibodies) "Multispecific antibodies" include antibodies that have specificity for more than one antigen (e.g., three types). It includes. The multispecific antibodies of the present invention may be full-length antibodies or fragments of such antibodies (e.g., F (ab')2 bispecific antibodies) are also possible. Bispecific antibodies are antibodies that combine two different antibody heavy and light chains ( They can be made by combining two different antibodies (HL pairs) or by combining two different antibodies that produce different monoclonal antibodies. Bispecific antibody-producing hybridomas can also be fused to produce bispecific antibody-producing hybridomas. (Millstein et al., Nature (1983) 3 05, p.537-539).

[0101] The antibody of the present invention may be a single chain antibody (also referred to as scFv). A heavy chain variable region and a light chain variable region are linked with a polypeptide linker. (Pluckthun,The Pharmacology of Monoclona Antibodies, 113 (eds. Rosenberg and Moore, Springer, Ger Verlag, New York, p. 269-315 (1994), Natu re Biotechnology (2005), 23, p.1126-1136). In addition, a BiscFv fragment is prepared by linking two scFvs with a polypeptide linker. They may also be used as bispecific antibodies.

[0102] Methods for producing single chain antibodies are well known in the art (see, e.g., U.S. Pat. No. 4,633,663). No. 946,778, U.S. Pat. No. 5,260,203, U.S. Pat. No. 5,091,513 (See U.S. Patent No. 5,455,030, etc.) In this scFv, the heavy chain variable region and The light chain variable region is preferably linked to a linker, preferably a polypeptide linker, that does not form a conjugate. (Huston, J.S. et al., Proc. Natl. .Acad.Sci.USA(1988), 85, p.5879-5883). s The heavy and light chain variable regions in a cFv may be derived from the same antibody or may be derived from separate The polypeptide linker linking the variable regions may be derived from an antibody. Any single chain peptide of .about.19 residues can be used.

[0103] The DNA encoding the scFv is DNA encoding the heavy chain or heavy chain variable region of the antibody. and DNA encoding the light chain or the light chain variable region, The DNA portion encoding the desired amino acid sequence is used as a template, and primers are used to define both ends of the template. The pair is then amplified by PCR, and a polypeptide linker portion is then further amplified. The DNA is combined with a primer pair that specifies that both ends of the DNA are linked to the heavy and light chains, respectively. It is obtained by combining and amplifying the

[0104] In addition, once DNA encoding scFvs has been prepared, expression vectors containing them can be used. and a host transformed with the expression vector can be obtained by a conventional method. By using the host, scFv can be obtained according to a conventional method. The fragment can be produced by a host by obtaining and expressing the gene in the same manner as described above. do.

[0105] The antibody of the present invention may be polymerized to increase affinity for an antigen. A single antibody may recognize multiple epitopes of the same antigen. A method for multimerizing antibodies may be to combine IgG CH3 domains and Binding to two scFvs, binding to streptavidin, helix-turn-helix Examples include the introduction of a motif. The antibody of the present invention is a mixture of multiple types of anti-SIRPα antibodies having different amino acid sequences. The antibody may be a polyclonal antibody. An example of a polyclonal antibody is an antibody having different CDRs. Examples of such polyclonal antibodies include mixtures of multiple types of antibodies. by culturing a mixture of cells producing different antibodies and using antibodies purified from the cultures. This is possible (see WO2004 / 061104).

[0106] Antibodies conjugated with various molecules such as polyethylene glycol (PEG) as antibody modifications can also be used.

[0107] The antibodies of the present invention may further be conjugated with other drugs ( Examples of such antibodies include antibodies that are Examples of such compounds include those bound to radioactive substances or compounds with pharmacological properties (Natur e Biotechnology (2005) 23, p. 1137-1146).

[0108] Alternatively, the full-length sequences of the heavy and light chains of an antibody may be linked together using an appropriate linker to produce a single-chain immunoglobulin. Those who want to obtain single chain immunoglobulin The method is also known (Lee, HS, et. al., Molecular Immuno logy(1999)36,p.61-71;Shirrmann,T.et.al., mAbs (2010), 2, (1) p. 1-4). Such single-chain immunoglobulins are By dimerizing, it is possible to maintain a structure and activity similar to that of antibodies that are originally tetramers. In addition, the antibody of the present invention may have a single heavy chain variable region and no light chain sequence. Such antibodies may be single domain antibodies. These are called sdAb (single-stranded antibody) or nanobodies. It has been reported that this has actually been observed in camels and llamas, and that antigen-binding ability is maintained. (Muyldemans S.et.al., Protein Eng.(1994) 7(9),1129-35,Hamers-Casterman C.et.al.,N The above antibody is It can also be interpreted as a type of antigen-binding fragment of an antibody.

[0109] Methods for producing antibodies The antibody of the present invention is a DNA encoding a heavy chain variable region or a DNA encoding a light chain variable region. A is inserted into an expression vector, and a host cell for expression is transformed with the vector, By culturing the cells, the cells can be made to produce a recombinant antibody.

[0110] The DNA encoding the antibody contains DNA encoding the heavy chain variable region and DNA encoding the heavy chain constant region. The DNA encoding the heavy chain is obtained by ligating the DNA encoding the light chain variable region. The light chain was encoded by linking the DNA encoding the light chain constant region to the DNA encoding the light chain constant region. The resulting DNA corresponds to the nucleotide sequence of the target gene.

[0111] The anti-SIRPα antibody of the present invention is a DNA encoding the heavy chain and a DNA encoding the light chain. The NA is inserted into an expression vector, a host cell is transformed with the vector, and the host cell is In this case, the DNA encoding the heavy chain and the DNA encoding the light chain are co-cultured. The DNA to be encoded is introduced into the same expression vector, and the vector is used to transform a host cell. Alternatively, the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into separate vectors. Alternatively, the two vectors may be used to transform host cells. The heavy chain variable region was inserted into a vector into which DNA encoding the light chain constant region and DNA encoding the light chain constant region had already been introduced. Alternatively, DNA encoding the light chain variable region and the light chain variable region may be introduced into the host cell. It may also contain DNA encoding a signal peptide that promotes secretion of the antibody from the cell. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are inflated. After the antibody is produced, the signal peptide is removed to obtain the antibody. The whole can be obtained as a mature protein.

[0112] In this case, DNA encoding the heavy chain variable region, DNA encoding the light chain variable region, DNA encoding the variable region and DNA encoding the heavy chain constant region linked together, The DNA encoding the variable region and the DNA encoding the light chain constant region are linked together to form a protease. It may be operably linked to elements such as motors, enhancers, polyadenylation signals, etc. Here, operative linkage refers to linking elements so that they can perform their functions. .

[0113] There are no particular limitations on the expression vector, so long as it can be replicated in a host such as an animal cell, a bacterium, or a yeast. Examples of the vectors used in the construction of expression vectors include known plasmids and phages. Examples of vectors that can be used include pcDNA (trademark) (ThermoFisher SCIENTIFIC), Flexi (registered trademark) Vector (Promega), pUC19 , pUEX2 (Amersham), pGEX-4T, and pKK233-2 (Pharmacia) Examples of host cells include pMAM-neo (manufactured by Clontech) and pMAM-neo (manufactured by Clontech). Prokaryotic cells such as bacteria and Bacillus subtilis, as well as eukaryotic cells such as yeast and animal cells, can be used. For example, human embryonic kidney cell line HEK2 is preferably used as an animal cell. 93 cells, Chinese hamster ovary (CHO) cells, etc. The vector may be introduced into a host cell by a known method to transform the host cell. Troporation method, calcium phosphate precipitation method, DEAE-dextran transfection The produced antibodies can be used in a variety of ways, including separation, immunoassay, and immunoassay. Purification methods can be used to purify the protein, for example, affinity chromatography. Other methods such as chromatography, filtration, ultrafiltration, salting out, and dialysis are appropriately selected to separate the components. Just combine them.

[0114] Antitumor Agents The present invention includes an antitumor agent containing the anti-SIRPα antibody of the present invention as an active ingredient. The heavy chain constant region of the anti-SIRPα antibody of the present invention is a heavy chain constant region of the IgG4 subclass. The heavy chain constant region IgG4proFALA has a Pro mutation and a FALA mutation. , which has no effector function and inhibits the binding of SIRPα to CD47. It only has the function of inhibiting "Don't-eat-me" signaling. Therefore, the anti-SIRPα antibody of the present invention alone cannot sufficiently damage tumor cells. Therefore, the present invention relates to a method for treating tumors by using other antitumor agents or tumor inhibitors that have effector functions and can attack and damage tumor cells. Combined with other antitumor drugs that block immune checkpoints on immune cells activated by tumor cells. Other antitumor agents used in this study bind to tumor cells and mediate the interaction between tumor cells and phagocytes such as macrophages. In this case, the anti-SIRPα antibody of the present invention binds to the CD47 of the tumor cell. By inhibiting the binding of IL-1 to SIRPα on phagocytes, the phagocytosis of tumor cells by phagocytes was suppressed. Thus, the anti-SIRPα antibody of the present invention and other anti-tumor The combined use of these antitumor agents can produce a synergistic antitumor effect.

[0115] Antitumor agents that can be used in combination with the anti-SIRPα antibody of the present invention include immune checkpoint inhibitors, Examples of such drugs include antibody drugs that specifically bind to cancer antigens and have ADCC and / or ADCP activity. Immune checkpoint inhibitors include those that inhibit PD-1 and its ligand, PD-L. 1 binding inhibitors, or CTLA4 inhibitors. Specifically, anti-PD-1 antibodies ( nivolumab, pembrolizumab, cemiplimab, spart alizumab, PDR-001, BI 754091), anti-PD-L1 antibody (ate zolizumab, avelumab, durbarumab), anti-CTLA4 antibody ( ipilimumab, tremelimumab, etc. Antibody drugs that specifically react with and have ADCC and / or ADCP activity include anti-CD2 0 antibody (rituximab), anti-HER2 antibody (trastuzumab), anti-EGF R antibody (cetuximab), anti-CD52 antibody (alemutuzumab), etc. can be done.

[0116] ADCC is a method to inhibit the proliferation and differentiation of nonspecific cytotoxic cells that express Fcγ receptors (e.g., NK cells, Neutrophils, macrophages, etc. recognize the antibodies bound to the target cells and then This refers to a cell-mediated reaction that causes lysis of target cells. FcγRIIC and FcγRIIIA are expressed on NK cells, whereas FcγRI is expressed on monocytes. , FcγRIIA, FcγRIIC, and FcγRIIIA. P is a phagocyte that expresses Fc receptors (e.g., macrophages, neutrophils, etc.) that binds to target cells. Cell-mediated phagocytosis by recognition of antibodies bound to the surface of the target cell and subsequent phagocytosis of the target cell The primary cells that carry ADCP are monocytes, which react with FcγRI and FcγRII. A, FcγRIIC and FcγRIIIA are expressed.

[0117] The present invention relates to an antitumor agent comprising an anti-SIRPα antibody as an active ingredient, This includes antitumor agents used in combination with anticancer agents.

[0118] Furthermore, the present invention relates to an antitumor agent containing an anti-SIRPα antibody as an active ingredient and the above-mentioned other antitumor The present invention also includes an anti-tumor agent or kit comprising both the agent and the anti-tumor agent.

[0119] The antitumor agent containing the anti-SIRPα antibody of the present invention as an active ingredient and the other antitumor agent described above can be used simultaneously. The administration order of the anti-SIRP inhibitors of the present invention is not limited. After administration of an antitumor agent containing an α antibody as an active ingredient, another antitumor agent may be administered. After administering the antitumor agent, an antitumor agent containing the anti-SIRPα antibody of the present invention as an active ingredient is administered. It may be administered.

[0120] The antitumor agent of the present invention is effective for treating carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, For one or more of the following: carcinoid, neuroblastoma, retinoblastoma, and nephroblastoma Specifically, the present invention can be used for the treatment of renal cancer, melanoma, squamous cell carcinoma, Basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer , esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, large intestine cancer, rectal cancer, appendix cancer, anal cancer Liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer , vaginal cancer, etc., and sarcoma includes liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, and euthyroidism. Ing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral nerve sheath tumor, retroperitoneal sarcoma These include sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, and epithelioid sarcoma. Examples of lymphoma include B-cell lymphoma, T / NK cell lymphoma, and Hodgkin's lymphoma. Leukemia includes myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, and myelodysplastic syndromes. Myeloma includes multiple myeloma, and germ cell tumors include testicular cancer and ovarian cancer. Examples of brain tumors include gliomas and meningiomas.

[0121] The anti-SIRPα antibody of the present invention enhances cellular immunity when used in combination with other antitumor agents. The present invention also includes a cellular immunity enhancing agent containing an anti-SIRPα antibody as an active ingredient. In the case of cellular immunity enhancing agents, the function of natural killer cells and / or T cells is enhanced. Enhances cell-mediated immunity.

[0122] The antitumor agent of the present invention comprises a therapeutically effective amount of an anti-SIRPα antibody, a pharma- ceutical acceptable carrier, a diluent, The carrier may include a pharmaceutical agent, a solubilizer, an emulsifier, a preservative, an auxiliary agent, etc. The "body" and "drugs" can be selected from a wide range of options depending on the type of target disease and the dosage form of the drug. The method of administration of the antitumor agent of the present invention can be appropriately selected. For example, the antitumor agent can be administered by injection. It can be used for local injection, intraperitoneal injection, selective intravenous injection, intravenous injection, subcutaneous injection, organ perfusion Injection of liquids can be used. The liquid for injection can be a salt solution, a glucose solution, or It is formulated using a carrier consisting of a mixture of saline and glucose solution, various buffer solutions, etc. Alternatively, the agent can be formulated in a powder form and mixed with the liquid carrier at the time of use to prepare an injection solution. This may be done.

[0123] Other administration methods can be appropriately selected along with the development of the formulation. For example, oral administration In certain cases, oral liquids, powders, pills, capsules, tablets, and the like can be applied. In the case of oral liquid preparations, such as suspensions and syrups, water, syrups, etc. Sugars such as glycerol, sorbitol, and fructose; oils such as sesame oil and soybean oil; preservatives such as alkyl parahydroxybenzoate; It can be made using flavors such as strawberry flavor, peppermint, etc. Powders, pills, capsules and tablets can be made with lactose, glucose, sucrose, Excipients such as mannitol, disintegrants such as starch and sodium alginate, magnesium ste Lubricants such as arates and talc, polyvinyl alcohol, hydroxypropyl cellulose, Using a binder such as gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, etc. Tablets and capsules are advantageous in that they are easy to administer. This is a preferred unit dosage form for the composition of the present invention. In the method, a solid manufacturing support is used.

[0124] The amount of antibody that is effective for treatment will vary depending on the nature of the condition being treated and the age and condition of the patient. The final dose is determined by the doctor. For example, 0.0001 mg per kg of body weight per dose The prescribed dosage may be administered once every 1 to 180 days, or once a day. The dose may be administered in two, three, four or more divided doses at appropriate intervals. EXAMPLES

[0125] The present invention will be described in detail with reference to the following examples. Not limited to this.

[0126] Example 1. Preparation of rat anti-SIRPA antibody 1)-1 Preparation of expression construct 1)-1-1 Construction of SIRPA_V1_ECD expression vector Human SIRPA_V1 (NCBI protein database accession number: NP _001035111) amino acid sequence 1 to 373 at the C-terminal side HHHHHH The DNA encoding the ligated polypeptide and the vector digested with the restriction enzymes XbaI and PmeI pcDNA3.3-TOPO / LaxZ(ThermoFisher SCIENT IFIC) to In‐Fusion HD Cloning Kit(CLONTECH) The SIRPA_V1_ECD expression vector was generated by ligating the vector using SI The amino acid sequence of RPA_V1_ECD is shown in SEQ ID NO: 45 of the sequence listing, and SIRPA_V1_E The nucleotide sequence encoding CD is shown in SEQ ID NO:44 in the sequence listing.

[0127] 1)-1-2 Construction of SIRPA_V1_IgV expression vector SIRPA_V1 (NCBI protein database ACCESSION number NP_00 1035111) amino acid sequence 1 to 149 to the C-terminal side of HHHHHH Using DNA encoding the polypeptide, SIRPA_V was synthesized in the same manner as in 1)-1-1. The amino acid sequence of SIRPA_V1_IgV was shown in the sequence table. The nucleotide sequence encoding SIRPA_V1_IgV is shown in SEQ ID NO: 47 of the sequence listing. Shown in sequence number 46.

[0128] 1)-1-3 Construction of SIRPA_V2_ECD expression vector SIRPA_V2 (V1 sequence, JBC Vol.289, No.14, 10024 (20 14) modified based on the amino acid sequence of HHHHHH linked to the C-terminal side of positions 1 to 372 Using DNA encoding the polypeptide, SIRPA was synthesized in the same manner as in 1)-1-1. The V2_ECD expression vector was constructed. The amino acid sequence of SIRPA_V2_ECD was determined by sequencing. SEQ ID NO: 49 in the table shows the nucleotide sequence encoding SIRPA_V2_ECD. The sequence is shown in sequence number 48.

[0129] 1)-1-4 Construction of SIRPA_V2_IgV expression vector HHHHHH is linked to the C-terminal side of amino acid 1 to 148 of the SIRPA_V2 sequence. Using DNA encoding the polypeptide, SIRPA was synthesized in the same manner as in 1)-1-1. The V2_IgV expression vector was constructed. The amino acid sequence of SIRPA_V2_IgV was determined. SEQ ID NO: 51 in the table shows the nucleotide sequence encoding SIRPA_V2_IgV. The sequence of the gene is shown in sequence number 50.

[0130] 1)-1-5 Construction of cSIRPA_ECD expression vector cSIRPA (NCBI protein database ACCESSION number NP_0017 A polypeptide in which HHHHHHH is linked to the C-terminal side of the 1st to 372nd amino acid sequence of 68) cSIRPA_ECD was expressed using DNA encoding the peptide in the same manner as in 1)-1-1. The amino acid sequence of cSIRPA_ECD is shown in SEQ ID NO:53 of the sequence listing. The nucleotide sequence encoding cSIRPA_ECD is shown in SEQ ID NO:52 in the sequence listing.

[0131] 1)-1-6 Construction of CD47-Fc expression vector Human CD47 (NCBI protein database accession number NP_0012 71679) using DNA encoding the polypeptide, in the same manner as in 1)-1-1. A CD47-Fc expression vector was constructed. The amino acid sequence of CD47-Fc is shown in the sequence listing. No. 55, the nucleotide sequence encoding SIRPA_V1_ECD is set forth in SEQ ID NO: Shown in 54.

[0132] 1)-2 Preparation of recombinant proteins 1)-2-1 Preparation of SIRPA_V1_ECD 1)-1-1 The SIRPA_V1_ECD expression vector prepared in Transfection was performed on 293F cells (ThermoFisher SCIENTIFIC). The culture supernatant was equilibrated with 3× PBS and then transfected with Hi After adding to sTrap excel (GE Healthcare Japan), The column was washed with 3× PBS, 500 mM Imidazole, pH 7.5. The collected SIRPA_V1_ECD fraction was eluted with HiLoad 26 / 600. SIRPA_V was measured using Superdex 75 pg (GE Healthcare Japan). 1_ECD was purified.

[0133] 1)-2-2 Preparation of SIRPA_V1_IgV 1)-1-2 The SIRPA_V1_IgV expression vector prepared in Transfection was performed on 293F cells (ThermoFisher SCIENTIFIC). The culture supernatant was equilibrated with 3× PBS and then transfected with Hi After adding to sTrap excel (GE Healthcare Japan), The column was washed with 3× PBS, 500 mM Imidazole, pH 7.5. The recovered SIRPA_V1_IgV fraction was eluted with HiLoad 26 / 600 SIRPA_V was measured using Superdex 75 pg (GE Healthcare Japan). 1_IgV was purified.

[0134] 1)-2-3 Preparation of SIRPA_V2_ECD Using the SIRPA_V2_ECD expression vector prepared in 1)-1-3, SIRPA_V2_ECD was purified in the same manner as above.

[0135] 1)-2-4 Preparation of SIRPA_V2_IgV 1)-2-2 Using the SIRPA_V2_ECD expression vector prepared in 1)-1-4 SIRPA_V2_ECD was purified in the same manner as above.

[0136] 1)-2-5 Preparation of cSIRPA_ECD The same procedure was performed as in 1)-2-1 using the cSIRPA_ECD expression vector prepared in 1)-1-5. cSIRPA_ECD was purified by the same method.

[0137] 1)-2-6 Preparation of CD47-Fc The CD47-Fc expression vector was transfected into FreeStyle 293F cells (Ther Transient transfection into moFisher SCIENTIFIC The culture supernatant was equilibrated with PBS and then incubated with MabSelectSuRe (GE Healthcare) After the entire volume was loaded into a PBS column (Asia Japan), the column was washed with PBS. The fractions containing CD47-Fc were collected by elution with hydrochloride solution (pH 4.0). CD47‐Fc fraction: HiLoad 26 / 600 Superdex 200 pg CD47-Fc was purified using a Purified Polyclonal Antibody (GE Healthcare Japan).

[0138] 1)-3 Immunity Female WKY / Izm rats (Japan SLC) were used for immunization. The antigen proteins SIRPA_V1_ECD, SIRPA_V1_IgV, and SIRPA_V 2_ECD, SIRPA_V2_IgV and Freund's Complete Lymph nodes of rats injected with a mixture of adjuvant (Wako Pure Chemical Industries, Ltd.) at the base of the tail The spleens were then harvested and used to prepare hybridomas.

[0139] 1)-4 Hybridoma production Lymph node cells or spleen cells and mouse myeloma SP2 / 0-ag14 cells (ATC C: CRL-1581) in an LF301 Cell Fusion Unit (BEX). Cells were electrofused using the Clona Cell-HY Selection Medium. The cells were diluted in umD (Stem Cell Technologies) and cultured. The resulting hybridoma colonies were collected to prepare monoclonal hybridomas. Each of the collected hybridoma colonies was cultured, and the resulting hybridoma culture supernatant was used to Screening of anti-SIRPA antibody-producing hybridomas was performed.

[0140] 1)-5 Construction of expression vector for screening antigen-binding antibodies 1)-5-1 Human SIRPA_V1 and V2 expression vector (pcDNA3.2 V5- Construction of DEST-SIRPA_V1_ECD and SIRPA_V2_ECD) Human SIRPA_V1 protein (NP_001035111), or human SIRPA_V 2 protein [NP_001035111, JBC Vol.289,No14,10024( The cDNA encoding the nucleotide sequence of the nucleotide sequence modified from that of the original nucleotide sequence (p-nucleotide sequence) was inserted into the pcDNA3.2 V5-DEST vector. The vectors were cloned into the vector pcDNA3.2 V5‐DEST and expressed respectively. -SIRPA_V1_ECD, V2_ECD (or pcDNA3.2 V5-DEST- The amino acid sequence of human SIRPA_V1 protein was determined. The amino acid sequence of human SIRPA_V2 protein is shown in SEQ ID NO:56 of the sequence table. 57 respectively.

[0141] 1)-5-2 Monkey SIRPA and mouse SIRPA expression vectors (pcDNA3.2 V5-DEST-monkey SIRPA, pFLAG V5-DEST-monkey SIRPA or p Construction of FLAG V5-DEST-mouse SIRPA Monkey SIRPA protein (NP_001271679) or mouse SIRPA protein (C 57BL6:NP_031573, BALB / c:BAA20376, 129:P977 97, Based on NOD SCID: Immunology, 143, 61-67, 2014 The cDNA encoding the modified nucleotide was inserted into the pcDNA3.2 V5-DEST vector or pFL Vectors expressing each protein cloned into the AG V5-DEST vector pcDNA3.2 V5-DEST-Monkey SIRPA, pFLAG V5-DEST-Monkey SIRPA pFLAG V5-DEST-mouse SIRPA (C57BL6, BA The amino acid sequence of monkey SIRPA was determined by the sequence shown in the sequence table. In the sequence number 58, the amino acid sequence of mouse SIRPA_C57BL / 6 is shown in SEQ ID NO: 59 of the sequence listing. The amino acid sequence of mouse SIRPA_BALB / c is shown in SEQ ID NO: 60 of the sequence listing, and the amino acid sequence of mouse SIRPA_BALB / c is shown in SEQ ID NO: 61 of the sequence listing. The amino acid sequence of SIRPA_129 is shown in SEQ ID NO: 61 of the sequence listing. The amino acid sequence of D is shown in SEQ ID NO:62 in the sequence listing.

[0142] 1)-6 Hybridoma screening 1)-6-1 Preparation of antigen gene expressing cells for Cell-ELISA HEK293α cells (HEK293 expressing integrin αv and integrin β3) The stable expression cell line derived from 10% FBS-containing DMEM medium was incubated at 7.5 × 10 5 cells / mL In contrast, Lipofectamine 2000 (Therm o Fisher Scientific) and transfection was performed using pcDNA 3.2 V5-DEST-SIRPA_V1 or pcDNA3.2 V5-DEST -SIRPA_V2 or pcDNA3.2 V5-DEST as a control Then, 50 μL was added to a 96-half area well plate (Corning). or 100 μL per well in a 96-well plate (Corning) The cells were then incubated in 10% FBS-containing DMEM medium at 37°C and 5% CO2 for 24-28 h. The cells were cultured for 7 hours. The resulting cells were used for Cell-ELISA while still in an adherent state. .

[0143] 1)-6-2 Binding evaluation to human SIRPA (Cell-ELISA) After removing the culture supernatant of the expression vector-introduced 293α cells prepared in Example 1)-6-1, cDNA3.2 V5‐DEST‐SIRPA_V1, V2 or pcDNA3.2 V5 Hybridoma culture supernatant was added to each of the -DEST-transfected 293α cells, and incubated at 4°C. The cells in the wells were washed twice with PBS containing 5% FBS, and then Anti-Rat IgG-Peroxidase a diluted 500-fold in PBS containing In addition to antibody produced in rabbit (SIGMA), The cells in the wells were washed twice with PBS containing 5% FBS and then incubated at 37 °C for 1 h. PD color development solution (OPD solution (0.05 M trisodium citrate, 0.1 M phosphoric acid water o-Phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) in sodium chloride 12 water (pH 4.5) ), and H2O2 were dissolved to 0.4 mg / mL and 0.6% (v / v), respectively. 0 μL / well. The color reaction was carried out with occasional stirring, and 50 μL of 1M HCL was added. After stopping the color reaction by adding 100 μl / well, the plate was read by a plate reader (ENVISION The absorbance at 490 nm was measured using a spectrophotometer (PerkinElmer). In order to select hybridomas that produce antibodies that specifically bind to SIRPA, Compared with the role of pcDNA3.2 V5‐DEST-transfected 293α cells, pcDNA3. 2. V5-DEST-SIRPA_V1 and SIRPA_V2 expression vectors introduced 293α The hybridomas that produced culture supernatants that showed higher absorbance than the cells were analyzed by anti-SIRPA antibody. It was selected as a positive production.

[0144] 1)-6-3 Evaluation of SIRPA-CD47 binding inhibitory activity After removing the culture supernatant of the expression vector-introduced 293α cells prepared in Example 1)-6-1, cDNA3.2 V5‐DEST‐SIRPA_V1, SIRPA_V2 or pcDNA 3.2 Hybridoma culture supernatant was added to each of the V5-DEST-transfected 293α cells. Immediately after adding the antibody, adjust the final concentration to 10,000 ng / mL with 5% FBS-containing PBS. Peroxidase labeled CD47‐Fc was added at 50 μL / well. The cells were left to stand at 4°C for 1 hour. The cells in the wells were washed twice with PBS containing 5% FBS and then PD color development solution (OPD solution (0.05 M trisodium citrate, 0.1 M phosphoric acid water o-Phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) in sodium chloride 12 water (pH 4.5) (Prepared by manufacturer) and H2O2 were dissolved to 0.4 mg / mL and 0.6% (v / v), respectively. 100 μL / well of 1M HCl was added. After stopping the color reaction by adding 0 μL / well, the plate was read by a plate reader (Spectra The absorbance at 490 nm was measured using a spectrophotometer (max: Molecular Devices). We produced an antibody that specifically inhibits the binding of SIRPA expressed on the surface of the vesicle to CD47-Fc. To select hybridomas that express pcDNA3, we compared the control group with the medium. .2 V5-DEST-SIRPA_V1 or SIRPA_V2 expression vector introduction29 Hybridomas producing culture supernatants exhibiting lower absorbance in 3α cells were selected as ligands. The mice were selected as positive for the production of anti-SIRPA antibodies with dodecyl sulfate binding inhibitory activity.

[0145] 1)-6-4 Cross-species reactivity evaluation for mouse and monkey SIRPA pcDNA3.2 V5-DEST-monkey SIRPA prepared in Example 1)-5-2; Or mouse SIRPA expression vector-transfected 293α cells, and pcDNA3.2 V5-D After removing the culture supernatant of EST-introduced 293α cells, the binding activity of human SIRPA was evaluated in the same manner. The binding to monkey or mouse SIRPA was evaluated using the above-mentioned human and animal species. Based on the binding activity and SIRPA-CD47 binding inhibitory activity, D13, F42, and F44 A total of seven clones, F47, F60, F63, and F86 antibodies, were selected.

[0146] 1)-7 Determination of antibody isotype Among the rat anti-SIRPA antibody-producing hybridomas obtained, we found that the antibody strongly and specifically expressed human SIRPA Binds to RPA_V1, SIRPA_V2, and monkey SIRPA, and has high SIRPA-C D13, F42, F44, F47, and F6 were suggested to have D47 binding inhibitory activity. Hybridomas producing 0, F63, and F86 antibodies were selected, and the antibody isotypes were identified. The isotype was determined using the Rat Immunoglobulin Isotyping The results were determined using an ELISA Kit (BD Pharmingen). Isotype of rat anti-SIRPA monoclonal antibodies D13, F42, F60, and F86 The isotype of F44 is IgG1 / κ chain, that of F47 is IgG2a / κ chain, and that of F63 is IgG1 / κ chain. The isotype was confirmed to be IgG2a / λ chain.

[0147] 1)-8 Preparation of monoclonal antibodies 1)-8-1 Preparation of culture supernatant Seven rat anti-SIRPA monoclonal antibodies were purified from hybridoma culture supernatants. First, each antibody-producing hybridoma was subjected to the ClonaCell-HY Selection Increase to a sufficient amount with Medium E (StemCell Technologies). After proliferation, the cells were supplemented with Ultra Low IgG FBS (Thermo Fisher Scientific). 5 μg / mL gentamicin (Thermo Fisher Scientific) containing Hybridoma SFM (Ther The medium was replaced with a new medium (Fisher Scientific) and cultured for 7 days. The supernatant was collected and sterilized through a 0.22 μm filter (Corning).

[0148] 1)-8-2 Purification of antibodies The antibody was extracted from the culture supernatant of the hybridoma prepared in Example 1)-8-1 using Protein The product was purified by Protein G affinity chromatography using a GE Protein G column (GE The antibody was adsorbed onto a column of 100 mM NaCl (Healthcare Biosciences), and the column was washed with PBS. After washing, the column was eluted with 0.1M glycine / HCl aqueous solution (pH 2.7). After adding is-HCl (pH 9.0) to adjust the pH to 7.0-7.5, Centrifugal separation was performed. Fugal UF Filter Device VIVASPIN20 (molecular weight cutoff U The buffer was replaced with PBS using F30K (Sartorius) and the antibody was The antibody concentration was adjusted to 2 mg / mL or more. The mixture was filtered through a S filter (Sartorius) to obtain a purified sample.

[0149] Example 2. In vitro evaluation of rat anti-human SIRPA antibodies (7 types) 2)-1 Construction of expression vector for screening antigen-binding antibodies 2)-1-1 FLAG-human SIRPA expression vector (pFLAG V5-DEST- Construction of SIRPA_V1-V10 Ten human SIRPA variant proteins (Nature Immunology The cDNA encoding the plasmid pFLAG V5 was used. - Vectors that are cloned into the DEST vector and express the respective variant proteins The pFLAG V5-DEST-SIRPA_V1-V10 was constructed. The amino acid sequence of human SIRPA_V3 is shown in SEQ ID NO: 63 of the sequence listing. The amino acid sequence of human SIRPA_V5 is shown in SEQ ID NO: 64 of the sequence listing. The amino acid sequence of human SIRPA_V6 is shown in SEQ ID NO:66 of the sequence listing. The amino acid sequence of human SIRPA_V7 is shown in SEQ ID NO: 67 of the sequence listing, and the amino acid sequence of human SIRPA_V The amino acid sequence of SIRPA_V9 is shown in SEQ ID NO:68 of the sequence listing. The amino acid sequence of human SIRPA_V10 is shown in SEQ ID NO: 69 of the sequence table and SEQ ID NO: 70 of the sequence table. are shown in the following.

[0150] 2)-1-2-1 Human SIRPA_ECD, IgV, and IgV_IgC1 expression vectors -(pFLAG V5-DEST-SIRPA_ECD, and IgVIgV_IgC1) Construction The full length amino acid sequence (1 to 504) of human SIRPA_V2 and the amino acid sequence (165 to 371) of human SIRPA_V2 are shown in Table 1. a deletion of the region (hereinafter referred to as "IgV body"), and a deletion of the region from 225 to 371 (hereinafter referred to as " The cDNA encoding IgV_IgC (referred to as "IgV_IgC") was inserted into the pFLAG V5-DEST vector. The mutant proteins were then cloned into the vector, and vectors expressing each mutant protein were constructed.

[0151] The amino acid sequence of human SIRPA_V2_IgV is shown in SEQ ID NO: 71 of the sequence listing. The amino acid sequence of RPA_V2_IgV_IgC1 is shown in SEQ ID NO: 72 of the sequence listing.

[0152] 2)-1-2-2 hmSIRPA_Δ0, Δ1, and Δ2 mouse SIRPA expression vectors Construction of pFLAG V5-DEST-hmSIRPA_Δ0, Δ1, and Δ2 SEQ ID NO: 60, which consists of the 81st to 85th amino acid residues of mouse SIRPA The FTGE sequence (SEQ ID NO: 78) was replaced with the NQKEG sequence (SEQ ID NO: 76), and the 126 to The RGSSE sequence (SEQ ID NO: 79) consisting of the 130th amino acid residue was replaced with the KGS sequence. The resulting SIRPA mutant was named hmSIRPA_Δ0. The SFTGE sequence consisting of amino acid residues 81 to 85 of SIRPA (SEQ ID NO: 78) The SIRPA mutant with the NQKEE sequence (SEQ ID NO: 77) was designated hmSIRPA_Δ1. Furthermore, the amino acids 81 to 85 of mouse SIRPA shown in SEQ ID NO:60 were named The SFTGE sequence (SEQ ID NO: 78) consisting of residues is replaced with the SFTEG sequence (SEQ ID NO: 80). The resulting SIRPA mutant was named hmSIRPA_Δ2. The cDNAs encoding the nucleotides were cloned into the pFLAG V5-DEST vector, respectively. We constructed vectors expressing SIRPA mutants. The amino acid sequence of hmSIRPA_Δ0 is shown in SEQ ID NO: 73 of the sequence listing. The amino acid sequence of hmSIRPA_Δ2 is shown in SEQ ID NO: 74 of the sequence listing. They are shown in column number 75 respectively.

[0153] 2)-2 Binding evaluation for human SIRPA variants V1 to V10 Introduction of expression vectors for 10 types of variant proteins prepared in Example 2)-1-1 After removing the culture supernatant of α cells, pFLAG V5‐DEST‐SIRPA_V1~V10 or The final concentration of each of the pFLAG V5-DEST-transfected 293α cells was 10,000 nM. Rat anti-human SIRPA purified antibody diluted in 5% FBS-containing PBS to 1 μg / mL 50 μL / well of each was added and left to stand at 4°C for 1 hour. The detection wells contained 5% FBS in PBS at a final concentration of 10,000 ng / mL. Add 50 μL of anti-FLAG M2 antibody (Sigma) diluted in The mixture was left to stand for 1 hour. The following procedure was performed in the same manner as for the human SIRPA binding activity titer shown in 1)-6-2. The binding activity against 10 types of human SIRPA variants was evaluated. The binding of the rat anti-human SIRPA antibody was normalized based on the expression of the FLAG tag. As shown in Table 1, all of the clones exhibited binding to all of the variants.

[0154] [Table 1]

[0155] 2)-3 Species cross-reactivity evaluation against mouse and monkey SIRPA pFLAG V5-DEST-monkey SIRPA prepared in Example 1)-5-2, or p FLAG V5-DEST-mouse SIRPA expression vector-transfected 293α cells and pF After removing the culture supernatant of LAG V5-DEST-transfected 293α cells, the binding activity of human SIRPA was The binding affinity to monkey or mouse SIRPA was evaluated in the same manner as in the evaluation of binding affinity. As shown in Table 2, all rat anti-human SIRPA antibodies had no binding activity to monkey SIRPA. However, it did not show any binding to mouse SIRPA.

[0156] [Table 2]

[0157] 2)-4 Epitope analysis 2)-4-1-1 Epitope analysis by Cell-ELISA method (1) 2) pFLAG V5-DEST-ECD body, IgV body, and 293α cells transfected with IgV_IgC1 expression vector and pFLAG V5-DEST After removing the culture supernatant of the transfected 293α cells, each cell was treated with 10,000 ng / mL of Dilute the rat anti-human SIRPA purified antibody in PBS containing 5% FBS to 5 mL. 0 μL / well and left at 4°C for 1 hour. The detection wells contained 5% FBS in PBS at a final concentration of 10,000 ng / mL. Add 50 μL of anti-FLAG M2 antibody (Sigma) diluted in The antibody was incubated for 1 hour. The following procedure was the same as for the evaluation of the binding activity of human SIRPA. The binding of each construct to each domain was evaluated. The binding of antibody A was normalized based on the expression of the FLAG tag. As shown in Figure 1A, rat anti-human SIRPA antibody bound to both constructs. The binding of these antibodies to the IgV domain was suggested to be involved in the recognition of the IgV domain.

[0158] 2)-4-1-2 Epitope analysis by Cell-ELISA method (2) 2) pFLAG V5-DEST-hmSIRPA_Δ0, Δ 1, Δ2 expression vector-transfected 293α cells, and pFLAG V5-DEST-transfected 2 After removing the culture supernatant of 93α cells, the final concentration of each cell was adjusted to 10,000 ng / mL. D13 humanized anti-human SIRPA antibodies diluted in 5% FBS-containing PBS to obtain Add 50 μL each of anti-human SIRPA antibody and chimeric anti-human SIRPA antibody to each well and leave at 4℃ for 1 hour. In addition, the wells for detecting the expression of each SIRPA construct contained 10,000 Anti-FLAG M2 antibody (SIG The plate was then incubated at 4°C for 1 hour. The binding to each construct was evaluated in the same manner as in the binding activity evaluation. The binding of the anti-human SIRPA antibody was normalized based on the expression of the FLAG tag. As shown in Figure 1B, hD13_H1L3, hD13_H1L4h, and hD13_H2L2 , hD13_H2L3, or cD13 corresponds to hmSIRPA_Δ0 having the NQKEG sequence. The binding was dependent on the antibody concentration added, but not on Δ1 or Δ2. No binding was observed at any of the concentrations. From the above, it is clear that the NQKEG sequence is necessary for the binding of hD13 and cD13. was suggested.

[0159] 2)-4-2 Epitope analysis by X-ray crystal structure analysis 2)-4-2-1 Crystallization of the complex The full-length cD13 antibody was digested with Lysyl Endopeptidase (Wak o) and purified using a BioAssist S cation exchange column (Tosoh Corporation). The Fab fragment of the cD13 antibody was isolated. _IgV and cD13 Fab fragments were mixed in a molar ratio of 1:1 and incubated on Superdex 75,1 The complex fraction was separated using a 0 / 300GL gel filtration column (GE Healthcare). After that, the buffer was replaced with 10 mM Tris HCl (pH 8.2) by ultrafiltration, and the The complex solution was concentrated to 0.5 μg / L. The complex solution was crystallized by vapor diffusion. L contains precipitant solution (0.2M potassium phosphate dibasic , 20%(w / v) Polyethylene Glycol 3350, pH 9. 2) was added to an equal amount of the solution, and the solution was placed in a sealed container containing 0.05 mL of precipitant solution. The sample was stored in a cool, dark place and left at 25°C. After one week, the sample was 0.2 mm x 0.2 mm x 0.05 m The resulting crystals were diluted with Glycol to about 1.4 times the precipitant solution. The samples were then immersed in a solution containing 100% ethanol and then frozen in liquid nitrogen. X-ray diffraction data were collected at the beamline PF BL-17A. Software XDS (Max Plank Institute for Medica The diffraction intensity was quantified using a NMR spectrometer (Digital Research) to determine the crystal structure factor. The cubic system has a space group of R32, and the crystal unit cell is a=b=149.61Å, c=155.61. Å, alpha=beta=90°, gamma=120°.

[0160] 2)-4-2-2 Structural analysis of the complex The homology model of the obtained structural elements, Fab fragment, and IgV domain of human SIRPA were The molecular replacement method was performed using the three-dimensional structural coordinates of the known structure of α-aminobutyric acid (PDBID: 2JJS), and the position was determined. The phase was determined using the software Phaser (CCP4: Collaborative Phaser). The crystals used were non- The symmetric unit contained one complex. Using the Laboratory Computational Project No. 4 The structure was refined using the FTIR method, and the model was modified using the software COOT. The work was repeated, and a final R value of 22% and a free R value of 25% were obtained at 2.4 Å resolution. The final model is the amino acid residues 1-213 of the light chain and the heavy chain of the Fab fragment of cD13. 1-225, and amino acid residues 33-143 of human SIRPA variant 2.

[0161] 2)-4-2-3 Structural analysis of the complex Identification of the epitope of D13 The amino acid residues of human SIRPA within 4 Å of the Fab fragment of cD13 (each amino acid The residue positions correspond to SEQ ID NO:57 in the sequence listing: Gly64 , Pro65, Leu78, Gln82, Lys83, Glu84, Gly85, His 86, Phe87, Thr91, Thr92, Glu95, Thr97, Lys98, L Figure 2 shows the ribbon model and surface of the entire complex, and Figure 3 shows the bet The interaction between the a5 region (A) and the beta5 region (B) and cD13. 3 is the beta4-5 loop, i.e., residue number 8, which has low sequence diversity in human SIRPA. Strong recognition of 2-87 allows strong binding to various variants On the other hand, the beta5-6 loop (residues 92-105) For example, the interaction in the region of Glu95, which has a variant, is located near the Fab, but the electron density of its side chain is not observed. This suggests that it does not contribute significantly to the interaction. "·" indicates an amino acid residue that is identical to SIRPA_V1, and the amino acid residue is listed. The marks indicate different amino acid residues.

[0162] 2)-5 Evaluation of human SIRPA-CD47 binding inhibitory activity 1)-5-1. The culture supernatant of human SIRPA expression vector-transduced 293α cells was removed. After that, pcDNA3.2 V5‐DEST‐SIRPA_V1, V2 or pcDNA3. 2. For each of V5-DEST-transfected 293α cells, the final concentration was 0 to 10,000 ng. Purified rat anti-human SIRPA antibody was diluted with 5% FBS-containing PBS to a concentration of 1 mL / mL. 50 μL / well was added. Immediately after, 5% FB was added to the final concentration of 10,000 ng / mL. Peroxidase labeled CD47‐Fc prepared in PBS containing S was added at 50 Add μL / well and let stand at 4°C for 1 hour. The following steps were performed in the same manner as in 1)-6-3 to inhibit binding. The toxicity was evaluated. As shown in Table 3, all rat anti-human SIRPA antibodies were found to bind human SIRPA-CD47. It showed binding inhibitory activity against

[0163] [Table 3]

[0164] 2)-6 ADCP activity of rat anti-human SIRPA antibody against cancer cell lines 2)-6-1 Preparation of target cells The human gastric cancer cell line AGS cells were treated with TrypLE Express (Life Technology After adding 10% FBS-containing RPM (manufactured by logy), the cells were incubated at 37°C for 5 minutes and then peeled off. Add I1640 medium (Life Technology) and wash twice, then add PBS. After washing twice, the number of viable cells was counted using the trypan blue dye exclusion test. 7 Cell division After extraction and centrifugation, PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling(S) The cells were suspended in 2 mL of Diluent C (manufactured by Igma). Immediately after diluting 10 mM PKH26 Linker with Diluent C to 10 μM, The cell suspension was mixed with an equal volume of PKH26 Linker solution and allowed to stand at room temperature for 5 minutes. 10 mL of RPMI1640 medium containing 10% FBS (Life Technologies, Inc.) ) was added, washed twice, and then 2 × 10 6 The target cells were resuspended at 1000 cells / mL. It was used.

[0165] 2)-6-2 Preparation of PBMC cells 25mL of healthy blood was added to 20mL of Ficoll Paque Plus (GE). After slowly layering, the mixture was centrifuged at room temperature at 1500 rpm for 30 minutes. The cell layer in the middle of the ue Plus was collected with a dropper and added 20 mL of 10% FBS The cells were suspended in RPMI 1640 medium (Life Technologies) containing 150 Centrifuge at 0 rpm for 5 minutes, remove supernatant, and add 20 mL RPMI1640 medium containing 10% FBS. The plate was washed twice with 1 mL of Robosep buffer (STEMCELL After suspending the cells in trypan blue dye exclusion test, the number of viable cells was counted and the effector cells were counted. It was used as a cell.

[0166] 2)-6-3 Preparation of effector cells The PBMC cells prepared in Example 2)-6-2 were stored in RoboSep buffer (STE MCELL) 5×10 7 The concentration was adjusted to 100 cells / mL. cell enrichment kit Without CD16 Deplet EasySep human Monocyte included with ion (manufactured by STEMCELL) Add 50 μL of enrichment cocktail per 1 mL of PBMC cell suspension. After incubation at 4°C for 10 minutes, the EasySep Magnetic Particl 50 μL of es was added per 1 mL of PBMC cell suspension. After incubation at 4°C for 5 minutes, Add RoboSep buffer (STEMCELL) to make E The supernatant was collected after 2 minutes and 30 seconds and then the mixture was stirred at 1200 rpm. The cells were centrifuged for 5 min in RPMI1 containing 10% FBS to separate the monocyte fraction. After adding 640 medium (Life Technology), washing once, 10ng / mL RPMI1640 medium containing 10% FBS and M-CSF (PEPROTEC) (Life Technology) was added and suspended in 225 cm 2 Flask (Sumitomo The cells were seeded on a Bakelite plate and cultured at 37°C in 5% CO2 for 10 days. The culture supernatant was removed and supplemented with 10 ng / mL IL-10, 10 ng / mL M-CSF (PEPRO RPMI1640 medium (Life Technologies, Inc.) containing 10% FBS After 12 days, differentiation-induced macrophages were The gel was supplemented with TrypLE Express (Life Technology). After incubation at 37°C for 40 minutes, the cells were detached. After washing twice, 5 × 10 5 It will be cells / mL Sea urchin, RPMI1640 medium containing 10% FBS (Life Technology, Inc.) ) and used as effector cells.

[0167] 2)-6-4 Evaluation of ADCP activity 50 μL / well of target cells prepared by the method of Example 2)-6-1 was placed on a 96-well plate with ultra-low attachment surface. The mixture was added to a U-bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.) at a final concentration of 0 to 10 RPMI 1640 medium (Life Technologies) containing 10% FBS at a concentration of 0.000 ng / mL. Rat anti-human SIRPA antibody 7 clones, Hu5F9, diluted with 100% IgG1 (manufactured by Chromology, Inc.) G4 (anti-human CD47 antibody: PloS ONE 10[9]:e0137345, US20 15183874), TTI‐621 (human SIRPA‐Fc: WO2014 / 094122) or various control IgG were added at 50 μL / well. In the single agent group, RPMI1640 medium containing 10% FBS (Life Technology In the combination group, 50 μL of 10 ... Dilute with RPMI1640 medium containing 100% FBS (Life Technologies). Trastuzumab (Roche) was added at 50 μL / well. Example 2 1×10 prepared in ‐6‐3 6 cells / mL, add 50 μL / well of effector cells. After heating, the mixture was left to stand at 37°C and 5% CO2 for 16 hours. After removing the supernatant, the cells were washed with 200 μL / well of PBS containing 5% FBS. 5 μL / well of 5% FBS in PBS, 5 μL / well of APC human CD1 1b (Becton Dickinson) was added and the mixture was allowed to stand at 4°C for 15 minutes. The plate was washed twice with 100 μL / well of 5% FBS-containing PBS. Stabilizing Fixative (Becton Dickinson) The cells were suspended in 0.5% CO and allowed to stand overnight at 4°C. The next day, flow cytometry (FACS CantoII) was performed. Measurements were performed using a Flowjo ( (TreeStar) was used. Development was performed using FSC (forward scattered light) / SSC (side scattered light). After that, the number of cells positive for PE (A) and positive for both APC and PE (B) was calculated. C and PE positive (B) cells are target cells that have been phagocytosed by macrophages The cell phagocytosis rate due to ADCP activity was calculated using the following formula. Cell phagocytosis rate (%)=B / (A+B)×100 As shown in Figure 5, rat anti-human SIRPA antibody was used to treat CD47-positive human gastric cancer cell line AGS. As for single antibody agents, Hu5F9G4 (anti-human CD47 antibody) and TTI-621 (human SI RPA-Fc) (Fig. 5A). When combined with umab, it showed ADCP activity equivalent to that of Hu5F9-G4 and TTI-621. Thus, anti-SIRPA antibody inhibited the binding of SIRPA-CD47. These results suggest that administration of IFN-γ enhances the phagocytic ability of macrophages.

[0168] Example 3. Cloning of the cDNA of the variable regions of rat anti-SIRPA antibodies (D13, F44, F63) Nucleotide sequence analysis and amino acid sequence determination 3)-1 Nucleotide sequence analysis of the cDNA of the variable region of D13 and determination of the amino acid sequence 3)-1-1 Preparation of total RNA from D13-producing hybridoma To amplify cDNA encoding the variable region of D13, a hybridoma producing D13 was used. Total RNA was prepared using TRIzol Reagent (Ambion). did.

[0169] 3)-1-2 Nucleotide cDNA of the light chain variable region of D13 by 5'-RACE PCR Otide sequence analysis and amino acid sequence determination The cDNA encoding the light chain variable region was amplified using the total cDNA prepared in Example 3)-1-1. Approximately 1 μg of RNA was used with the SMARTer RACE 5' / 3' Kit (Clontech) The cDNA encoding the variable region of the light chain gene of D13 was cloned into PCR The primers used for amplification in R were UPM (Universal Primer A Mix: Included in the SMARTer RACE 5' / 3' Kit) and a known Primers designed from the sequence of the constant region of the human light chain were used. The cDNA encoding the light chain variable region amplified by 5'-RACE PCR was inserted into the plasmid The nucleotide sequence of the cDNA encoding the light chain variable region is then sequenced. A sequence analysis was performed. The amino acid sequence of the D13 light chain variable region encoded by the determined cDNA The amino acid sequence is an amino acid sequence consisting of amino acid residues 21 to 126 of SEQ ID NO: 23 in the sequence listing. The amino acid sequences of CDRL1, CDRL2, and CDRL3 of D13 are shown in the sequence table. The amino acids of these CDRs are also shown in FIG. The amino acid sequence of each CDR is defined as follows: Cheetham, JC and Rees, AR (1989) P roc. Natl Acad. Sci. USA, 86,9268-9272) It is stated based on.

[0170] 3)-1-3 Nucleotide cDNA of the heavy chain variable region of D13 by 5'-RACE PCR Otide sequence analysis and amino acid sequence determination The cDNA encoding the heavy chain variable region was amplified using the total Approximately 1 μg of RNA was used with the SMARTer RACE 5' / 3' Kit (Clontech) The cDNA encoding the variable region of the heavy chain gene of D13 was The primers used for amplification in R were UPM (Universal Primer A Mix: Included in the SMARTer RACE 5' / 3' Kit) and known rat Primers designed from the sequence of the heavy chain constant region were used. The cDNA encoding the heavy chain variable region amplified by 5'-RACE PCR was inserted into the plasmid The cDNA encoding the heavy chain variable region is then cloned into A sequence analysis was performed. The amino acid sequence of the variable region of the heavy chain of D13 encoded by the determined cDNA The amino acid sequence is an amino acid sequence consisting of amino acid residues 20 to 139 of SEQ ID NO: 25 in the sequence listing. The amino acid sequences of CDRH1, CDRH2, and CDRH3 of D13 are shown in the sequence table. The amino acid sequences of these CDRs are also shown in FIG.

[0171] 3)-2 Nucleotide sequence analysis of the cDNA of the variable region of F44 and determination of the amino acid sequence The same procedure as in Example 3)-1 was carried out. The amino acid sequence of the variable region of the light chain of F44 to be loaded is 21-12 of SEQ ID NO: 27 in the sequence listing. The amino acid sequence corresponds to the 7th amino acid residue. The amino acid sequence of the heavy chain variable region of F44 encoded by the nucleotide sequence is shown in the sequence listing under SEQ ID NO: It corresponds to the amino acid sequence consisting of amino acid residues 20 to 138 of No. 29. Amino acid sequences of DRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 The amino acid sequences of these CDRs are shown in SEQ ID NOs: 7 to 12 in the sequence listing. As shown.

[0172] 3)-3 Nucleotide sequence analysis of the cDNA of the variable region of F63 and determination of the amino acid sequence The same procedure as in Example 3)-1 was carried out. The amino acid sequence of the variable region of the light chain of F63 to be loaded is 21 to 13 of SEQ ID NO: 31 in the sequence listing. The amino acid sequence corresponds to the amino acid residue at position 0. The amino acid sequence of the heavy chain variable region of F63 encoded by the nucleotide sequence is SEQ ID NO:33. It corresponds to the amino acid sequence consisting of amino acid residues 20 to 143 of F63. The amino acid sequences of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 are shown in The amino acid sequences of these CDRs are shown in SEQ ID NOs: 13 to 18 in the Table of Contents. is.

[0173] Example 4. Generation of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) 4)-1 Construction of human chimeric and humanized κ-type light chain expression vector pCMA-LK Restriction of the plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) A fragment of about 5.4 kb obtained by digestion with the enzymes XbaI and PmeI and the sequence of SEQ ID NO: D containing a DNA sequence encoding the human light chain signal sequence and the human κ chain constant region shown in The NA fragment was cloning using the In-Fusion HD PCR cloning kit (Clontech, ) to generate pcDNA3.3 / LK. pCMA was created by removing the neomycin expression unit from pcDNA3.3 / LK. -LK was constructed.

[0174] 4)-2 Construction of human chimeric and humanized .LAMBDA.-type light chain expression vector pCMA-LL pCMA-LK was digested with XbaI and PmeI to remove the light chain signal sequence and the human kappa chain constant sequence. A DNA fragment from which the region has been removed, a human light chain signal sequence shown in SEQ ID NO: 20, and a human A DNA fragment containing the DNA sequence encoding the λ chain constant region was added to In-Fusion HD P The plasmid pCMA-LL was constructed by ligating the plasmid pCMA-LL using the CR cloning kit (Clontech). Built it.

[0175] 4)-3 Human chimeric and humanized IgG4 proFALA type heavy chain expression vector pC Construction of MA-G4PFALA The human heavy chain signal sequence shown in SEQ ID NO: 21 and the human IgG4 PFALA constant region Using a DNA fragment containing a DNA sequence encoding an amino acid, a method similar to that of Example 4)-2 was carried out. The pCMA-G4proFALA was constructed by this method.

[0176] 4)-4 Construction of expression vector for cD13 4)-4-1 Construction of IgG4proFALA type heavy chain expression vector of cD13 The cDNA encoding the variable region of the D13 heavy chain obtained in Example 3)-1 was used as a template. We decided to perform PCR using primers designed for in-fusion cloning. A DNA fragment containing the cDNA encoding the heavy chain variable region was amplified. pCMA-G4 In-Fusion HD PC Insert the amplified DNA fragment using the R cloning kit (Clontech). The cD13 heavy chain expression vector was constructed by the above procedure. The sequence of the nucleotide sequence is shown in SEQ ID NO: 24 in the sequence listing. The nucleotide sequence is the signal sequence, which is the nucleotide sequence from 58 to 417. The nucleotide sequence from nucleotides 418 to 1398 represents the variable region, and the nucleotide sequence from nucleotides 418 to 1398 represents the constant region. The amino acid sequence of the cD13 heavy chain is shown in SEQ ID NO: 25 of the sequence listing. The amino acid sequence consisting of amino acid residues 1 to 19 is the signal sequence, and the amino acid sequence consisting of amino acid residues 20 to 19 is the The amino acid sequence consisting of the 39th amino acid residue is in the variable region, and the amino acid sequence consisting of the 140th to 466th amino acids is in the The amino acid sequences consisting of the 5'-amino acid residues correspond to the constant region. The sequence numbered 25 is also shown in FIG.

[0177] 4)-4-2 Construction of the light chain expression vector of cD13 The cDNA encoding the variable region of the D13 light chain obtained in Example 3)-1 was used as a template. We decided to perform PCR using primers designed for in-fusion cloning. A DNA fragment containing the cDNA encoding the light chain variable region was amplified. pCMA-LK The In-Fusion HD PCR cloning was performed at the site cut with the restriction enzyme BsiWI. The amplified DNA fragment was inserted into the cDNA library using a gene expression kit (Clontech). A D13 light chain expression vector was constructed. The nucleotide sequence encoding the D13 light chain was The nucleotide sequence consisting of nucleotides 1 to 60 is shown in SEQ ID NO: 22 in the table below. The nucleotide sequence from 61 to 378 represents the variable region. The nucleotide sequence consisting of nucleotides 379 to 699 of the constant region is The amino acid sequence of the cD13 light chain is shown in SEQ ID NO: 23 of the sequence listing. The amino acid sequence consisting of amino acid residue 0 is the signal sequence, and amino acids 21 to 126 are The amino acid sequence consisting of amino acid residues from 127 to 233 in the variable region is The amino acid sequences of SEQ ID NO: 22 and SEQ ID NO: 23 correspond to the constant region. is also shown in FIG.

[0178] 4)-5 Construction of expression vector for cF44 4)-5-1 Construction of IgG4proFALA type heavy chain expression vector of cF44 The cDNA encoding the variable region of the F44 heavy chain obtained in Example 3)-2 was used as a template. As a result, a cF44 heavy chain expression vector was constructed in the same manner as in Example 4)-4-1. The nucleotide sequence encoding the 44 heavy chain is shown in SEQ ID NO: 28 of the sequence listing. The nucleotide sequence of the signal sequence is The nucleotide sequence consisting of the nucleotides from 415 to 1395 defines the variable region as The nucleotide sequences encoding the constant regions of the cF44 heavy chain are The amino acid sequence is shown in SEQ ID NO: 29 in the sequence listing. The sequence is a signal sequence, and the amino acid sequence consisting of amino acid residues 20 to 138 is a variable region. The amino acid sequence consisting of amino acid residues 139 to 465 is in the constant region, The sequences of SEQ ID NO:28 and SEQ ID NO:29 are also shown in FIG.

[0179] 4)-5-2 Construction of cF44 light chain expression vector The cDNA encoding the variable region of the F44 light chain obtained in Example 3)-2 was used as a template. As a result, a cF44 light chain expression vector was constructed in the same manner as in Example 4)-4-2. The nucleotide sequence encoding the 44 light chain is shown in SEQ ID NO: 26 of the sequence listing. The nucleotide sequence of the signal sequence is The nucleotide sequence of the variable region, consisting of nucleotides 382-702, The nucleotide sequences encoding the constant regions of the cF44 light chain are: The sequence is shown in SEQ ID NO: 27 in the sequence listing. The sequence is the signal sequence, and the amino acid sequence from 21st to 127th amino acid residues is the variable region. The amino acid sequence consisting of amino acid residues 128 to 234 corresponds to the constant region. The sequences of SEQ ID NO:26 and SEQ ID NO:27 are also shown in FIG.

[0180] 4)-6 Construction of expression vector for cF63 4)-6-1 Construction of IgG4proFALA type heavy chain expression vector of cF63 The cDNA encoding the variable region of the F63 heavy chain obtained in Example 3)-3 was used as a template. As a result, a cF63 heavy chain expression vector was constructed in the same manner as in Example 4)-4-1. The nucleotide sequence encoding the 63 heavy chain is shown in SEQ ID NO: 32 in the sequence listing. The nucleotide sequence of the signal sequence is The nucleotide sequence consisting of nucleotides from 430 to 1410 defines the variable region as The nucleotide sequences encoding the constant regions of the cF63 heavy chain are The amino acid sequence is shown in SEQ ID NO: 33 in the sequence listing. The sequence is a signal sequence, and the amino acid sequence consisting of amino acid residues 20 to 143 is a variable region. The amino acid sequence consisting of amino acid residues 144 to 470 in the constant region is The sequences of SEQ ID NO:32 and SEQ ID NO:33 are also shown in FIG.

[0181] 4)-6-2 Construction of cF63 light chain expression vector The cDNA encoding the variable region of the F63 light chain obtained in Example 3)-3 was used as a template. We decided to perform PCR using primers designed for in-fusion cloning. A DNA fragment containing the cDNA encoding the light chain variable region was amplified. pCMA-LL The DNA was cut with the restriction enzymes BsiWI and HpaI and then subjected to In-Fusion HD PCR. The amplified DNA fragment was inserted using a cloning kit (Clontech). The cF63 light chain expression vector was constructed by the following: The sequence is shown in SEQ ID NO: 30 in the sequence listing. The nucleotide sequence from nucleotides 61 to 390 corresponds to the signal sequence. The nucleotide sequence from 391 to 708 represents the variable region and the constant region. The amino acid sequence of the cF63 light chain is shown in SEQ ID NO: 31 of the sequence listing. The amino acid sequence consisting of amino acid residues 1 to 20 corresponds to the signal sequence, and the amino acid sequence consisting of amino acid residues 21 to 130 corresponds to the The amino acid sequence consisting of amino acid residues 131 to 236 is in the variable region. The amino acid sequences consisting of residues 1, 2, 3 and 4 correspond to the constant region, respectively. The sequence of 31 is also shown in FIG.

[0182] 4)-7 Preparation of cD13, cF44, and cF63 4)-7-1 Production of cD13, cF44, and cF63 FreeStyle 293F cells (Invitrogen) were cultured according to the manufacturer's instructions. Subculture and culture were performed. 1.2 × 10 9 FreeStyle 293 F cells (Invitrogen) were cultured in 3L Fernbach Erlenmeyer The seeds were seeded in a Flask (Corning) and then cooled with a FreeStyle 293 expres Dilute with sion medium (Invitrogen) to 2.0 × 10 6 cells / m The volume was adjusted to 40 L. 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector, and 1.8 mg of Po Add lyethyleneimine (Polyscience #24765) and gently The mixture was stirred gently and left for another 5 minutes before being added to the FreeStyle 293F cells. After 4 hours of shaking culture at 90 rpm in an 8% CO2 incubator at 37°C, L EX-CELL VPRO medium (SAFC Biosciences), 18 mL GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ul Trafiltrate (GIBCO) was added and the cells were incubated in a 37℃, 8% CO2 incubator. The culture supernatant obtained by shaking at 90 rpm for 7 days was placed in Disposable C Filter with apsule filter (Advantec #CCS-045-E1H) Ta.

[0183] 4)-7-2 Purification of cD13, cF44, and cF63 The antibody was isolated from the culture supernatant obtained in Example 4)-7-1 by rProtein A affinity chromatography. The culture supernatant was equilibrated with PBS and purified by a single chromatography step. A column packed with ElectSuRe (GE Healthcare Biosciences) After applying the sample to a column (manufactured by nce), the column was washed with PBS that was at least twice the column volume. Next, the antibody was eluted with 2M arginine hydrochloride solution (pH 4.0) and the fractions containing the antibody were collected. The fraction was dialyzed (Thermo Scientific, Slide-A-Lys). Buffer replacement with PBS(-) was performed using the PBS Dialysis Cassette. Centrifugal UF Filter Device VIVASPI The antibody was concentrated with N20 (molecular weight cutoff UF10K, Sartorius) to obtain the IgG concentration. The solution was adjusted to 10 mg / mL or more. Finally, the solution was filtered using the Minisart‐Plus filter (S The mixture was filtered through a filter (Artorius) to obtain a purified sample.

[0184] Example 5. In vivo analysis of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) itro evaluation 5)-1 Binding to human SIRPA 5)-1-1 Binding evaluation to human SIRPA (Cell-ELISA) 293α cells [described in Example 1)-6] were cultured at 5x10 in DMEM medium containing 10% FBS. 5 The cells were adjusted to 100 cells / mL. pFLAG V5‐DEST‐SIRPA_V1 , V2, or pFLAG V5-DEST was introduced into a 96-well plate ( After dispensing 100 μL into each of the 10% FBS-containing DMEM medium The cells were then cultured overnight at 37°C in 5% CO2. After removing the culture supernatant, pFLAG V5-DEST- For SIRPA_V1, V2, or pFLAG V5-DEST transfected cells, The cD13 (IgG2, IgG4pf) and cF44 ( IgG1, IgG2, IgG4p, IgG4pf), cF63(IgG2, IgG4pf) ) Add antibody at a final concentration of 0 to 10,000 ng / mL at 50 μL / well and incubate at 4° C. for 1 hour. In addition, each well for detecting SIRPA construct expression was filled with 100 Anti-FLAG M2 antibody (S) diluted in 5% FBS-containing PBS to 0.1 ng / mL 50 μL of IGMA (manufactured by IGMA) was added to each well and the plate was left to stand at 4° C. for 1 hour. After washing once with PBS containing 5% FBS, the cells were incubated with PBS containing 5% FBS diluted 1000-fold. eroxidase AffiniPure F(ab')2Fragment Goat Anti-Human IgG, Fcγ Fragment Specific(J Then, a 50 mM NaCl solution (manufactured by ackson ImmunoResearch) was added and the mixture was left to stand at 4°C for 1 hour. The cells in the wells were washed five times with PBS containing 5% FBS, and then the cells were incubated with OPD coloring solution (OPD dissolving solution). Solution (0.05M trisodium citrate, 0.1M disodium hydrogen phosphate, 12H2O pH 4.5) with o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) and H2O2, Add 100μL of 0.4mg / mL (dissolved to 0.6% (v / v)) per well The color reaction was carried out with occasional stirring, and 100 μL / well of 1M HCl was added. After the color reaction was stopped, the plate was read using a plate reader (ARVO: PerkinElmer). The absorbance at 490 nm was measured. The binding of antibody A was normalized based on the expression of the FLAG tag. As shown in Figure 6, the cD13, cF44, and cF63 antibodies bind to SIRPA_V1 and SIRPA _V2 (Fig. 6A, B), and the binding affinity was almost equivalent between the isotypes (Fig. 6C, D).

[0185] 5)-1-2 Evaluation of binding of human chimeric antibodies to human SIRPA Human SIRP produced in Example 1 of cD13, cF44, and cF63 produced in Example 4 The dissociation constant for A_V1_IgV was measured using a Biacore T200 (GE Heal The antibody was used as a ligand for the human chimeric antibody. The running buffer was then captured by the capture method, in which the antigen was measured as an analyte. HBS-EP+ (GE Healthcare Bioscience) ), and CM5 (GE Healthcare Bioscience) as the sensor chip. A human chimeric antibody (manufactured by the company) was applied to the chip at 1 μg / mL for 60 seconds at 10 μL / min. After addition, a dilution series of human SIRPA protein (0.5-8 μg / mL) was added as an antigen. was added at a flow rate of 30 μL / min for 120 seconds, followed by monitoring the dissociation phase for 600 seconds. As a regeneration solution, 3M magnesium chloride (GE Healthcare (manufactured by Bioscience) was added at a flow rate of 20 μL / min for 30 seconds. A 1:1 binding model was used for the analysis, and the binding rate constant ka, dissociation rate constant kd, and dissociation constant (KD; KD=kd / ka) was calculated. The results are shown in Table 4.

[0186] [Table 4]

[0187] 5)-2 Cross-species reactivity evaluation for monkey SIRPA 5x10 293α cells in DMEM medium containing 10% FBS 5 Adjust to 100 cells / mL In contrast, Lipofectamine LTX (Invitrogen) was used. ) to construct pFLAG V5-DEST-monkey SIRPA or pFLAG V5 -DEST was introduced and 100 μL was added to a 96-well plate (Corning). After dispensing, the cells were incubated in 10% FBS-containing DMEM medium at 37°C under 5% CO2 conditions. The transfected cells were cultured overnight at 4°C for 10 min. The cells were then used in Cell-ELISA while still attached to the medium. After removing the culture supernatant, the binding activity to monkey SIRPA was measured in the same manner as for human SIRPA. The binding activity against the antibodies was evaluated. As shown in Figure 7, the cD13, cF44, and cF63 antibodies showed binding to monkey SIRPA. did.

[0188] 5)-3 Evaluation of human or monkey SIRPA-CD47 binding inhibitory activity Human SIRPA or monkey SIRPA expression vector prepared in Examples 5)-1 and 5)-2 After removing the culture supernatant of the transfected 293α cells, pcDNA3.2 V5-DEST-SIRP A_V1, pcDNA3.2 V5-DEST-monkey SIRPA or pcDNA3.2 For V5-DEST-transfected 293α cells, the final concentrations were 0 to 10,000 ng / ml cD13 (IgG2, IgG4pf) diluted with 5% FBS-containing PBS to L cF44 (four types of constant regions: IgG1, IgG2, IgG4p, and IgG4pf), and cF 63 (IgG2, IgG4pf) was added at 50 μL / well. Peroxidase la was prepared at 0 ng / mL in 5% FBS-containing PBS. Beled CD47-Fc was added at 50μL / well and incubated at 4℃ for 1 hour. 1)-6-3 was used to evaluate SIRPA-CD47 binding inhibitory activity. As shown in FIG. 8, cF44, cF63, and cD13 bind to human and monkey SIRPA-CD4 7 binding inhibitory activity (Figure 8A(i), (ii), (iii)), and the activity was almost the same among the isotypes. The results were comparable (Figure 8B(i), (ii), and (iii)).

[0189] 5)-4 ADCP activity of human chimeric anti-human SIRPA antibody against cancer cell lines 5)-4-1 Preparation of target cells CD47-positive human Burkitt's lymphoma cell line Raji cells were harvested. After washing twice with PBS, the number of viable cells was counted using a trypan blue exclusion test. Target cells were prepared in the same manner as in -6-1.

[0190] 5)-4-2 Preparation of PBMC cells PBMC cells were prepared in the same manner as in 2)-6-2.

[0191] 5)-4-3 Preparation of effector cells Effector cells were prepared in the same manner as in 2)-6-3.

[0192] 5)-4-4 Evaluation of ADCP activity 50 μL / well of target cells prepared by the method of Example 5)-4-1 was plated on a 96-well plate with ultra-low attachment surface. The mixture was added to a U-bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.) at a final concentration of 0 to 10 RPMI 1640 medium (Life Technologies) containing 10% FBS at a concentration of 0.000 ng / mL. cD13, cF44, cF63, Hu5F9G4, and T TI-621 or various control human IgGs were added at 50 μL / well. In the treatment group, RPMI1640 medium containing 10% FBS (Life Technology (Production) was added at 50 μL / well, and in the combination group, 10% was added at a final concentration of 400 ng / mL. Diluted with FBS-containing RPMI1640 medium (Life Technology) Rituximab (Zenyaku Kogyo Co., Ltd.) was added at 50 μL per well. ADCP activity was evaluated in a similar manner. As shown in FIG. 9, cD13, cF44, and cF63 were isolated from CD47-positive human Burkitt The drug alone did not show ADCP activity against Raji cells, a lymphoma cell line (Figure 1). 9A), when combined with Rituximab, the same The ADCP activity was approximately 100% (Fig. 9B).

[0193] 5)-5 Toxicity of human chimeric anti-human SIRPA antibody to PBMCs and macrophages Sexual evaluation 5)-5-1 Preparation of PBMCs and macrophages as target cells In the same manner as in 2)-6-2 (PBMC) and 2)-6-3 (macrophages), The target cells were prepared. Each of the collected cells was fluorescently labeled in the same manner as in 2)-6-1. It was used as a cell.

[0194] 5)-5-2 Preparation of effector cells Effector cells were prepared in the same manner as in 2)-6-3.

[0195] 5)-5-3 Evaluation of ADCP activity PBMC or macrophages prepared according to the method of Example 5)-4-2, 50 μL / well was added to a 96-well U-bottom microplate with an ultra-low attachment surface (Sumitomo Bakelite Co., Ltd.). RPMI1 containing 10% FBS at a final concentration of 0.64 to 10,000 ng / mL. cD13 (IgG4pf ), cF44 (four types of constant regions: IgG1, IgG2, IgG4p, and IgG4pf), cF 63 (IgG4pf), Hu5F9G4, TTI‐621 or various control Huma IgG was added at 50 μL / well. RPMI1640 medium containing 10% FBS (L 50μL / well of 100μL ... The ADCP activity was evaluated in the same manner. The ratio is the number of macrophages counted when each antibody was added to the control antibody. It was calculated by dividing by the number. As shown in Figure 10, the ADCP activity of cD13, cF44, and cF63 against PBMCs was The results were almost the same as those of the control IgG (Figure 10A). When comparing antibodies, IgG1 and IgG4p types increased ADCP levels depending on the antibody concentration added. In contrast, IgG2 and IgG4pf did not show ADCP activity (Figure 10B). On the other hand, the abundance ratio of macrophages 16 hours after antibody addition was compared with cF When comparing the 44 antibodies, the IgG4pf type had the lowest rate of macrophage reduction, This suggests that the toxicity induced by antibody addition to SIRPA-positive cells is the lowest. (Figure 10C).

[0196] Example 6 Design of humanized anti-SIRPA antibodies 6)-1 Molecular modeling of the variable region of chimeric antibody cD13 Molecular modeling of the variable region of cD13 was performed using a method known as homology modeling [M Methods in Enzymology, 203, 121-153 (1991)] The protein cDNA was used, which has high sequence identity to the heavy and light chain variable regions of cD13. in Data Bank[Nuc.Acid Res.35,D301‐D303(2 The structure registered in the

[007] (PDB ID: 3CSY) was used as a template. The solid structure analysis program BioLuminate (Schrodinger) was used. He left.

[0197] 6)-2 Design of humanized amino acid sequence cD13 is a CDR-grafted DNA [Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)]. INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM, http: / / www.imgt.org) The rmline sequences IGHV3‐30*13, IGHJ3*01, and IGKV1‐6* 01 and IGKJ2*01, and Kabat et al. Proteins of Immunological Interest,5th Ed.Public Health Service National Institute Published in the American College of Health, Bethesda, MD (1991)] The consensus sequence for human kappa chain subgroup 4 is located in the framework region of cD13. It was selected as the acceptor because it has high identity to the region. The donor residues to be transferred onto the nuclease are determined according to the method described by Queen et al. [Proc. Natl. Aca d. Sci. USA 86, 10029-10033 (1989)] The selection was made by analyzing three-dimensional models with reference to criteria and other factors.

[0198] 6)-3 Humanization of cD13 heavy chain The two heavy chains designed were named hH1 and hH2. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:41 is set forth in SEQ ID NO:41. The amino acid sequence of the heavy chain of hH2 is shown in SEQ ID NO: 40 of the sequence listing. The sequence is shown in SEQ ID NO: 43 in the table below. Nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 The sequence is shown in SEQ ID NO: 42 in the sequence listing. In SEQ ID NOs: 41 and 43, the 1st to 19th The amino acid sequence consists of the 20th to 139th amino acids of the signal sequence. The amino acid sequence consists of amino acid residues 140 to 466 in the variable region. The amino acid sequences correspond to the constant regions. The nucleotide sequence consisting of nucleotides ~57 corresponds to the signal sequence, and nucleotides 58-417 correspond to the The nucleotide sequence of the variable region is nucleotides 418 to 1398. The nucleotide sequences of the polypeptides each encode the constant region. The sequences of SEQ ID NO: 42 and SEQ ID NO: 43 are also shown in FIG. 27. It is also shown in. cD13_H, the heavy chain of the human chimeric anti-SIRPA antibody cD13, and humanized antibody heavy chain h A comparison of the amino acid sequences of hH1 and hH2 is shown in Figure 11. In the above, "·" indicates the same amino acid residue as c013_H, and where an amino acid residue is listed, indicates the substituted amino acid residue.

[0199] 6)-4 Humanization of cD13 light chain The three designed light chains were named hL2, hL3, and hL4. The amino acid sequence is shown in SEQ ID NO: 35 of the sequence listing. The nucleotide sequence of the light chain of hL3 is shown in SEQ ID NO: 34 of the sequence listing. The sequence is set forth in SEQ ID NO: 37 of the sequence listing. The nucleotide sequence is set forth in SEQ ID NO: 36. The full-length amino acid sequence of the light chain of hL4 is set forth in SEQ ID NO: 36. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:39 is shown in SEQ ID NO:39. In SEQ ID NO: 35, 37 and 39, the 1st to 20th amino acids are shown in SEQ ID NO: 38. The signal sequence is composed of amino acid residues 21 to 127. The amino acid sequence of the variable region is the amino acid sequence of 128 to 234 amino acid residues. In addition, in SEQ ID NOs: 34, 36 and 38, 1 to The nucleotide sequence consisting of the 60th nucleotide constitutes the signal sequence, and the nucleotide sequence consisting of the 61st to 381st nucleotides constitutes the The nucleotide sequence of the variable region is nucleotides 382 to 702. The nucleotide sequences consisting of SEQ ID NO: 34 and SEQ ID NO: 35 encode the constant region, respectively. The sequences of SEQ ID NO:36 and SEQ ID NO:37 are also shown in FIG. 24. The sequences of SEQ ID NO:38 and SEQ ID NO:39 are also shown in FIG. 25, respectively. cD13_L, the light chain of the human chimeric anti-SIRPA antibody cD13; humanized antibody light chain h A comparison of the amino acid sequences of hL2, hL3 and hL4 is shown in Figure 12. In the sequence of L4, "·" indicates the same amino acid residue as in cD13_L, and indicates the substituted amino acid residue.

[0200] 6)-5 Design of humanized antibodies by combining heavy and light chains An antibody consisting of hH1 and hL3 is referred to as "H1L3 antibody" or "H1L3". hH1 An antibody consisting of hH2 and hL4 is referred to as "H1L4 antibody" or "H1L4". An antibody consisting of hH2 and hL3 is called an "H2L2 antibody" or "H2L2." The antibody is referred to as "H2L3 antibody" or "H2L3."

[0201] Example 7. Preparation of humanized anti-SIRPA antibody 7)-1 Construction of humanized antibody heavy chain expression vector 7)-1-1 Construction of hH1 expression vector Nucleotide numbers 36 to 4 of the nucleotide sequence of hH1 shown in SEQ ID NO:40 in the Sequence Listing The DNA fragment shown in Figure 34 was synthesized (GENEART). In-Fusion HD Using a PCR cloning kit (Clontech), ALA was synthesized by inserting a synthetic DNA fragment into the site cut with the restriction enzyme BlpI. An H1 expression vector was constructed.

[0202] 7)-1-2 Construction of hH2 expression vector Nucleotide numbers 36 to 4 of the nucleotide sequence of hH2 shown in SEQ ID NO:42 in the Sequence Listing The DNA fragment shown in Figure 34 was synthesized (GENEART). The hH2 expression vector was constructed by the method described above.

[0203] 7)-2 Construction of humanized antibody light chain expression vector 7)-2-1 Construction of hL2 expression vector Nucleotide numbers 37 to 4 of the nucleotide sequence of hL2 shown in SEQ ID NO:34 of the Sequence Listing The DNA fragment shown in Figure 02 was synthesized (GENEART). In-Fusion HD Using a PCR cloning kit (Clontech), pCMA-LK was cloned using restriction enzymes. The hL2 expression vector was created by inserting a synthetic DNA fragment into the BsiWI cleavage site. We built a new system.

[0204] 7)-2-2 Construction of hL3 expression vector Nucleotide numbers 37 to 4 of the nucleotide sequence of hL3 shown in SEQ ID NO:36 in the Sequence Listing The DNA fragment shown in Figure 02 was synthesized (GENEART). The hL3 expression vector was constructed by the method described above.

[0205] 7)-2-3 Construction of hL4 expression vector Nucleotide numbers 37 to 4 of the nucleotide sequence of hL4 shown in SEQ ID NO:38 of the Sequence Listing The DNA fragment shown in Figure 02 was synthesized (GENEART). The hL4 expression vector was constructed by the method described above.

[0206] 7)-3 Preparation of humanized antibodies 7)-3-1 Production of humanized antibodies It was produced in the same manner as in Example 4)-7-1. By combining H-chain and L-chain expression vectors that correspond to the combination of Got the body.

[0207] 7)-3-2 Preparation of humanized antibodies The culture supernatant obtained in Example 7)-3-1 was subjected to rProtein A affinity chromatography. The culture supernatant was purified in a two-step process using HPLC and ceramic hydroxyapatite. A column packed with MabSelectSuRe (GE Healthcare) equilibrated with PBS was After applying the solution to a column (Human Care Biosciences), the volume of the column was increased by more than twice the volume of the column. The column was washed with PBS. Then, the antibody was dissolved in 2 M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was dialyzed (Thermo Scientific, Slide Buffer to PBS using eA-Lyzer Dialysis Cassette -Replace with 5mM sodium phosphate / 50mM MES / pH7.0 buffer. After 5-fold dilution, 5mM NaPi / 50mM MES / 30mM NaCl / pH7 A ceramic hydroxyapatite column (Nihon BioLa) was equilibrated with a buffer solution of 0.0. , Bio-Scale CHT Type-1 Hydroxyapatite C A linear gradient elution with sodium chloride was performed to obtain the antibody. The fractions containing were collected. The fractions were dialyzed (Thermo Scientific, S lide-A-Lyzer Dialysis Cassette) for HBSor( The buffer was replaced with 25mM histidine / 5% sorbitol, pH 6.0. . Centrifugal UF Filter Device VIVASPIN20 The antibody was concentrated using UF10K (molecular weight cutoff, Sartorius) to adjust the IgG concentration to 50 Finally, the solution was filtered through a Minisart-Plus filter (Sarto The mixture was filtered through a filtration filter (by Rius Co., Ltd.) to obtain a purified sample.

[0208] Example 8. Humanized anti-SIRPA antibodies (hD13_H1L3, hD13_H1L4h, hD In vitro evaluation of 13_H2L2, hD13_H2L3) 8)-1 Binding activity of humanized anti-SIRPA antibodies to human, monkey, and mouse SIRPA 8)-1-1 Binding activity of humanized anti-SIRPA antibodies to human, monkey, and mouse SIRPA Sex (Cell‐ELISA) 293α cells [described in Example 1-6] were cultured at 5x10 5 The cells were adjusted to 100 cells / mL. pFLAG V5‐DEST‐SIRPA_V1 ‐V10, pFLAG V5‐DEST‐Monkey SIRPA, pFLAG V5‐DEST - Mouse SIRPA or pFLAG V5-DEST was introduced into 96-well plates. After dispensing 100 μL of each into a 10% FBS-containing D The cells were cultured overnight in MEM medium at 37°C and 5% CO2. The resulting cells were allowed to adhere to the cells. The cells were used in Cell-ELISA as they were. After removing the culture supernatant, various SIRPA genes were detected. For each of the transfected cells, hD13_H1L3, hD13_H1L3, and hD13_H1L3 prepared in Examples 6 and 7 were used. D13_H1L4h, hD13_H2L2, hD13_H2L3, or cD13 and con The control antibody was added at a final concentration of 0 to 10,000 ng / mL, 50 μL per well, and incubated at 4°C for 1 h. The binding to human SIRPA was evaluated in the same manner as in Example 5-1. Ta. As shown in Figures 13A-C, hD13_H1L3, hD13_H1L4h, and hD13_H 2L2 and hD13_H2L3 antibodies are SIRPA variants (V1‐V10), monkey SIR The binding activity to PA was equal to or higher than that of the cD13 antibody. Neither the humanized antibody nor the human chimeric antibody showed binding to mouse SIRPA. Ta.

[0209] 8)-1-2 Evaluation of binding of humanized antibodies to SIRPA hD13_H1L3, hD13_H1L4, hD13_H2L2 and and hD13_H2L3, human SIRPA_V1_IgV and monkey S The dissociation constant for IRPA_ECD was measured using a Biacore T200 (GE Heal Human Antibody Capture Kit (GE Healthcare Bioscience) Humanized antibody to immobilized Anti-Human IgG(Fc) antibody The capture method was used, in which the ligand was captured and the antigen was measured as an analyte. HBS-EP+ (GE Healthcare Biosciences) was used as the running buffer. ience), and CM5 (GE Healthcare Biosciences) as the sensor chip. A humanized antibody (manufactured by Science) was applied to the chip at 1 μg / mL at 10 μL / min. After 60 seconds, a dilution series of human SIRPA protein (0.5 to 8 μg / mL) or a dilution series of monkey SIRPA protein (1-16 μg / mL) was run at a flow rate of 30 μL. The regeneration solution was added at 100 mL / min for 120 seconds, and the dissociation phase was then monitored for 600 seconds. 3M magnesium chloride(GE Healthcare Bi A 1: Using a one-binding model, the association rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD; KD = kd / ka) was calculated. The results are shown in Table 5.

[0210] [Table 5]

[0211] 8)-2 Inhibitory activity of humanized anti-SIRPA antibodies against human or monkey SIRPA-CD47 binding evaluation Example 8)-1: Human SIRPA or monkey SIRPA expression vector introduced After removing the culture supernatant of 3α cells, pcDNA3.2 V5‐DEST‐SIRPA_V1, V2, pcDNA3.2 V5-DEST-monkey SIRPA or pcDNA3.2 V5 -DEST-transfected 293α cells were each treated with a final concentration of 0 to 10,000 ng / mL. hD13_H1L3 and hD13_H1L4 were diluted with PBS containing 5% FBS to obtain Add 50 μL of hD13_H2L2 or hD13_H2L3 to each well. Peroxidase labeled prepared at 1μg / mL in PBS containing 100% FBS CD47-Fc was added and incubated at 4°C for 1 hour. The following procedure was repeated in the same manner as in 1)-6-3. The inhibitory activity of SIRPA-CD47 binding was evaluated. As shown in Figure 15, hD13_H1L3, hD13_H1L4h, hD13_H2L2 The hD13_H2L3 antibody inhibited SIRPA_V1 (Figure 15A) and SIRPA_V2 (Figure 1 5B) and monkey SIRPA (Fig. 15C), exhibited binding inhibitory activity equal to or greater than that of the cD13 antibody. did.

[0212] 8)-3 ADCP activity of humanized anti-human SIRPA antibody against cancer cell lines 8)-3-1 Preparation of target cells CD47-positive human Burkitt's lymphoma cell line Raji cells, or R Amos cells were harvested, washed twice with PBS, and the viable cell count was determined by trypan blue exclusion test. The measurement was made using 4×10 7 After separating and centrifuging the cells, Red Fluorescent Cell Linker Kit (Sigma) The cells were suspended in 2 mL of the attached Diluent C. The labeling solution was 1 mM Cell Vue Claret Dye was diluted to 10 μM with Diluent C and immediately applied to the cells. Mix the cell suspension with an equal volume of CellVue Claret Dye solution and incubate at room temperature for 15 minutes. The cells were placed in 25 mL of RPMI1640 medium (Life Technologies, After washing twice, 2 × 10 6 Resuspended at 100 cells / mL The target cells were prepared in the same manner as in 2)-6-1.

[0213] 8)-3-2 Preparation of PBMC cells PBMC cells were prepared in the same manner as in 2)-6-2.

[0214] 8)-3-3 Preparation of effector cells 2) Prepare effector cells in the same manner as in 6-3, wash twice with PBS, and then resuspend in 1× 10 6 The cells were resuspended in PBS at 1 μL / 10 cells / mL. 6 cell CFSE solution (ThermoFisher) was added at 1 / mL and allowed to stand at room temperature for 10 minutes. 20 ml of RPMI1640 medium (Life Technologies, After washing twice, 1 × 10 6 The cells were resuspended at 1000 cells / mL and then The cells were used as vector cells.

[0215] 8)-3-4 Evaluation of ADCP activity 50 μL / well of target cells prepared by the method of Example 8)-3-1 was plated on a 96-well plate with ultra-low attachment surface. The final concentration of the solution was 0 to 100. RPMI 1640 medium (Life Technologies) containing 10% FBS at a concentration of 0.100 ng / mL. hD13_H1L3, hD13_H1L4h, and hD1 3_H2L2, hD13_H2L3, or cD13 antibody, Hu5F9G4, TTI‐62 1 or various control human IgGs were added at 50 μL / well. RPMI1640 medium (Life Technologies) containing 0% FBS was added for 50 min. In the combination group, 10% FBS was added to the final concentration of 400ng / mL. Rituximab diluted in RPMI 1640 medium (Life Technologies) Imab (Zenyaku Kogyo Co., Ltd.) was added at 50 μL / well. 1×10 6 cells / mL, 50 μL / well of effector cells were added, and then incubated at 37°C, 5% The mixture was left to stand for 16 hours under CO2 conditions. Centrifuge at 1200 rpm for 5 minutes at 4°C, remove the supernatant, and then Wash with 200 μL / well of 5% FBS-containing PBS. Stabilizing Fixative (Becton Dickinson) The cells were suspended in 0.5% CO and allowed to stand overnight at 4°C. The next day, flow cytometry (FACS CantoII) was performed. Measurements were performed using a Flowjo ( (TreeStar) was used. Development was performed using FSC (forward scattered light) / SSC (side scattered light). After that, the number of cells that were APC positive (A) and APC and FITC positive (B) was calculated. The cells that are positive for both APC and FITC (B) are phagocytosed by macrophages. The cell phagocytosis rate due to ADCP activity was calculated using the following formula. Cell phagocytosis rate (%)=B / (A+B)×100 As shown in Figure 16, hD13_H1L3, hD13_H1L4h, hD13_H2L2 The hD13_H2L3 or cD13 antibody-added groups were treated with CD47-positive human Burkitt's ADCP activity in lymphoma cell lines Raji and Ramos cells when used alone When combined with Rituximab, the antibody concentration was not dependent on the antibody. The humanized antibody clones showed typical ADCP activity (Fig. 16B, D). The ADCP activity was equal to or greater than that of the antibody clone.

[0216] Example 9. In vitro evaluation of various anti-SIRPA antibodies 9)-1 Evaluation of binding of various anti-SIRPA antibodies to SIRPA The hD13_H1L3 antibody prepared in Example 7 and OSE-172 (International Publication No. WO1 7 / 178653), KWAR23 (prepared with reference to International Publication No. WO18 / 02660 0), or ADU-1805 (prepared with reference to International Publication No. WO18 / 190719) Human SIRPA_V1_IgV and human SIRP prepared in Example 1 (prepared with reference The dissociation constant for A_V2_IgV was measured. The sequence is shown in SEQ ID NO: 81 of the sequence listing, and the amino acid sequence of the light chain of OSE-172 is shown in SEQ ID NO: 82. The amino acid sequence of the heavy chain of KWAR23 is set forth in SEQ ID NO:83; the amino acid sequence of the light chain of KWAR23 is set forth in SEQ ID NO:84; The sequence is shown in SEQ ID NO: 84, the amino acid sequence of the heavy chain of ADU-1805 is shown in SEQ ID NO: 85, The amino acid sequence of the light chain of U-1805 is shown in SEQ ID NO: 86. Dissociation constant The measurement was performed using a Biacore T200 (GE Healthcare Biosciences) nce) and Human Antibody Capture Kit (GE Anti-H was immobilized using the antibody (manufactured by Healthcare Biosciences, Inc.). Each antibody is captured as a ligand by the human IgG (Fc) antibody, and the antigen is then attached to the The measurement was performed using the capture method, in which the amount of nalyte was measured. HBS was used as the running buffer. -EP+ (GE Healthcare Bioscience), sensor chip CM5 (GE Healthcare Bioscience) was used as the After adding 2 μg / mL of each antibody to the chip at 10 μL / min for 30 seconds, human A dilution series of SIRPA protein (0.25-16 nM) was run at a flow rate of 30 μL / min for 120 s. The regeneration solution was added for 600 seconds, and the dissociation phase was monitored for 600 seconds. nesium chloride(GE Healthcare Bioscience A 1:1 binding model was used for data analysis. The binding rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD; KD = kd / ka) are The results are shown in Table 6.

[0217] [Table 6]

[0218] 9)-2 Evaluation of the inhibitory activity of various anti-human SIRPA antibodies against human SIRPA-CD47 binding Example 9)-1: Human SIRPA_V1 or V2 expression vector-introduced 293α cells After removing the culture supernatant of the cells, pcDNA3.2 V5-DEST-SIRPA_V1, V2 derivatives were For each of the 293α cells, 5% was added to give a final concentration of 0 to 10,000 ng / mL. Various anti-SIRPA antibodies diluted in FBS-containing PBS or various control Human Add 50 μL of IgG per well and immediately adjust the concentration to 1 μg / mL with 5% FBS-containing PBS. Add 50μL / well of the prepared Peroxidase labeled CD47-Fc. The following procedure was used to isolate SIRPA-CD47. The binding inhibitory activity was evaluated. As shown in Figure 31A, hD13_H1L3, OSE-172, KWAR23, and AD The U-1805 antibody showed binding inhibitory activity against SIRPA_V1-CD47. As shown in FIG. 31B, hD13_H1L3, K WAR23 and ADU-1805 showed binding activity, while OSE-172 showed binding inhibitory activity. In addition, as shown in FIG. 31C, hD13_H1L3 showed no effect at the lowest concentration. inhibited binding.

[0219] 9)-3 Evaluation of the binding activity of various anti-human SIRPA antibodies to human SIRPB and human SIRPG Price SIRPβ1(signal regulatory protein β1:Ref The amino acid sequence is published under the Seq accession number NP_006056. and SIRPγ (signal regulatory protein γ:Ref The amino acid sequence is published under the Seq accession number NP_061026. In the present invention, "SIRPα" is referred to as "SIRPα". A, "SIRPβ1" is called "SIRPB1" and "SIRPγ" is called "SIRPG." CHO-K1 cells were cultured in Ham's F-12K medium containing 10% FBS for 3. 3x10 5 The cells / mL were adjusted and incubated overnight at 37°C, 5% CO2. In contrast, Lipofectamine LTX (Invitrogen) was used. pFLAG V5-DEST-human SIRPB, pFLAG V5-DEST-human pFLAG V5-DEST or pSIRPG was introduced into the culture medium containing 10% FBS. The cells were cultured in F-12K medium at 37°C and 5% CO2 for 24 hours. The transfected cells were collected and seeded on a 96-well plate. After removing the culture supernatant, Each of the gene-transfected cells was treated with various anti-human SIRPA antibodies or various control H uman IgG was added at a final concentration of 0 to 10,000 ng / mL, 100 μL / well, The mixture was left to stand at ° C. for 25 minutes. After centrifugation, the supernatant was removed, and the mixture was washed twice with PBS containing 5% FBS. After centrifugation, the supernatant was removed and incubated with PE Mouse anti-Human IgG antibody (Bio Add 50 μL / well of a 1 / 400 diluted solution of 100% ethanol (Legend) and incubate at 4°C for 25 minutes. After centrifugation, the supernatant was removed and the cells were washed twice with PBS containing 5% FBS. After centrifugation, remove the supernatant and add 100 μL / well of 1x BD Stabilizing F The cells were suspended in ixative (Becton Dickinson) and analyzed by flow cytometry. Measurements were performed using a FACS CantoII (Becton Dickinson). Data analysis was performed using Flowjo (TreeStar). FSC (forward scattered light) After development with / SSC (side scattered light), the average fluorescence intensity of PE was measured. By normalizing the mean fluorescence intensity of the reacted samples, the family classification of various antibodies was The connectivity to the offspring was calculated. As shown in Figure 32A and B, various anti-human SIRPA antibodies inhibited human SIRPB and human S The binding to IRPG was concentration-dependent, whereas OSE-172 was not binding to human SIRPG. No binding was observed.

[0220] 9)-4 ADCP activity of various anti-human SIRPA antibodies against cancer cell lines 9)-4-1 Preparation of target cells CD47-positive human Burkitt's lymphoma cell line Raji cells were harvested. After washing twice with PBS, the number of viable cells was counted using a trypan blue exclusion test. 6 The cells were resuspended in PBS at 1 μL / 10 cells / mL. 6 cells / m L of Cell Trace Far Red solution (ThermoFisher) was added. The mixture was then left to stand at room temperature for 10 minutes. Ife Technology) was added, washed twice, and 2 × 10 6 In cells / mL The target cells were resuspended in the same manner as in 2)-6-1. Cells were prepared.

[0221] 9)-4-2 Preparation of PBMC cells PBMC cells were prepared in the same manner as in 2)-6-2.

[0222] 9)-4-3 Preparation of effector cells 2) Prepare effector cells in the same manner as in 6-3, wash twice with PBS, and then resuspend in 1× 10 6 The cells were resuspended in PBS at 1 μL / 10 cells / mL. 6 cell CFSE solution (ThermoFisher) was added at 1 / mL and allowed to stand at room temperature for 10 minutes. 20 ml of RPMI1640 medium (Life Technologies, After washing twice, 1 × 10 6 The cells were resuspended at 1000 cells / mL and then The cells were used as vector cells.

[0223] 9)-4-4 Evaluation of ADCP activity The target cells prepared by the method of Example 8-3-1 were placed in a 96-well U-well plate with an ultra-low attachment surface at 50 μL / well. The final concentration of the solution was 0 to 1000. RPMI1640 medium (Life Technologies) containing 10% FBS was added at 0 ng / mL. Various anti-SIRPA antibodies diluted with IgG (Humanology) or various control antibodies IgG was added at 50 μL / well. RPMI1640 medium (Life Technology, Inc.) containing 10% FBS was used to Rituximab (Zenyaku Kogyo Co., Ltd.) diluted with 50 μL / well was added. 1 x 10 prepared in 8-3-3 6 cells / mL, 50 μL / well of effector cells After addition, the mixture was left to stand for 2 to 16 hours under conditions of 37°C and 5% CO2. After centrifugation for 5 minutes, the supernatant was removed and the plate was washed with 200 μL / well of PBS containing 5% FBS. 0 μL / well of 1× BD Stabilizing Fixative (Becton The cells were suspended in a 500 ml flask (Dickison) and analyzed by flow cytometry (FACS CantoII Measurements were performed using a Flowjo ( (TreeStar) was used. Development was performed using FSC (forward scattered light) / SSC (side scattered light). After that, the number of cells that were APC positive (A) and APC and FITC positive (B) was calculated. The cells that are positive for both APC and FITC (B) are phagocytosed by macrophages. The cell phagocytosis rate due to ADCP activity was calculated using the following formula. Cell phagocytosis rate (%)=B / (A+B)×100 As shown in Figures 33A-C, hD13_H1L3, OSE-172, KWAR23, and The ADU-1805 antibody-added group was CD47-positive human Burkitt's lymphoma aADCP activity was observed against the cell line Raji when administered in combination with Rituximab. After 2 hours of reaction, the strength of ADCP enhancing activity was hD13_H1L3, ADU‐1805, and KW The order was AR23, OSE-172 (A), and after 16 hours of reaction, ADU-1805, hD The order was 13_H1L3, KWAR23, and OSE-172 (B). The reactivity is assumed to be at saturation of phagocytic activity. Concentration dependence after 2 hours of reaction and ADCP In terms of activity comparison, hD13_H1L3 showed the highest activity even at the lowest concentration, and ADU-1 805 and KWAR23 were almost equal, followed by OSE-172. From the above results, hD1 3_H1L3 enhanced ADCP activity in a short time and at the lowest concentration.

[0224] 9)-4-5 Phagocytosis between macrophages by various anti-human SIRPA antibodies (Sel f-ADCP activity) evaluation 50 μL / well of effector cells prepared by the method of Example 8-3-3 was placed on the ultra-low attachment surface. The mixture was added to a 96-well U-bottom microplate (Sumitomo Bakelite Co., Ltd.) at a final concentration of 0 to 100 mM NaCl. RPMI1640 medium (Life Technologies, Inc.) containing 10% FBS at a concentration of 5000 ng / mL Anti-SIRPA antibodies diluted with various anti-SIRPA antibodies or various control antibodies 50 μL of human IgG was added per well. The plate was incubated at 37° C. for 5 min at room temperature for 10 min. The mixture was left to stand for 16-20 hours under conditions of 10% CO2. Centrifuge at 1200 rpm for 5 minutes at 4°C. After removing the supernatant, the plate was washed with 200 μL / well of PBS containing 5% FBS. 1 x BD Stabilizing Fixative (Becton Dickinson The cells were suspended in a Becton flask and analyzed by flow cytometry (FACS CantoII: Becton). The measurements were performed using Flowjo (TreeSta After development using FSC (forward scattered light) / SSC (side scattered light), The number of FITC-positive cells in the wells was calculated (A). By normalizing with the ITC-positive cell count (B), the decrease in each sample was The self-ADCP activity was calculated using the following formula: Self-ADCP(%)=(A / B)×100 As shown in Figure 33B, hD13_H1L3, OSE-172, KWAR23, and AD Addition of U-1805 antibody inhibited the self-immunization of macrophages, which are effector cells. -ADCP activity was observed. The highest reduction rates were observed in OSE-172, KWAR23, and ADU-18. The order of decrease was hD13_H1L3, followed by hD13_H1L3. The decrease rate was higher in Sel 05, which was caused by anti-SIRPA antibodies. This phenomenon was observed when each anti-SIRPA antibody was administered. This suggests that SIRPA-positive cells such as macrophages and dendritic cells may be reduced or depleted. This is an indicator of side effects on the immune system.

[0225] Example 10. In vivo evaluation of various anti-SIRPA antibodies Since SIRPA is a target expressed on host immune cells, it can be targeted by human SIRPA antibodies. To evaluate the antitumor effect of this drug, it is necessary to study it in mice expressing human SIRPA. [Ring et al. PNAS, 2017 (114) 49, E10578‐ On the other hand, to evaluate the contribution of the immune system to the antitumor effect, It is important to use immunocompetent mice rather than whole mice [Yanagita et al. t al. JCI, 2017 (2) 1, 1-15]. Human SIRPA alone, or We have developed a genetically engineered mouse model in which both human SIRPA and human CD47 were introduced into immunocompetent mice. A mouse cancer cell line transfected with human CD47 was transplanted into the mouse, and the tumor volume was 100 mm 3 These mice were then divided into groups. Anti-CD47 biologics, such as CD47 antibodies or SIRPA-Fc fusion proteins, or negative As a control group, PBS etc. is administered 1-3 times a week for 1-3 weeks. When considering the additive effect, chemotherapy drugs, antibody drugs, and molecular targets are used for each of these groups. The tumor diameter (long diameter / short diameter) in each administration group was measured with an electronic caliper every 2-3 days. The tumor volume was calculated from the tumor volumes of the antibody-administered groups and the negative control group. By calculating the growth inhibition rate, it is possible to compare and evaluate the in vivo efficacy of each drug. The tumor volume and tumor growth inhibition rate are expressed by the following formula. Tumor volume (mm 3 )=(major axis x minor axis x minor axis) / 2 Tumor growth inhibition rate (%) = (1 - tumor volume of each treatment group / tumor volume of negative control group) x 100 [Industrial Applicability]

[0226] The anti-SIRPα antibody of the present invention can be used in combination with other antibody drugs or immune chains having other effector functions. It can be used as an antibody drug to be used in combination with other antibody drugs that have a checkpoint inhibitory effect. This can be done. [Sequence List Free Text]

[0227] SEQ ID NO:1: Amino acid sequence of D13 CDR-L1 SEQ ID NO:2: Amino acid sequence of D13 CDR-L2 SEQ ID NO:3: Amino acid sequence of D13 CDR-L3 SEQ ID NO:4: Amino acid sequence of D13 CDR-H1 SEQ ID NO:5: Amino acid sequence of D13 CDR-H2 SEQ ID NO:6: Amino acid sequence of D13 CDR-H3 SEQ ID NO: 7: Amino acid sequence of F44 CDR-L1 SEQ ID NO: 8: Amino acid sequence of F44 CDR-L2 SEQ ID NO: 9: Amino acid sequence of F44 CDR-L3 SEQ ID NO: 10: Amino acid sequence of F44 CDR-H1 SEQ ID NO: 11: Amino acid sequence of F44 CDR-H2 SEQ ID NO: 12: Amino acid sequence of F44 CDR-H3 SEQ ID NO: 13: Amino acid sequence of F63 CDR-L1 SEQ ID NO: 14: Amino acid sequence of F63 CDR-L2 SEQ ID NO: 15: Amino acid sequence of F63 CDR-L3 SEQ ID NO: 16: Amino acid sequence of F63 CDR-H1 SEQ ID NO: 17: Amino acid sequence of F63 CDR-H2 SEQ ID NO: 18: Amino acid sequence of F63 CDR-H3 SEQ ID NO: 19: Nucleotide sequence encoding the human light chain signal sequence and the constant region of the human kappa light chain DNA fragment containing the sequence SEQ ID NO: 20: Nucleotide sequence encoding the human light chain signal sequence and the constant region of the human λ light chain DNA fragment containing the sequence SEQ ID NO: 21: Human heavy chain signal sequence and human IgG4 ProFALA heavy chain constant region A DNA fragment containing the nucleotide sequence to be read SEQ ID NO: 22: Nucleotide sequence encoding the light chain of human chimeric antibody D13 SEQ ID NO: 23: Amino acid sequence of the light chain of human chimeric antibody D13 SEQ ID NO: 24: Nucleotide sequence encoding the heavy chain of human chimeric antibody D13 SEQ ID NO: 25: Amino acid sequence of the heavy chain of human chimeric antibody D13 SEQ ID NO: 26: Nucleotide sequence encoding the light chain of human chimeric antibody F44 SEQ ID NO: 27: Amino acid sequence of the light chain of human chimeric antibody F44 SEQ ID NO: 28: Nucleotide sequence encoding the heavy chain of human chimeric antibody F44 SEQ ID NO: 29: Amino acid sequence of the heavy chain of human chimeric antibody F44 SEQ ID NO: 30: Nucleotide sequence encoding the light chain of the human chimeric antibody F63 SEQ ID NO: 31: Amino acid sequence of the light chain of human chimeric antibody F63 SEQ ID NO: 32: Nucleotide sequence encoding the heavy chain of the human chimeric antibody F63 SEQ ID NO: 33: Amino acid sequence of the heavy chain of human chimeric antibody F63 SEQ ID NO: 34: Nucleotide sequence encoding the hL2 light chain of humanized D13 SEQ ID NO: 35: Amino acid sequence of the hL2 light chain of humanized D13 SEQ ID NO: 36: Nucleotide sequence encoding the hL3 light chain of humanized D13 SEQ ID NO: 37: Amino acid sequence of the hL3 light chain of humanized D13 SEQ ID NO: 38: Nucleotide sequence encoding the hL4 light chain of humanized D13 SEQ ID NO: 39: Amino acid sequence of the hL4 light chain of humanized D13 SEQ ID NO: 40: Nucleotide sequence encoding the hH1 heavy chain of humanized D13 SEQ ID NO: 41: Amino acid sequence of the hH1 heavy chain of humanized D13 SEQ ID NO: 42: Nucleotide sequence encoding the hH2 heavy chain of humanized D13 SEQ ID NO: 43: Amino acid sequence of the hH2 heavy chain of humanized D13 SEQ ID NO: 44: Nucleotide sequence encoding the ECD of human SIRPA variant 1 SEQ ID NO: 45: Amino acid sequence of the ECD of human SIRPA variant 1 SEQ ID NO: 46: Nucleotide sequence encoding IgV of human SIRPA variant 1 SEQ ID NO: 47: Amino acid sequence of IgV of human SIRPA variant 1 SEQ ID NO: 48: Nucleotide sequence encoding the ECD of human SIRPA variant 2 SEQ ID NO: 49: Amino acid sequence of the ECD of human SIRPA variant 2 SEQ ID NO: 50: Nucleotide sequence encoding IgV of human SIRPA variant 2 SEQ ID NO: 51: Amino acid sequence of IgV of human SIRPA variant 2 SEQ ID NO: 52: Nucleotide sequence encoding the ECD of monkey SIRPA SEQ ID NO: 53: Amino acid sequence of the ECD of monkey SIRPA SEQ ID NO: 54: Nucleotide sequence encoding human CD47-Fc SEQ ID NO: 55: Amino acid sequence of IgV of human CD47-Fc SEQ ID NO: 56: Amino acid sequence of human SIRPA variant 1 SEQ ID NO: 57: Amino acid sequence of human SIRPA variant 2 SEQ ID NO: 58: Amino acid sequence of monkey SIRPA SEQ ID NO: 59: Amino acid sequence of C57 BL / 6 mouse SIRPA SEQ ID NO: 60: Amino acid sequence of BALB / C mouse SIRPA SEQ ID NO: 61:129 Amino acid sequence of mouse SIRPA SEQ ID NO: 62: Amino acid sequence of NOD mouse SIRPA SEQ ID NO: 63: Amino acid sequence of human SIRPA variant 3 SEQ ID NO: 64: Amino acid sequence of human SIRPA variant 4 SEQ ID NO: 65: Amino acid sequence of human SIRPA variant 5 SEQ ID NO: 66: Amino acid sequence of human SIRPA variant 6 SEQ ID NO: 67: Amino acid sequence of human SIRPA variant 7 SEQ ID NO: 68: Amino acid sequence of human SIRPA variant 8 SEQ ID NO: 69: Amino acid sequence of human SIRPA variant 9 SEQ ID NO: 70: Amino acid sequence of human SIRPA variant 10 SEQ ID NO: 71: Amino acid sequence of human SIRPA_V2_IgV SEQ ID NO: 72: Amino acid sequence of human SIRPA_V2_IgV_IgC1 SEQ ID NO: 73: Amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ0 SEQ ID NO: 74: Amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ1 SEQ ID NO: 75: Amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ2 SEQ ID NO: 76: Amino acid sequence 81 of mouse SIRPA mutant hmSIRPA_Δ0 Amino acid sequence from 85th to 86th SEQ ID NO: 77: amino acid 81 in the amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ1 Amino acid sequence from 85th to 86th SEQ ID NO: 78: 81st to 85th amino acids in the amino acid sequence of BALB / C mouse SIRPA Amino acid sequence SEQ ID NO: 79: Amino acid sequence 126 to 130 of BALB / C mouse SIRPA Eye amino acid sequence SEQ ID NO: 80: amino acid sequence 81 of mouse SIRPA mutant hmSIRPA_Δ2 Amino acid sequence from 85th to 86th SEQ ID NO: 81: Amino acid sequence of OSE-172 antibody heavy chain (OSE-172_hG4Pro) column SEQ ID NO: 82: Amino acid sequence of OSE-172 antibody light chain (OSE-172_hK) SEQ ID NO: 83: Amino acid sequence of KWAR23 antibody heavy chain (KWAR23_hG4Pro) SEQ ID NO: 84: Amino acid sequence of KWAR23 antibody light chain (KWAR23_hK) SEQ ID NO: 85: Amino acid sequence of the ADU-1805 antibody heavy chain (ADU-1805_hG2) SEQ ID NO: 86: Amino acid sequence of ADU-1805 antibody light chain (ADU-1805_hK)

[0228] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. It shall be incorporated.

Claims

1. Any of the following (1) to (8): (a) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO:1; (b) a light chain CDRL2 consisting of the amino acid sequence represented by SEQ ID NO:2; (c) a light chain CDRL3 consisting of the amino acid sequence represented by SEQ ID NO:3; (d) a heavy chain CDRH1 consisting of the amino acid sequence represented by SEQ ID NO:4; (e) a heavy chain CDRH2 consisting of the amino acid sequence represented by SEQ ID NO:5; and (f) An antibody comprising a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO:6: (1) An antibody that specifically binds to human SIRPα and is composed of a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 139 of SEQ ID NO: 41 and a light chain comprising a light chain variable region consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 127 of SEQ ID NO: 37, and inhibits the binding of human SIRPα to CD47; (2) an antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47, the antibody being composed of a heavy chain comprising a heavy chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues at positions 20 to 139 of SEQ ID NO: 41 and a light chain comprising a light chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues at positions 21 to 127 of SEQ ID NO: 37; (3) An antibody that specifically binds to human SIRPα and that has a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 139 of SEQ ID NO: 41 and a light chain comprising a light chain variable region consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 127 of SEQ ID NO: 39, and inhibits the binding of human SIRPα to CD47; (4) An antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47, the antibody being composed of a heavy chain comprising a heavy chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues at positions 20 to 139 of SEQ ID NO: 41 and a light chain comprising a light chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues at positions 21 to 127 of SEQ ID NO: 39; (5) An antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47; and the antibody is composed of a heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues at positions 20 to 139 of SEQ ID NO: 43 and a light chain having a light chain variable region consisting of an amino acid sequence consisting of amino acid residues at positions 21 to 127 of SEQ ID NO: 35; and (6) An antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47, the antibody being composed of a heavy chain comprising a heavy chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues 20 to 139 of SEQ ID NO: 43 and a light chain comprising a light chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO: 35; (7) An antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47; and the antibody is composed of a heavy chain having a heavy chain variable region consisting of an amino acid sequence consisting of amino acid residues 20 to 139 of SEQ ID NO: 43 and a light chain having a light chain variable region consisting of an amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO: 37; and (8) An antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47, the antibody being composed of a heavy chain including a heavy chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues 20 to 139 of SEQ ID NO: 43, and a light chain including a light chain variable region having an amino acid sequence having 95% or more sequence identity with an amino acid sequence consisting of amino acid residues 21 to 127 of SEQ ID NO:

37.

2. The antibody of claim 1, which has reduced ADCC and / or ADCP activity.

3. The antibody according to claim 1 or 2, which enhances the phagocytic activity of macrophages.

4. The antibody according to any one of claims 1 to 3, wherein the lysine residue at the carboxyl terminus of the heavy chain is deleted.

5. An antigen-binding fragment of the antibody according to any one of claims 1 to 4.

6. An antigen-binding fragment of the antibody of claim 5 selected from the group consisting of Fab, F(ab')2, Fab' and scFv.

7. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 4 or an antigen-binding fragment of the antibody according to claim 5 or 6 as an active ingredient.

8. The pharmaceutical composition according to claim 7, which is an antitumor agent.

9. The pharmaceutical composition according to claim 8, further comprising an immune checkpoint inhibitor and / or an antibody drug that specifically reacts with a cancer antigen and has ADCC and / or ADCP activity as an active ingredient of an antitumor agent.

10. A pharmaceutical composition which is an antitumor agent used in combination with an immune checkpoint inhibitor and / or an antibody drug having ADCC and / or ADCP activity by specifically reacting with a cancer antigen, comprising the antibody according to any one of claims 1 to 4 or an antigen-binding fragment of the antibody according to claim 5 or 6 as an active ingredient.

11. The pharmaceutical composition according to claim 9 or 10, wherein the immune checkpoint inhibitor is a binding inhibitor between PD-L1 and PD-1, or a CTLA4 inhibitor.

12. The pharmaceutical composition according to claim 9 or 10, wherein the antibody drug that specifically reacts with a cancer antigen and has ADCC and / or ADCP activity is selected from the group consisting of an anti-CD20 antibody, an anti-HER2 antibody, and an anti-EGFR antibody.

13. The pharmaceutical composition according to any one of claims 8 to 12, wherein the tumor is one or more types of tumors selected from the group consisting of carcinoma, sarcoma, lymphoma, leukemia, myeloma, germinoma, brain tumor, carcinoid, neuroblastoma, retinoblastoma, and nephroblastoma.

14. Tumors include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, appendix cancer, anal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer, vaginal cancer, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, and undifferentiated multiforme. The pharmaceutical composition according to claim 13, wherein the tumor is one or more types selected from the group consisting of myelosarcoma, myxoid fibrosarcoma, malignant peripheral nerve sheath tumor, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, epithelioid sarcoma, B cell lymphoma, T / NK cell lymphoma, Hodgkin's lymphoma, myeloid leukemia, lymphocytic leukemia, myeloproliferative disease, myelodysplastic syndrome, multiple myeloma, testicular cancer, ovarian cancer, glioma, and meningioma.

15. A polynucleotide consisting of a nucleotide sequence encoding the amino acid sequence of the heavy and light chains of the antibody according to any one of claims 1 to 4, or the antigen-binding fragment of the antibody according to claim 5 or 6.

16. A vector comprising the polynucleotide of claim 15.

17. 17. A host cell comprising the polynucleotide of claim 15 or the vector of claim 16.

18. A method for producing the antibody according to any one of claims 1 to 4, comprising culturing the host cell according to claim 17 and purifying the antibody from the culture.

19. 20. An antibody produced by the method of claim 18.

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