anti-SIRPα antibody

Abstract

The present invention relates to an anti-SIRPα antibody that can be used as an antitumor agent, and an antitumor agent containing the antibody as an active ingredient. , , , , It aims to provide an antitumor agent. 【Solution means】The present invention includes (a) a light chain CDRL1 consisting of the amino acid sequence represented by SEQ ID NO: 1, 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) a heavy chain CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 6. The heavy chain constant region is the heavy chain constant region of human IgG4, and the phenylalanine at position 234 indicated by the EU index by Kabat et al. is substituted with alanine, the leucine at position 235 is substituted with alanine, and further the serine at position 228 is substituted with proline. It is an antibody that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. That's right. There is.

Description

[Technical Field]

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

[0002] SIRPα (SHPS-1) is used in 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, C Regarding the IgV domain, which is the binding site for D47, there are 10 variants in humans. This has been reported (Non-Patent Document 2). On the other hand, the intracellular region is immunoreceptor Includes tyrosine-based inhibition motifs (ITIM) Furthermore, through binding with CD47, the tyrosine dephosphorylation enzymes SHP-1 and SHP-2 Binding to is induced, and an inhibitory signal is transmitted.

[0003] Physiological phenomena resulting from SIRPα-CD47 interaction include SIRP on macrophages. CD47 on red blood cells binds to alpha, transmitting the "Don't eat me" signal. It has been shown that this avoids unnecessary phagocytosis of red blood cells (Non-Patent Literature 3). On the other hand, tumor micro Even in micro-environments, SIRPα is highly expressed on macrophages and dendritic cells, as well as on tumor cells. It has been suggested that the binding of CD47 suppresses the phagocytic activity of tumor cells. Suppression of phagocytic activity leads to the subsequent suppression of tumor antigen presentation to T cells, and further to the suppression of the tumor immune response. It is expected that they will be connected. Therefore, the immune phenomenon of phagocytosis by tumor cells is the uptake of tumor antigens. This can be considered a checkpoint for entry.

[0004] To date, antibodies against CD47, a ligand for SIRPα, have been used to study SIRPα-CD4 It has been reported that inhibiting the interaction enhances the phagocytic ability against tumor cells. (Non-patent document 4) This is because even when using anti-SIRPα antibodies, tumor cells are immune cells A similar phenomenon was observed under conditions of concomitant use with anti-cancer antibodies that possess effector activity that attracts to it. (Non-patent documents 5 and 6) are available. In addition, in an allogeneic mouse tumor model using anti-CD47 antibodies... It has been suggested that it not only has an antitumor effect but also induces tumor immunity (Non-Patent Document 7). Similarly, anti-SIRPα antibodies can be expected to have a similar effect when used in combination with anti-cancer antibodies.

[0005] On the other hand, immune checkpoint inhibitors include those that target T cells such as PD-1 / PD-L1. Several antibodies against disease-suppressing molecules have been developed, and their effectiveness has been demonstrated in clinical settings. Non-patent documents 8 and 9). SIRPα-CD47 is currently the only proven phagocytic inhibitory molecule. Therefore, inhibitory antibodies against this molecule are novel checkpoints against targets other than T cells. It is expected to have potential as an inhibitor of immune checkpoints, and is used in patients resistant to conventional immune checkpoint inhibitors. It also has the potential to be widely effective against [these conditions].

[0006] To date, human Burkitt's has been studied using anti-mouse SIRPα antibody (MY-1). Studies using a lymphoma subcutaneous transplant model have shown that combination therapy with rituximab is effective against tumors. The effect has been demonstrated. In addition, in a mouse colorectal cancer model, the combination with PD-1 antibody showed an antitumor effect. Results have been observed (Non-Patent Document 5). In addition, different clones of anti-mSIRPα antibodies ( In the study using P84), antitumor and life-prolonging effects have also been observed in a mouse liver cancer model even when combined with an anti-PD-L1 antibody or an anti-4-1BB antibody. When the same tumor cells were re-implanted into the mice that showed a life-prolonging effect, further antitumor and life-prolonging effects were obtained. Therefore, it is shown that it is possible to induce a strong tumor immune response by inhibiting different immune checkpoints (Patent Document 1). These results are examples showing a combined effect not only in the combination with an anticancer antibody having the conventionally predicted effector activity but also in the combination with an immune checkpoint inhibitor targeting T cells. Similar effects can also be expected for an anti-human SIRPα antibody. In recent years, reports of patents on anti-SIRPα antibodies have been successively made by various companies (Patent Documents 1, 2 and 3). For example, OSE-172 is an IgG4Pro type antibody and shows binding affinity to the V1 type of SIRPα and SIRPβ1, but does not show binding affinity to the V2 type of SIRPα and SIRPγ. KWAR23 is an IgG1N279A type antibody and shows binding affinity to 10 types of SIRP α variants as well as SIRPβ1 and SIRPγ. ADU-1805 is an I gG2 type antibody and shows binding affinity to 10 types of SIRPα variants and SIRPγ. It is unclear which antibody is the most appropriate as a medicine, and efforts to obtain excellent antibodies have been continuing. Furthermore, in the study using an anti-CD47 antibody, in addition to the antibody medicines as described above, it has been reported that sufficient antitumor and life-prolonging effects are also shown in the combination with a chemotherapeutic agent widely used as a conventional SOC (Standard of Care: Standard of Care) and radiotherapy.

[0007] (Standard of Care) <000… In particular, in cases of combination therapy with chemotherapy agents, the chemotherapy agent is administered first, followed by the anti-CD47 antibody. Administering this drug provides a stronger antitumor effect and prolongs life compared to the simultaneous administration of chemotherapy agents and anti-CD47 antibodies. Since it shows effectiveness (Non-Patent Document 7), pre-administration of chemotherapy agents facilitates the uptake of tumor antigens. By preparing such an environment, antigen uptake can be inhibited through the SIRPα-CD47 interaction. This suggests the possibility of enhancing the effect of immune activation.

[0008] Based on the above, anti-SIRPα antibodies, when used in combination with various antitumor agents, can lead to stronger tumor immunity. It can be inferred that this drug may induce an epidemic 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 [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention provides an anti-SIRPα antibody that can be used as an antitumor agent, and a method for effectively using the antibody. The purpose is to provide an antitumor agent that is included as part of the product. [Means for solving the problem]

[0012] The inventors have identified SIRPα expressed in phagocytic cells with phagocytic ability and SIRPα expressed in tumor cells. The interaction with CD47 is inhibited by an anti-SIRPα antibody, allowing tumor cells to switch to phagocytic cells. It inhibits the transmission of the "Don't-eat-me" signal, preventing phagocytic cells from destroying tumor cells. We investigated methods to enhance the phagocytic activity of cells. The inventors investigated methods to enhance SIRPα We are attempting to create an antibody with higher affinity and a stronger inhibitory effect on the interaction between SIRPα and CD47. Furthermore, anti-SIRPα antibodies possess effector functions such as ADCC or ADCP. Considering that this could potentially attack one's own immune cells, the effector We also investigated the production of anti-SIRPα antibodies that do not possess the effector function. To reduce the effect, the antibody subclass was set to IgG4, and the effector function was further reduced. A mutation was introduced into the Fc region of the antibody to induce this interaction. As a result, the interaction between SIRPα and CD47 was observed. To create an anti-SIRPα antibody that strongly inhibits but has reduced effector function. This was achieved. This antibody does not bind to the Fc receptor on effector cells and does not exert effector function. Since it does not exert its full effect, it does not have sufficient antitumor activity on its own. Therefore, other ingredients with effector functions are used. When used in combination with antibody drugs or other antibody drugs that have immune checkpoint inhibitory effects, The discovery of a favorable antitumor effect led to the completion of this invention.

[0013] In other words, the present invention is as follows. [1](a) Light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 1, (b) Light chain CDRL2 consisting of the amino acid sequence represented by Sequence ID No. 2, (c) Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 3, (d) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 4, (e) Heavy chain CDRH2 consisting of the amino acid sequence represented by Sequence ID No. 5, and (f) Heavy chain CDRH3 consisting of the amino acid sequence represented by Sequence ID No. 6 It contains a compound that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. antibody. [2] The heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADCP An antibody of [1] containing a mutation that results in reduced activity. [3] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al. found that the EU The phenylalanine at position 234, as shown by DEX, is substituted with alanine, and at position 235 Leucine is replaced by alanine, and serine at position 228 is replaced by proline. Antibodies of [1] or [2]. [4] The amino acid sequence of the heavy chain constant region is the same as the amino acid sequence of positions 140-466 of SEQ ID NO: 25. An antibody with amino acid sequence [3] consisting of the group.

[0014] [5] (ai) From the amino acid sequence consisting of amino acid residues 21-126 of sequence number 23 The light chain variable region or (aii) The amino acid sequence consisting of amino acid residues 21-126 of sequence number 23 and 95% It consists of an amino acid sequence having sequence identity as shown above, and has binding activity to human SIRPα. Chain variable region, and (bi) Heavy chain consisting of amino acid sequence from amino acid residues 20 to 139 of SEQ ID NO: 25 Variable region or (bii) The amino acid sequence consisting of amino acid residues 20-139 of SEQ ID NO: 25 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. Chain variable region It includes, and the heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADC Mutations that result in reduced P activity, specifically bind to human SIRPα, and human SIRPα An antibody that inhibits the binding of CD47 to it. [6] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al.'s EU-1 The phenylalanine at position 234, as shown by DEX, is substituted with alanine, and at position 235 Leucine is replaced by alanine, and serine at position 228 is replaced by proline. [5] Antibody. [7] The amino acid sequence of the heavy chain constant region is the same as the amino acid sequence of positions 140-466 of SEQ ID NO: 25. An antibody with amino acid sequence [6] consisting of the group.

[0015] [8](a) Light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 7, (b) Light chain CDRL2 consisting of the amino acid sequence represented by Sequence ID No. 8, (c) Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 9, (d) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 10, (e) Heavy chain CDRH2 consisting of the amino acid sequence represented by Sequence ID No. 11, and (f) Heavy chain CDRH3 consisting of the amino acid sequence represented by Sequence ID No. 12 It contains a compound that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. antibody. [9] The heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADCP An antibody [8] containing a mutation that results in reduced activity.

[10] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al.'s EU The phenylalanine at position 234, indicated by INDEX, is replaced with alanine, and at position 235, The leucine is replaced with alanine, and the serine at position 228 is replaced with proline. [8] or [9] specifically binds to human SIRPα, and the binding of human SIRPα to CD47 is impaired. Antibodies that inhibit this process.

[11] The amino acid sequence of the heavy chain constant region is the amino acid sequence of sequence number 139-465 of sequence number 29. An antibody

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

[0016]

[12] (ai) From the amino acid sequence consisting of amino acid residues 21-127 of sequence number 27 A light chain variable region or (aii) The amino acid sequence consisting of amino acid residues 21-127 of sequence number 27 and 95% It consists of an amino acid sequence having sequence identity as shown above, and has binding activity to human SIRPα. Chain variable region, and (bi) Heavy chain consisting of amino acid sequence from amino acid residues 20 to 138 of SEQ ID NO: 29 Variable region or (bii) The amino acid sequence consisting of amino acid residues 20-138 of sequence number 29 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. Chain variable region It includes, and the heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADC Mutations that result in reduced P activity, specifically bind to human SIRPα, and human SIRPα An antibody that inhibits the binding of CD47 to it.

[13] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al.'s EU study The phenylalanine at position 234, indicated by INDEX, is replaced with alanine, and at position 235, The leucine is replaced with alanine, and the serine at position 228 is replaced with proline. It specifically binds to human SIRPα in

[12] and inhibits the binding of human SIRPα to CD47. Antibodies.

[14] The amino acid sequence of the heavy chain constant region is the amino acid sequence of sequence number 139-465 of sequence number 29. An antibody

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

[0017]

[15] (a) Light chain CDRL1 consisting of the amino acid sequence represented by Sequence ID No. 13, (b) Light chain CDRL2 consisting of the amino acid sequence represented by Sequence ID No. 14, (c) Light chain CDRL3 consisting of the amino acid sequence represented by Sequence ID No. 15, (d) Heavy chain CDRH1 consisting of the amino acid sequence represented by Sequence ID No. 16, (e) Heavy chain CDRH2 consisting of the amino acid sequence represented by Sequence ID No. 17, and (f) Heavy chain CDRH3 consisting of the amino acid sequence represented by Sequence ID No. 18 It contains a compound that specifically binds to human SIRPα and inhibits the binding of human SIRPα to CD47. antibody.

[16] The heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADC An antibody containing a mutation that results in reduced P activity,

[15] .

[17] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al.'s EU study The phenylalanine at position 234, indicated by INDEX, is replaced with alanine, and at position 235, The leucine is replaced with alanine, and the serine at position 228 is replaced with proline.

[15] or

[16] specifically binds to human SIRPα and binds human SIRPα to CD47. Antibodies that inhibit the synthesis of antibodies.

[18] The amino acid sequence of the heavy chain constant region is the amino acid sequence of sequence number 144-470 of sequence number 33. An antibody having an amino acid sequence consisting of residues,

[17] .

[0018]

[19] (ai) From the amino acid sequence consisting of amino acid residues 21-130 of sequence number 31 A light chain variable region or (aii) The amino acid sequence consisting of amino acid residues 21-130 of sequence number 31 and 95% It consists of an amino acid sequence having sequence identity as shown above, and has binding activity to human SIRPα. Chain variable region, and (bi) Heavy chain consisting of amino acid sequence from amino acid residues 20 to 143 of SEQ ID NO: 33 Variable region or (bii) The amino acid sequence consisting of amino acid residues 20-143 of sequence number 33 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. Chain variable region It includes, and the heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADC Mutations that result in reduced P activity, specifically bind to human SIRPα, and human SIRPα An antibody that inhibits the binding of CD47 to it.

[20] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al.'s EU The phenylalanine at position 234, indicated by INDEX, is replaced with alanine, and at position 235, The leucine is replaced with alanine, and the serine at position 228 is replaced with proline. It specifically binds to human SIRPα in

[19] and inhibits the binding of human SIRPα to CD47. Antibodies.

[21] The amino acid sequence of the heavy chain constant region is the amino acid sequence of position 144-470 of SEQ ID NO: 33 An antibody with amino acid sequence

[20] consisting of residues.

[0019]

[22] Any of the antibodies in (1) to (8) below, or any of [1] to [4] below: (1) A heavy amino acid sequence consisting of amino acid residues 20 to 466 of Sequence ID No. 41 The chain and the amino acid sequence consisting of amino acid residues 21-234 of Sequence ID No. 37 It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. antibodies; (2) The amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 41 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. The chain and amino acid residues 21-234 of Sequence ID No. 37 have more than 95% sequence identity. Human SIR consists of a light chain with an amino acid sequence that has binding activity to human SIRPα. An antibody that specifically binds to Pα and inhibits the binding of human SIRPα to CD47; (3) The amino acid sequence consisting of amino acid residues 20 to 466 of SEQ ID NO: 41 The chain and the amino acid sequence consisting of amino acid residues 21-234 of Sequence ID No. 39 It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. antibodies; (4) The amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 41 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. The amino acid sequence consisting of the chain and amino acid residues 21-234 of SEQ ID NO: 39 is 95% or It consists of an amino acid sequence having sequence identity as shown above, and has binding activity to human SIRPα. It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. antibodies; (5) The amino acid sequence consisting of amino acid residues 20 to 466 of SEQ ID NO: 43 The chain and the amino acid sequence consisting of amino acid residues 21-234 of Sequence ID No. 35 It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. antibodies; (6) The amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 43 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. The amino acid sequence consisting of the chain and amino acid residues 21-234 of SEQ ID NO: 35 is 95% or It consists of an amino acid sequence having sequence identity as shown above, and has binding activity to human SIRPα. It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. antibodies; (7) The amino acid sequence consisting of amino acid residues from position 20 to 466 of sequence number 43 The chain and the amino acid sequence consisting of amino acid residues 21-234 of Sequence ID No. 37 It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. antibodies; and (8) The amino acid sequence consisting of amino acid residues 20-466 of SEQ ID NO: 43 and 95% The amino acid sequence has the same sequence identity as shown above and has binding activity to human SIRPα. The amino acid sequence consisting of the chain and amino acid residues 21-234 of SEQ ID NO: 37 is 95% or It consists of an amino acid sequence having sequence identity as shown above, and has binding activity to human SIRPα. It specifically binds to human SIRPα, which is composed of chains, and inhibits the binding of human SIRPα to CD47. Antibodies.

[23] An antibody of

[22] in which ADCC and / or ADCP activity is reduced.

[0020]

[24] The 82nd Gln and 83rd Lys of human SIRPα represented by sequence number 57 , which binds to the epitopes including Glu at position 84 and Gly at position 85 in human SIRPα An antibody that specifically binds and inhibits the binding of human SIRPα to CD47.

[25] The heavy chain constant region is the heavy chain constant region of human IgG4, and ADCC and / or ADC

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

[26] The heavy chain constant region is the heavy chain constant region of human IgG4, and Kabat et al.'s EU study The phenylalanine at position 234, indicated by INDEX, is replaced with alanine, and at position 235, The leucine is replaced with alanine, and the serine at position 228 is replaced with proline. Antibodies of

[24] or

[25] .

[27] The amino acid sequence of the heavy chain constant region is the amino acid sequence of sequence number 140-466 of sequence number 25. An antibody with amino acid sequence

[26] consisting of residues.

[28] Any of the antibodies [1] to

[27] that enhance the phagocytic activity of macrophages.

[29] An antibody of any of [1]~

[28] , wherein the lysine residue at the heavy chain carboxyl terminus is A missing antibody.

[0021]

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

[29] .

[31] Antigen-binding fragments of the antibody of

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

[32] Antibody of any of [1] to

[29] or antigen-binding fragment of antibody

[30] or

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

[33] A pharmaceutical composition of

[32] which is an antitumor agent.

[34] As an active ingredient in antitumor agents, further as an immune checkpoint inhibitor and / or cancer A pharmaceutical composition of

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

[35] Antibody of any of [1] to

[29] or antigen-binding fragment of antibody

[30] or

[31] Contains as an active ingredient an immune checkpoint inhibitor and / or a drug that specifically reacts to cancer antigens. 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 to PD-1, or C A pharmaceutical composition of

[34] or

[35] which is a TLA4 inhibitor.

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

[0034] or

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

[38] Tumors include carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, carcinoma One or more selected from the group consisting of noid, neuroblastoma, retinoblastoma, and nephroblastoma. A tumor, any of the pharmaceutical compositions

[33] to

[37] .

[39] Tumors include kidney cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, conjunctival cancer, and oral cancer. Hmm, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, duodenal cancer Hmm, small intestine cancer, large intestine cancer, rectal cancer, appendiceal 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 schwannoma, retroperitoneal sarcoma, synovial sarcoma, uterine sarcoma, gastrointestinal stromal tumor, leiomyosarcoma, etc. Episarcoma, B-cell lymphoma, T- and NK-cell lymphoma, Hodgkin lymphoma, myeloid leukemia, Lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndrome, multiple myeloma, testicular cancer, ovarian cancer n, one or more tumors selected from the group consisting of gliomas and meningiomas,

[38] A pharmaceutical composition.

[40] [1]~

[29] Nuclei encoding the amino acid sequences of the heavy and light chains of any of these antibodies A polynucleotide consisting of a rheotide sequence. A vector containing polynucleotides

[41]

[40] .

[42] Host cells containing polynucleotides of

[40] or vectors of

[41] . A method for producing an antibody according to any of [1] to

[0029] , comprising culturing the host cells of

[43]

[42] and purifying the antibody from the culture. Antibodies produced by the methods of

[44] and

[43] .

[0022] This specification discloses the information of Japanese Patent Application No. 2018-131116, which forms the basis of the priority claim of this application. To include. [Effects of the Invention]

[0023] The anti-SIRPα antibody of the present invention is expressed in SIRPα expressed in phagocytic cells and tumor cells. It strongly inhibits the interaction with CD47, and prevents tumor cells from transferring to phagocytic cells. While inhibiting the transmission of the "t-me" signal, it does not have any effects functionality. Therefore, it is safe as it does not attack the body's own immune cells.

[0024] The anti-SIRPα antibody of the present invention is used in conjunction with other antibody drugs and immunoassays that have effector functions. When used in combination with other antibody drugs that exhibit point inhibitory activity, it exhibits excellent antitumor effects. It is possible. [Brief explanation of the drawing]

[0025] [Figure 1A] Figure (A) shows the structure of the SIRPA construct used for epitope analysis of the anti-SIRPA antibody, and Figure (B) shows the reactivity of the rat anti-human SIRPA antibody to each construct. [Figure 1B] Figures (i)-(iv) show the reactivity of the humanized anti-human SIRPA antibody to the hmSIRPA construct used for epitope analysis of the anti-SIRPA antibody, and figure (v) shows the amino acid sequence of the SIRPA construct used for epitope analysis of the anti-SIRPA antibody. [Figure 2] This figure shows a ribbon model of the entire complex of the anti-SIRPA antibody Fab fragment and SIRPA_V2_IgV. [Figure 3] This figure shows the interaction between the pre-beta5 (A) and post-beta5 (B) regions of human SIRPA and the SIRPA antibody (antibody D13). [Figure 4]This figure shows a comparison of the amino acid sequences of each variant in the beta5-6 loop region of human SIRPA. [Figure 5] This figure shows the ADCP activity against gastric cancer cell lines when an anti-SIRPA antibody is used as a monotherapy (A) and when an anti-SIRPA antibody is used in combination with trastuzumab (B). [Figure 6] This figure shows the results of the evaluation of the binding affinity of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) to human SIRPA. [Figure 7] This figure shows the results of the binding affinity evaluation of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) to monkey SIRPA. [Figure 8A] This figure shows the results of evaluating the binding inhibitory activity of human or monkey SIRPA to CD47 by human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) ((i): SIRPA_V1, (ii): SIRPA_V2, (iii): Monkey SIRPA). [Figure 8B] This figure shows the results of evaluating the binding inhibitory activity of human or monkey SIRPA to CD47 by human chimeric anti-SIRPA antibodies (each isotype of cF44) ((i): SIRPA_V1, (ii): SIRPA_V2, (iii): Monkey SIRPA). [Figure 9] This figure shows the ADCP activity against Burkitt's lymphoma cell line when an anti-SIRPA antibody is used as a monotherapy (A) and when an anti-SIRPA antibody is used in combination with rituximab (B). [Figure 10] This figure shows the results of toxicity evaluations of human chimeric anti-human SIRPA antibodies against PBMCs and macrophages. A shows the ADCP activity against PBMCs of cD13, cF44, and cF63, B shows the ADCP activity of cF44 antibodies with different constant regions, and C shows the abundance of macrophages. [Figure 11] This figure 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]This figure shows a comparison of the amino acid sequences of the variable region of the light chain of antibody D13, the variable region of the humanized antibody light chain hL2, the variable region of the humanized antibody heavy chain hL3, and the variable region of the humanized antibody light chain hL4. [Figure 13A] This figure shows the binding activity of humanized anti-SIRPA antibodies to human SIRPA variants ((i):V1, (ii):V2, (iii):V3, and (iv):V4). [Figure 13B] This figure shows the binding activity of humanized anti-SIRPA antibodies to human SIRPA variants ((i):V5, (ii):V6, (iii):V7, and (iv):V8). [Figure 13C] This figure 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] This figure shows the binding activity of humanized anti-SIRPA antibodies to mouse SIRPA ((i):C57BL / 6, (ii):Balb / c, (iii):129sv). [Figure 14B] This figure shows the binding activity of humanized anti-SIRPA antibodies to mouse SIRPA ((i):NOD, (ii):Mock). [Figure 15] This figure shows the results of evaluating the inhibitory activity of humanized anti-SIRPA antibodies on the binding of human (A and B) or monkey SIRPA (C) to CD47. [Figure 16] This figure shows the ADCP activity against cancer cell lines (A and B: Raji strain, C and D: Ramos strain) when a humanized anti-human SIRPA antibody is used as a monotherapy (A and C) and when a humanized anti-human SIRPA antibody is used in combination with Rituximab (B and D). [Figure 17] This figure shows the nucleotide sequence encoding the cD13 light chain and the amino acid sequence of the cD13 light chain. [Figure 18] This figure shows the nucleotide sequence encoding the cD13 heavy chain and the amino acid sequence of the cD13 heavy chain. [Figure 19] This figure shows the nucleotide sequence encoding the cF44 light chain and the amino acid sequence of the cF44 light chain. [Figure 20] This figure shows the nucleotide sequence encoding the cF44 heavy chain and the amino acid sequence of the cF44 heavy chain. [Figure 21] This figure shows the nucleotide sequence encoding the cF63 light chain and the amino acid sequence of the cF63 light chain. [Figure 22] This figure shows the nucleotide sequence encoding the cF63 heavy chain and the amino acid sequence of the cF63 heavy chain. [Figure 23] This figure shows the nucleotide sequence encoding hL2 and the amino acid sequence of hL2. [Figure 24] This figure shows the nucleotide sequence encoding hL3 and the amino acid sequence of hL3. [Figure 25] This figure shows the nucleotide sequence encoding hL4 and the amino acid sequence of hL4. [Figure 26] This figure shows the nucleotide sequence encoding hH1 and the amino acid sequence of hH1. [Figure 27] This figure shows the nucleotide sequence encoding hH2 and the amino acid sequence of hH2. [Figure 28] This figure shows the CDR sequence of antibody D13. [Figure 29] This figure shows the CDR sequence of antibody F44. [Figure 30] This figure shows the CDR sequence of antibody F63. [Figure 31] This figure 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 ​​for inhibition of human SIRPA_V1 / CD47 or SIRPA_V2 / CD47 binding activity by various antibodies. [Figure 32] This figure shows the results of evaluating the binding affinity 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). [Figure 33]This figure shows the ADCP activity in Burkitt's lymphoma cell line (Raji) when various anti-human SIRPA antibodies were combined with rituximab: a comparison of reactivity at 10 μg / mL for each donor, specifically the concentration-dependent comparison results at reaction times of 2 hours (A), 16 hours (B), and 2 hours (C), as well as the self-ADCP activity (phagocytosis of macrophages) induced by various anti-human SIRPA antibodies (D). In each figure, "Ab-" indicates a negative control without antibody addition, and "+ Rmab" indicates the simultaneous addition of rituximab. [Figure 34] This figure shows the amino acid sequences of the OSE-172 antibody heavy chain (OSE-172_hG4Pro) and light chain (OSE-172_hK). [Figure 35] This figure shows the amino acid sequences of the KWAR23 antibody heavy chain (KWAR23_hG4Pro) and light chain (KWAR23_hK). [Figure 36] This figure shows the amino acid sequences of the ADU-1805 antibody heavy chain (ADU-1805_hG2) and light chain (ADU-1805_hK). [Modes for carrying out the invention]

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

[0027] Characteristics of anti-SIRPα antibodies This invention relates to an anti-SIRPα protein that recognizes and binds to the extracellular IgV domain of the SIRPα protein. It is the body.

[0028] SIRPα (signal regulatory protein α) is macro Ig cells present in myeloid cells such as phages, dendritic cells, and neutrophils, as well as glial cells. It is a per-family single-pass transmembrane molecule. The extracellular domain consists of one IgV domain and two It consists of an IgC domain, and the IgV domain, which is the binding site for CD47, is human Ten variants, V1 to V10, have been reported. Extracellular SIRPα protein The IgV domain is one of the 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 invention includes all variants, including the major variants V1 and V2. It binds to the riant. In this invention, "SIRPα" may be referred to as "SIRPA". be.

[0029] The amino acid sequence of the human SIRPα protein is, GenBank Accession No. It is disclosed in .:NP_001035111.

[0030] The monoclonal antibodies used in this invention are from mice, rats, rabbits, hamsters, and guinea pigs. Mammals such as horses, monkeys, dogs, pigs, cows, goats, and sheep are treated with SIRPα or its The fragments were used as immunogens to immunize the cells, and spleen cells and other tissues were fused with myeloma to create hybridomas. And it can be obtained as an antibody produced and secreted by hybridomas. Hybridomas are known It can be manufactured using the following method.

[0031] SIRPα as an immunogen can also be chemically synthesized based on sequence information, and it is also a protein. Based on the DNA sequence information encoding the protein, recombinant proteins are obtained using known methods. It can also be obtained this way.

[0032] Antibody screening can be performed by any method, but preferably, SIRPα Cell-ELISA using animal cells transfected with encoding DNA Screening is sufficient. The amino acid sequence of the human SIRPα V1 protein is the sequence of the sequence listing. Number 56 shows the amino acid sequence of the human SIRPα V2 protein as sequence number 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 in phagocytic cells that have phagocytic ability. Pα and CD47 bind and interact, causing phagocytic cells to "Don't-eat-m" It transmits the e" signal, escaping phagocytosis by phagocytic cells. Anti-SIRPα antibodies are SI By inhibiting the binding of RPα and CD47, tumor cells can transmit "Don't-" to phagocytic cells. It inhibits the transmission of the "eat-me" signal, thereby suppressing the phagocytic action of phagocytic cells on tumor cells. It enhances the function. As a result, it may exert an antitumor effect. As a phagocytic cell with phagocytic ability, type M1 macrophages such as M2 macrophages and dendritic cells such as imDCs (immature dendritic cells) Examples include cells, etc.

[0034] In this process, the anti-SIRPα antibody has an effector function, acting on phagocytic cells such as macrophages and Fc receptors such as Fcγ receptors on effector cells such as natural killer cells and T cells When it binds to the cell, ADCC (Antibody Dependent Cellular) r Cytotoxicity: Antibody-dependent cytotoxicity) and ADCP (Antidoby cytotoxicity) Dependent Cellular Phagocytosis: Antibody-dependent cellular phagocytosis Through phagocytosis, the body's own effector cells such as PBMCs (peripheral blood mononuclear cells) and macrophages are transformed. I'll attack.

[0035] To avoid attacking one's own cells, the anti-SIRPα antibody of the present invention has an effector function It is reduced. As a result, the anti-SIRPα antibody of the present invention is simply SIRPα and CD47 It only has the effect of inhibiting binding, and does not bind to the Fc receptor of effector cells. The filter function does not work.

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

[0037] However, the anti-SIRPα antibody of the present invention has reduced effector function, therefore, it cannot be used alone. Therefore, it does not exhibit sufficient antitumor effects. For this reason, as described later, it is used in combination with other antitumor agents. Use.

[0038] To reduce the effector function, the Fc portion of the anti-SIRPα antibody is macrophage It is necessary that it does not bind to the Fc receptors on T cells. Therefore, the anti-SI of the present invention The RPα antibody subclass has been substituted with one derived from IgG4. Generally, human IgG subclass Within the class, IgG4 exhibits effects such as ADCC activity, CDC activity and / or ADCP activity. It is known as a subclass with low cognitive function (Bruggemann et al.). (J.Exp.Med., 1351-1361, 1987). Therapeutic antibodies cause abnormalities in normal organs. When targeting molecules that exhibit this behavior, avoid toxicity caused by cytotoxicity mediated by effector functions. It is used as one of the IgG formats for (e.g., Opdivo). However, IgG Even though the effects functions of the 4 subclasses are said to be low, they are not completely absent. The anti-SIRPα antibody of the invention modifies the heavy chain constant region to further reduce effector function. Alterations, i.e., substitutions of one or more amino acids that result in a reduction in ADCC and / or ADCP activity. The mutation has been introduced. One such mutation is the EU index by Kabat et al. Kabat et.al., Sequences of proteins of im munological interest,1991 Fifth edition) The substitution of phenylalanine at position 234 to alanine (F234A) and 23 The fifth most common substitution is leucine-alanine substitution (L235A) (Parekh et al.) al., mAbs, 310-318, 2012). Such antibody mutations are called FALA mutations. It is called phenylalanine at position 234, as shown by the EU index by Kabat et al. It is sometimes referred to as phenylalanine, EU number 234.

[0039] Furthermore, IgG4 has unstable disulfide bond formation between antibody heavy chains, so we need to improve its stability. Therefore, mutations that promote the formation of disulfide bonds between antibody heavy chains are introduced. And, according to the EU Index by Kabat et al., serine is ranked 228th, and proline is... Substitution (S228P) is one example (ANGAL et.al., Molecular (Immunology, 105-108, 1993). This antibody mutation is called a PRO mutation. Bu.

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

[0041] The constant region of the antibody heavy chain consists of the CH1, hinge, CH2, and CH3 regions, with CH1 being the EU region. Index 118 to 215, hinge is EU index 216 to 230, CH2 is EU indices 231 to 340 are defined as EU indices 341 to 446, while CH3 is defined as EU indices 341 to 446. It is said that the phenylalanine at position 234, as shown by the EU index by Kabat et al. The alanine substituted from n is the 25th amino acid in SEQ ID NO: 25, which shows the heavy chain amino acid sequence of antibody D13. The third alanine, the 252nd amino acid in Sequence ID No. 29, which shows the heavy chain amino acid sequence of antibody F44. Alanine and the heavy chain amino acid sequence of antibody F63, shown in SEQ ID NO: 33, at the 257th alanine Alanine, which corresponds to n and is substituted from leucine at position 235, is the 254th position in Sequence ID No. 25. alanine, alanine 253 in SEQ ID NO: 29 and alanine 258 in SEQ ID NO: 33 It corresponds to alanine. Also, 228 is shown by the EU Index by Kabat et al. The proline substituted from the serine position is the 247th proline in SEQ ID NO: 25, SEQ ID NO: This corresponds to the 246th proline in sequence number 29 and the 251st proline in sequence number 33.

[0042] The amino acid sequence of the constant region of the "IgG4proFALA" type heavy chain is as shown in SEQ ID NO: 25. Amino acid sequence consisting of amino acid residues 140-466, sequence number 29, 139-46 The amino acid sequence consisting of the 5th amino acid residue and the 144th to 470th amino acid residues of SEQ ID NO: 33 It is an amino acid sequence consisting of amino acid residues.

[0043] Human IgG1, among the human IgG subclasses, exhibits complement-mediated CDC activity and antibody activity. It has very strong effector functions, such as dependent cytotoxic activity (Bruggemann et al., J.Exp.Med., 1351-1361, 1987), therapeutic antibodies When targeting molecules highly expressed in cancer cells, cancer cells are affected by cytotoxicity mediated by effector functions. It is used as an IgG format that shows therapeutic effects by promoting cell death induction in cells (tr (e.g., astuzumab, rituximab). I as the isotype of the antibody of the present invention. When using gG1, by substituting some of the amino acid residues in the constant region, effervescence can be achieved. It is possible to adjust the function (WO88 / 007089, W094 / 2802) 7. See W094 / 29351). A mutant of IgG1 with reduced effector function. Therefore, IgG1 LALA (IgG1-L234A, L235A), IgG1 LAGA Examples include (IgG1-L235A, G237A), etc. The constant region of the antibody of the present invention is It is also possible to use the IgG1 heavy chain constant region into which these mutations have been introduced.

[0044] Human IgG2, among the human IgG subclasses, exhibits complement-mediated CDC activity and antibody activity. The effector function, such as dependent cytotoxic activity, is very weak (Bruggemann et al., J.Exp.Med., 1351-1361, 1987), therapeutic antibodies When targeting molecules expressed in normal organs, toxicity occurs through cytotoxicity mediated by effector function. It is used as one of the IgG formats for sex avoidance (denosumab, (e.g., evolocumab, brodalumab). As the constant region of the antibody of the present invention, It is also possible to use the IgG double-chain constant region. Regarding species cross-reactivity, the anti-SIRPα antibody of the present invention cross-reactive with human SIRPα and monkey (Cyn) It binds to *Omolgus monkey* but not to mouse SIRPα.

[0045] Human chimeric antibodies and humanized antibodies The anti-SIRPα antibody of the present invention is modified to reduce heterologous antigenicity against humans. This includes tochimeric antibodies and humanized antibodies. Humanized antibodies are also called CDR transplant antibodies.

[0046] Human chimeric antibodies Human chimeric antibodies combine the light chain variable region and heavy chain variable region of antibodies from non-human animals with those of human antibodies. This refers to an antibody consisting of a light chain constant region and a heavy chain constant region of the body. Human chimeric antibodies are anti-SI antibodies. cDNA encoding the light chain variable region and heavy chain variable region were obtained from hybridomas that produce RPα antibodies. cDNA encoding the variant region was collected, and the light chain constant region and heavy chain constant region of the human antibody were coded. By inserting the cDNA into an expression vector containing the desired expression, a human chimeric antibody expression vector is constructed. It can be produced by introducing it into host cells and expressing it.

[0047] The heavy chain constant region consists of three domains C H 1, C H 2 and C H It consists of 3 parts. In the present, as described above, the human heavy chain constant region of the chimeric antibody is the IgG4 subclass. Heavy chain constant region having Pro mutations and FALA mutations IgG4pro It is FALA. Also, the light chain constant region only needs to belong to human Ig, and the κ or λ constant region It is a region.

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

[0049] antibody cD13 The nucleotide sequence of the cDNA encoding the light chain variable region of antibody cD13 is as follows (see sequence listing). This is a nucleotide sequence consisting of nucleotides 61-378 in column number 22 (Figure 17). The amino acid sequence of the light chain variable region of antibody cD13 is from 21 to 12 of sequence number 23 in the sequence listing. This is an amino acid sequence consisting of the sixth amino acid residue (Figure 17).

[0050] Furthermore, the nucleotide sequence of the cDNA encoding the heavy chain variable region of antibody cD13 is: This is a nucleotide sequence consisting of nucleotides 58-417 of sequence number 24 in the table ( (Figure 18) The amino acid sequence of the heavy chain variable region of antibody cD13 is 20 of sequence number 25 in the sequence listing. This is an amino acid sequence consisting of amino acid residues up to the 139th (Figure 18).

[0051] In other words, the anti-SIRPα antibody of the present invention is the amino acids 21-126 of SEQ ID NO: 23. The light chain variable region consisting of amino acid sequences made up of residues and the 20th to 139th of SEQ ID NO: 25 A heavy chain variable region consisting of an amino acid sequence composed of amino acid residues, which binds to human SIRPα. It is an anti-human SIRPα antibody.

[0052] Furthermore, the nucleotide sequence consisting of nucleotides 61-378 of sequence number 22 mentioned above A nucleotide sequence consisting of nucleotides 58-417 of column or sequence number 24 and CLUS TAL W (Alignment Tool), etc. (for example, default, i.e., initial parameters) When calculated using, at least 85%, preferably 90%, and even more preferably The sequence content should be 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more. DNA consisting of a nucleotide sequence having uniformity, wherein the light chain variable region or heavy chain of an antibody This protein encodes a protein that possesses activity in its chain variable region, i.e., binding activity to human SIRPα. The DNA is also included in the DNA that encodes the light chain variable region or heavy chain variable region of the antibody of the present invention. .

[0053] Furthermore, the nucleotide sequence consisting of nucleotides 61-378 of sequence number 22 mentioned above Complementary to the nucleotide sequence consisting of nucleotides 58-417 of the column or sequence number 24. It can hybridize with DNA consisting of specific sequences under stringent conditions. DNA, specifically the activity of the light chain variable region or heavy chain variable region of an antibody, i.e., human SIRPα DNA encoding a protein having binding activity is also the light chain variable region or heavy chain variable region of the present invention. It is contained within the DNA that codes for a region.

[0054] Furthermore, the above light chain variable region or heavy chain variable region is the 21st to 126th A of Sequence ID No. 23. An amino acid sequence consisting of amino acid residues or amino acid residues 20-139 of SEQ ID NO: 25 Not only the light chain variable region or heavy chain variable region consisting of the amino acid sequence, but also the amino acid sequence In this, one or several, for example, 1 to 10, preferably 1 to 5, more preferably This refers to an amino acid in which one or two, more preferably one, amino acids are deleted, substituted, or added. It consists of a sequence, and the activity of the heavy chain variable region or light chain variable region of the antibody, i.e., human SIRPα This also includes a light chain variable region or a heavy chain variable region consisting of a protein having binding activity.

[0055] An amino acid sequence consisting of amino acid residues 21-126 of sequence number 23, or In the amino acid sequence consisting of amino acid residues 20-139 of SEQ ID NO: 25, 1 or This is an amino acid sequence in which several amino acids are deleted, substituted, or added, as in SEQ ID NO: 23. An amino acid sequence consisting of amino acid residues 21-126 or sequences 20-139 of SEQ ID NO: 25 An amino acid sequence consisting of the nth amino acid residue, and CLUSTAL W (alignment tool), etc. (example) For example, when calculated using the default (i.e., initial) parameters, at least 85% or more, preferably 90% or more, more preferably 95% or more, and especially preferably 97% Examples include sequences with a sequence identity of % or more, 98% or more, or 99% or more.

[0056] An amino acid sequence consisting of amino acid residues 21-126 of sequence number 23, or In the amino acid sequence consisting of amino acid residues 20-139 of SEQ ID NO: 25, 1 or Proteins that have an amino acid sequence in which several amino acids are deleted, substituted, or added are The amino acid sequence consisting of amino acid residues 21-126 of row number 23 or sequence number 25 A protein having an amino acid sequence consisting of amino acid residues from position 20 to 139 is substantially the same as It is one.

[0057] Furthermore, antibody cD13 has a CDR (complementarity-determining region) in its light chain variable region, as indicated by the sequence number. CDRL1 and SEQ ID NO: 2 consist of the amino acid sequence (GASKSVRTYMH) represented by 1. CDRL2, represented by the amino acid sequence (SASNLEA) shown in Sequence ID No. 3 It contains CDRL3 consisting of the amino acid sequence (QQSNEPPYT), and further, a heavy chain variable region. The CDR consists of the amino acid sequence (GFTFSDYGMI) represented by SEQ ID NO: 4. CDRH1, represented by the amino acid sequence (SISSSSSYIY) in Sequence ID No. 5, consists of CD CDR consisting of the amino acid sequence (RYYGFNYPFDY) represented by RH2, SEQ ID NO: 6 Includes H3 (Figure 28).

[0058] In other words, the anti-SIRPα antibody of the present invention consists of the amino acid sequence represented by SEQ ID NO: 1. CDRL2, represented by sequence number 3, consists of the amino acid sequence represented by CDRL1, sequence number 2. It contains CDRL3 consisting of a specific amino acid sequence, and further, as the CDR of the heavy chain variable region, CDRH1, consisting of the amino acid sequence represented by sequence number 4, and the amino acid sequence represented by sequence number 5 Antibody containing CDRH2 consisting of a sequence and CDRH3 consisting of the amino acid sequence represented by SEQ ID NO: 6 It is the body.

[0059] Each of the above CDRs has one or several amino acids in the amino acid sequence it represents, preferably Alternatively, one or two amino acids, more preferably one amino acid, may be deleted, substituted, or added. This also includes CDRs consisting of amino acid sequences composed of no acid sequences.

[0060] Chimeric or humanized D13 antibodies are SIRs consisting of the amino acid sequence represented by SEQ ID NO: 73. The Pα variant binds to the SI, which consists of the amino acid sequence represented by SEQ ID NO: 74 or 75. It does not bind to the RPα mutant. The NQKEG sequence in the amino acid sequence represented by SEQ ID NO: 73 (Sequence ID 76) is the same as the NQKEE sequence (Sequence ID 77) in Sequence ID 74, In 5, the SFTEG sequence (SEQ ID NO: 80) is substituted, and it is used as a chimeric or humanized D13 antibody. It has become clear that the NQKEG sequence (sequence number 76) is required for the binding of SIRPα. Furthermore, X-ray crystallography revealed that antibody cD13 is the human SIR represented by sequence number 57. Pα variant 2 amino acid residues Gln82, Lys83, Glu84, Gly85, H is86, Phe87 (The positions of each amino acid residue correspond to sequence number 57 in the sequence listing.) It has been suggested that it binds to SIRPα via ), and Gln82, Lys83, Gl The sequence consisting of u84 and Gly85 corresponds to the QKEG portion of the NQKEG sequence mentioned above. Therefore, the NQKEG sequence is an essential epitope for the binding of the D13 antibody to human SIRPα. An antibody that specifically recognizes the NQKEG sequence (SEQ ID NO: 76), i.e., the NQKEG sequence ( SIRPα variant consisting of the amino acid sequence represented by SEQ ID NO: 73 (SEQ ID NO: 76) It consists of an amino acid sequence represented by SEQ ID NO: 74 or 75 that binds to but does not have the sequence. By selecting an antibody that does not bind to the SIRPα mutant, the same epidermal structure as the D13 antibody can be achieved. Antibodies containing the peculiar properties can be selected.

[0061] Antibody cF44 The nucleotide sequence of the cDNA encoding the light chain variable region of antibody cF44 is as follows (see sequence listing). This is a nucleotide sequence consisting of nucleotides 61-381 in column number 26 (Figure 19). ), the amino acid sequence of the light chain variable region of antibody cF44 is as follows: 21-12 of sequence number 27 in the sequence listing. This is an amino acid sequence consisting of the seventh amino acid residue (Figure 19).

[0062] Furthermore, the nucleotide sequence of the cDNA encoding the heavy chain variable region of the antibody cF44 is: This is a nucleotide sequence consisting of nucleotides 58-414 of sequence number 28 in the table ( Figure 20) The amino acid sequence of the heavy chain variable region of antibody cF44 is as follows: Sequence ID No. 29 of the sequence listing. This is an amino acid sequence consisting of amino acid residues up to the 138th (Figure 20).

[0063] In other words, the anti-SIRPα antibody of the present invention is the amino acids 21-127 of SEQ ID NO: 27. The light chain variable region consisting of amino acid sequences made up of residues and the 20th to 138th of SEQ ID NO: 29 A heavy chain variable region consisting of an amino acid sequence composed of amino acid residues, which binds to human SIRPα. It is an anti-human SIRPα antibody.

[0064] Furthermore, the nucleotide sequence consisting of nucleotides 61 to 381 of sequence number 26 mentioned above A nucleotide sequence consisting of nucleotides 58-414 of column or sequence number 28 and CLUS TAL W (Alignment Tool), etc. (for example, default, i.e., initial parameters) When calculated using, at least 85%, preferably 90%, and even more preferably The sequence content should be 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more. DNA consisting of a nucleotide sequence having uniformity, wherein the light chain variable region or heavy chain of an antibody This protein encodes a protein that possesses activity in its chain variable region, i.e., binding activity to human SIRPα. The DNA is also included in the DNA that encodes the light chain variable region or heavy chain variable region of the antibody of the present invention. .

[0065] Furthermore, the nucleotide sequence consisting of nucleotides 61 to 381 of sequence number 26 mentioned above Complementary to the nucleotide sequence consisting of nucleotides 58-414 of the column or sequence number 28. It can hybridize with DNA consisting of specific sequences under stringent conditions. DNA, specifically the activity of the light chain variable region or heavy chain variable region of an antibody, i.e., human SIRPα DNA encoding a protein having binding activity is also the light chain variable region or heavy chain variable region of the present invention. It is contained within the DNA that codes for a region.

[0066] Furthermore, the above light chain variable region or heavy chain variable region is the 21st to 127th A of Sequence ID No. 27. An amino acid sequence consisting of amino acid residues or amino acid residues 20-138 of SEQ ID NO: 29 Not only the light chain variable region or heavy chain variable region consisting of the amino acid sequence, but also the amino acid sequence In this, one or several, for example, 1 to 10, preferably 1 to 5, more preferably This refers to an amino acid in which one or two, more preferably one, amino acids are deleted, substituted, or added. It consists of a sequence, and the activity of the heavy chain variable region or light chain variable region of the antibody, i.e., human SIRPα This also includes a light chain variable region or a heavy chain variable region consisting of a protein having binding activity.

[0067] An amino acid sequence consisting of amino acid residues 21-127 of sequence number 27, or In the amino acid sequence consisting of amino acid residues 20-138 of SEQ ID NO: 29, 1 or This is an amino acid sequence in which several amino acids are deleted, substituted, or added, as in SEQ ID NO: 27. The amino acid sequence consisting of amino acid residues 21-127 or sequences 20-138 of SEQ ID NO: 29 An amino acid sequence consisting of the nth amino acid residue, and CLUSTAL W (alignment tool), etc. (example) For example, when calculated using the default (i.e., initial) parameters, at least 85% or more, preferably 90% or more, more preferably 95% or more, and especially preferably 97% Examples include sequences with a sequence identity of % or more, 98% or more, or 99% or more.

[0068] An amino acid sequence consisting of amino acid residues 21-127 of sequence number 27, or In the amino acid sequence consisting of amino acid residues 20-138 of SEQ ID NO: 29, 1 or Proteins that have an amino acid sequence in which several amino acids are deleted, substituted, or added are The amino acid sequence consisting of amino acid residues 21-127 of sequence number 27 or sequence number 29 A protein having an amino acid sequence consisting of amino acid residues 20 to 138 is substantially the same It is one.

[0069] Furthermore, antibody cF44 has a CDR (Complementarity Determining Region) in its light chain variable region, as indicated by the sequence number. CDRL1, consisting of the amino acid sequence (KASKSISKYLA) represented by 7, and sequence number 8 CDRL2, represented by the amino acid sequence (SGSTLQS) shown in sequence number 9 It contains CDRL3 consisting of the amino acid sequence (QQHNEYPPT), and further, a heavy chain variable region. The CDR is derived from the amino acid sequence (GFTFSNYYMA) represented by sequence number 10. CDRH1 consists of the amino acid sequence (YITTGGGSTY) represented by SEQ ID NO: 11. CDRH2, represented by the amino acid sequence (ANYGGSYFDY) in Sequence ID No. 12, is a C Includes DRH3 (Figure 29).

[0070] In other words, the anti-SIRPα antibody of the present invention consists of the amino acid sequence represented by SEQ ID NO: 7. CDRL2, represented by sequence number 9, consists of the amino acid sequence represented by CDRL1, sequence number 8. It contains CDRL3 consisting of a specific amino acid sequence, and further, as the CDR of the heavy chain variable region, CDRH1, consisting of the amino acid sequence represented by sequence number 10, and the amino acid represented by sequence number 11. CDRH2 consists of an acid sequence, and CDRH3 consists of an amino acid sequence represented by SEQ ID NO: 12. It contains antibodies.

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

[0072] Antibody cF63 The nucleotide sequence of the cDNA encoding the light chain variable region of antibody cF63 is a nucleotide sequence consisting of nucleotides 61 to 390 of SEQ ID NO: 30 in the Sequence Listing (FIG. 21 ), and the amino acid sequence of the light chain variable region of antibody F63 is an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 in the Sequence Listing (FIG. 21). ) and the amino acid sequence of the light chain variable region of antibody F63 is an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 in the Sequence Listing (FIG. 21). ).

[0073] Further, the nucleotide sequence of the cDNA encoding the heavy chain variable region of antibody cF63 is a nucleotide sequence consisting of nucleotides 5 to 429 of SEQ ID NO: 32 in the Sequence Listing ( FIG. 22), and the amino acid sequence of the heavy chain variable region of antibody cF63 is an amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33 in the Sequence Listing (FIG. 22). ).

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

[0075] Further, the nucleotide sequence consisting of nucleotides 61 to 390 of SEQ ID NO: 30 or the nucleotide sequence consisting of nucleotides 58 to 429 of SEQ ID NO: 32 and CLUS DNA consisting of a nucleotide sequence having at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more sequence identity when calculated using TAL W (alignment tool) etc. (for example, default or initial set parameters), and encoding a protein having the activity of the variable region of the light chain or heavy chain of an antibody, that is, the binding activity to human SIRPα is also included in the DNA encoding the variable region of the light chain or heavy chain of the antibody of the present invention. When calculated using [specific tool or parameter], it has at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, 98% or more, or 99% or more sequence identity, and is a DNA consisting of a nucleotide sequence encoding a protein having the activity of the variable region of the light chain or heavy chain of an antibody, that is, the binding activity to human SIRPα. Also included in the DNA encoding the variable region of the light chain or heavy chain of the antibody of the present invention is DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence consisting of nucleotides 61 to 390 of SEQ ID NO: 30 or the nucleotide sequence consisting of nucleotides 58 to 429 of SEQ ID NO: 32 and encodes a protein having the activity of the variable region of the light chain or heavy chain of an antibody, that is, the binding activity to human SIRPα. The variable region of the light chain or heavy chain of the antibody has an amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 or an amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33, and not only the variable region of the light chain or heavy chain consisting of this amino acid sequence, but also in this amino acid sequence, one or several, for example, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid is deleted, substituted, or added, and has the activity of the variable region of the heavy chain or light chain of the antibody, that is, the binding activity to human SIRPα. .

[0076] In addition, the above-mentioned nucleotide sequence consisting of nucleotides 61 to 390 of SEQ ID NO: 30 or the nucleotide sequence consisting of nucleotides 58 to 429 of SEQ ID NO: 32 and DNA consisting of a complementary sequence can hybridize under stringent conditions. The DNA encoding a protein having the activity of the variable region of the light chain or heavy chain of an antibody, that is, the binding activity to human SIRPα, is also included in the DNA encoding the variable region of the light chain or heavy chain of the present invention. Moreover, the above-mentioned variable region of the light chain or heavy chain is not only the variable region of the light chain or heavy chain consisting of the amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 or the amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33, but also in this amino acid sequence, one or several, for example, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid is deleted, substituted, or added, and has the activity of the variable region of the heavy chain or light chain of the antibody, that is, the binding activity to human SIRPα. [[ID=Z5]] .

[0077] In addition, the above-mentioned variable region of the light chain or heavy chain is not only the variable region of the light chain or heavy chain consisting of the amino acid sequence consisting of amino acid residues 21 to 130 of SEQ ID NO: 31 or the amino acid sequence consisting of amino acid residues 20 to 143 of SEQ ID NO: 33, but also in this amino acid sequence, one or several, for example, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid is deleted, substituted, or added, and has the activity of the variable region of the heavy chain or light chain of the antibody, that is, the binding activity to human SIRPα. In the amino acid sequence, one or several, for example, 1 to 10, preferably 1 to 5, more preferably 1 or 2, and even more preferably 1 amino acid is deleted, substituted, or added, and the amino acid sequence consisting thereof has the activity of the variable region of the heavy chain or light chain of the antibody, that is, the binding activity to human SIRPα. In the amino acid sequence, one or several, for example, 1 to 10, preferably 1 to 5, more preferably This also includes a light chain variable region or a heavy chain variable region consisting of a protein having binding activity.

[0078] Such an amino acid sequence consisting of amino acid residues 21 to 130 of sequence number 31 or In the amino acid sequence consisting of amino acid residues 20-143 of SEQ ID NO: 33, 1 or This is an amino acid sequence in which several amino acids are deleted, substituted, or added, as in SEQ ID NO: 31. The amino acid sequence consisting of amino acid residues 21-130 or 20-143 of SEQ ID NO: 33 An amino acid sequence consisting of the nth amino acid residue, and CLUSTAL W (alignment tool), etc. (example) For example, when calculated using the default (i.e., initial) parameters, at least 85% or more, preferably 90% or more, more preferably 95% or more, and especially preferably 97% Examples include sequences with a sequence identity of % or more, 98% or more, or 99% or more.

[0079] Such an amino acid sequence consisting of amino acid residues 21 to 130 of sequence number 31 or In the amino acid sequence consisting of amino acid residues 20-143 of SEQ ID NO: 33, 1 or Proteins that have an amino acid sequence in which several amino acids are deleted, substituted, or added are The amino acid sequence consisting of amino acid residues 21-130 of sequence number 31 or sequence number 33 A protein having an amino acid sequence consisting of amino acid residues 20 to 143 is substantially the same It is one.

[0080] Furthermore, antibody cF63 has a CDR (Complementarity Determining Region) in its light chain variable region, as indicated by the sequence number. CDRL1 consists of the amino acid sequence (ERSSGDIGDSYVS) represented by 13, sequence CDRL2, sequence number 15, consists of the amino acid sequence (ADDQRPS) represented by number 14. It contains CDRL3 consisting of the amino acid sequence (QSYDSKIDI) represented by, and further, the heavy chain variable region includes CDRH1 consisting of the amino acid sequence (GFSLASYSL S) represented by SEQ ID NO: 16, CDRH2 consisting of the amino acid sequence (RMYYDGDTA) represented by SEQ ID NO: 17, and CDRH3 consisting of the amino acid sequence (DRSMFGTDYPHW YFDF) represented by SEQ ID NO: 18 (Figure 30).

[0081] That is, the anti-SIRPα antibody of the present invention consists of the amino acid sequence represented by SEQ ID NO: 13 and includes CDRL1, CDRL2 consisting of the amino acid sequence represented by SEQ ID NO: 14, and CDRL3 consisting of the amino acid sequence represented by SEQ ID NO: 15. Further, as CDRs of the heavy chain variable region it includes CDRH1 consisting of the amino acid sequence represented by SEQ ID NO: 16, CDRH2 represented by SEQ ID NO: 17 consisting of the amino acid sequence, and CDR H3 consisting of the amino acid sequence represented by SEQ ID NO: 18.

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

[0083] Humanized antibody A humanized antibody (CDR grafted antibody) refers to an antibody in which the amino acid sequences of CDRs of the light chain variable region and heavy chain variable region of an antibody from a non-human animal are grafted to appropriate positions in the light chain variable region and heavy chain variable region of a human antibody.

[0084] The humanized anti-SIRPα antibody of the present invention binds to human SIRPα and, and SIRPα and C By inhibiting D47 binding, monoclonal cells enhance the phagocytic activity of macrophages. The light chain variable region of antibodies from non-human animals produced from antibody-producing hybridomas and The amino acid sequence of the CDR in the heavy chain variable region of any human antibody is used in the light chain variable region and heavy chain variable region. This code represents the variable region that has been ported to the framework (FR) domain. The cDNA was constructed and contains genes encoding the light chain constant region and heavy chain constant region of a human antibody. Humanized antibody expression vectors are constructed by inserting them into various animal cell expression vectors. It can be expressed and manufactured by introducing it into cells.

[0085] Specifically, the CDR of antibody D13, antibody F44, or antibody F63 and the framework of human antibodies You just need to synthesize a DNA sequence designed to link the 'c' region. Linking via CDR. The framework region of the human antibody is designed so that the CDR forms a good antigen-binding site. Selected. Also, if necessary, the CDR of the humanized antibody forms the appropriate antigen-binding site. Thus, amino acids in the framework region of the antibody's variable region may be substituted. Humanized antibodies with R transplanted can be produced using known CDR grafting techniques. Cut.

[0086] The CDR of the heavy chain variable region of antibody D13 (the amino acids shown in SEQ ID NOs. 1-6) A heavy chain of a humanized antibody having six CDRs, and a framework region of the variable region Humanized antibody heavy chain hH1 and humanized antibody heavy chain are examples of heavy chains in which some amino acids in the region are substituted. hH2 is one example. Also, the light chain variable region CDR of antibody D13 is a humanized antibody. A chain, in which some amino acids in the framework region of the variable region are substituted as a light chain, Examples include humanized antibody light chain hL2, humanized antibody light chain hL3, and 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 the sequence number. This is shown in number 41. Also, the full-length nucleotide sequence of the humanized antibody heavy chain hH2 is shown in sequence number 42. The amino acid sequence is shown in SEQ ID NO: 43. In SEQ ID NOs: 40 and 42, the amino acid sequences from 1 to 57th A nucleotide sequence consisting of nucleotides is the signal sequence, nucleos 58-417. The nucleotide sequence consisting of nucleotides has a variable region starting from nucleotides 418 to 1398. The nucleotide sequences respectively encode the constant region. Also, sequence number 41 and In 43, the amino acid sequence consisting of amino acid residues 1 to 19 is the signal sequence, 20 The amino acid sequence consisting of amino acid residues ~139 is a variable region, and the amino acid sequence from 140 to 466 is a The amino acid sequence consisting of amino acid residues is the amino acid sequence of the constant region. Figure 11 shows antibody D13. The variable region of the heavy chain, the 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 the amino acid residue of SEQ ID NO: 41 or 43, specifically the residues of amino acids 20 to 139. It has a heavy chain variable region consisting of 3 It contains antibodies that do this.

[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 the sequence number. This is shown in number 35. Additionally, the full-length nucleotide sequence of the humanized antibody light chain hL3 is shown in sequence number 36. The amino acid sequence is shown in Sequence ID No. 37. Furthermore, the full-length nucleotide of the humanized antibody light chain hL4 is also shown. The d-sequence is shown in SEQ ID NO: 38, and the amino acid sequence is shown in SEQ ID NO: 39. SEQ ID NOs: 34, 36 and In 38, the nucleotide sequence consisting of nucleotides 1 to 60 is the signal sequence. The nucleotide sequence consisting of nucleotides 61 to 381 has a variable region, 382 to 7 The nucleotide sequence consisting of the second nucleotide codes for the constant region. In SEQ ID NOs. 35, 37, and 39, amino acids consisting of amino acid residues 1 to 20 are present. The acid sequence is the signal sequence, and the amino acid sequence consisting of amino acid residues 21-127 is the variable region. The amino acid sequence consisting of amino acid residues 128-234 is the amino acid sequence of the constant region. Figure 12 shows the variable region of the light chain of antibody D13 and the variable region of the humanized antibody light chain hL2. 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) This shows a comparison (including arrays).

[0090] The anti-SIRPα antibody of the present invention corresponds to the 21st to 127th amines of SEQ ID NOs. 35, 37, and 39. A variable region consisting of no acid residues and a light chain constant region consisting of amino acid residues 128-234 Contains antibodies.

[0091] The heavy chain constant region of humanized antibodies is the heavy chain constant region of the IgG4 subclass and has a Pro mutation. And the heavy chain constant region IgG4proFALA has a 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 efficacy, an antibody consisting of a humanized antibody heavy chain hH1 and a humanized antibody light chain hL3 (hD 13_H1L3 antibody), an antibody consisting of humanized antibody heavy chain hH1 and humanized antibody light chain hL4 ( (hD13_H1L4 antibody), consisting of humanized antibody heavy chain hH2 and humanized antibody light chain hL2 From the body (hD13_H2L2 antibody) and the humanized antibody heavy chain hH2 and humanized antibody light chain hL3 One example is the antibody (hD13_H2L3 antibody).

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

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

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

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

[0097] Furthermore, the lysine residue at the carboxyl terminus of the heavy chain of antibodies produced in mammalian cultured cells is deleted. It is known that (Tsubaki et.al., Int.J.Biol.Ma) (Cromol, 139-147, 2013). However, this deletion of the heavy chain sequence is an antibody It does not affect the genobinding capacity or effector function (such as complement activation or antibody-dependent cell-mediated cytotoxicity). It does not affect. Therefore, the present invention also includes antibodies in which the lysine residue at the heavy chain carboxyl terminus is deleted. It is included.

[0098] Other antibodies The antibody of the present invention is an antigen-binding fragment of an antibody having an antigen-binding portion, or a modified version thereof. You can do that. Treat the antibody with a proteolytic enzyme such as papain or pepsin, or the antibody By modifying genes using genetic engineering techniques and expressing them in suitable cultured cells, This allows us to obtain a fragment of the antibody. Among such antibody fragments, the full-length antibody molecule A fragment that retains all or part of the function can be called an antigen-binding fragment of an antibody. The functions of antibodies generally include antigen-binding activity, activity to neutralize antigen activity, and activity to neutralize antigen activity. Activity that enhances antibody-dependent cytotoxicity, complement-dependent cytotoxicity, and complement-dependent cells Sexual cytotoxic activity can be cited. The antigen-binding fragment of the antibody in this invention retains Its function is its binding activity to SIRPα.

[0099] For example, antibody fragments may include Fab, F(ab')2, variable region (Fv), or heavy chain. And a single chain Fv (scFv) formed by linking light chain Fvs with appropriate linkers, di abody (diabodies), linear antibodies, and polyspecific antibodies formed from antibody fragments Examples include the body. Also, the variable of antibodies treated with F(ab')2 under reducing conditions. Fab', a monovalent fragment of the region, is also included in antibody fragments.

[0100] Furthermore, the antibody of the present invention is highly specific, having specificity for at least two different antigens. It may also be a sex antibody. Usually such molecules bind to two types of antigens (immediately In this invention, a bispecific antibody is a " A "multispecific antibody" is an antibody that has specificity for more than three (for example) different antigens. It includes. The polyspecific antibody of the present invention is an antibody consisting of its full length, or a fragment of such an antibody (e.g., F (ab')2 bispecific antibodies may also be used. A bispecific antibody consists of the heavy chain and light chain of two types of antibodies. It can also be produced by conjugating HL pairs, or by producing different monoclonal antibodies. By fusing ibridomas to create bispecific antibody-producing fusion cells, it is also possible to produce It can be manufactured (Millstein et al., Nature (1983) 3 (05, pp. 537-539).

[0101] The antibody of the present invention may also be a single-chain antibody (also referred to as scFv). A single-chain antibody is an antibody Obtained by linking the heavy chain variable region and the light chain variable region with a polypeptide linker. (Pluckthun,The Pharmacology of Monoclona l Antibodies, 113 (edited by Rosenberg and Moore, Spring) Ger Verlag, New York, p. 269-315 (1994), Natu re Biotechnology (2005), 23, pp. 1126-1136). Furthermore, a BiscFv fragment is created by linking two scFvs with a polypeptide linker. It can also be used as a bispecific antibody.

[0102] Methods for producing single-chain antibodies are well known in the art (for example, U.S. 4, U.S. Patent No. 946,778, U.S. Patent No. 5,260,203, U.S. Patent 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 a linker that does not form a conjugate, preferably a polypeptide chain. Linked via a linker (Huston, JSet al., Proc.Natl .Acad.Sci.USA(1988), 85, p.5879-5883). s The heavy chain variable region and light chain variable region in cFv may originate from the same antibody, or they may be separate. It may be derived from an antibody. For example, 12 polypeptide linkers can be used to link the variable regions. Any single-chain peptide consisting of approximately 19 residues can be used.

[0103] The DNA encoding scFv is the DNA encoding the heavy chain or heavy chain variable region of the antibody. , and DNA encoding the light chain or light chain variable region, all or of their sequences A DNA portion encoding a desired amino acid sequence is used as a template, and primers define both ends of it. The pairs are amplified by PCR, and then the polypeptide linker portion is further encoded. The DNA and a primer pair are assembled so that both ends are linked to the heavy and light strands, respectively. It is obtained by combining and amplifying them.

[0104] Furthermore, once the DNA encoding scFv is created, an expression vector containing them is created. - and a host transformed with the expression vector can be obtained by conventional methods, By using the host, scFv can be obtained according to the standard method. These antibodies Somatic fragments can be produced by a host after obtaining and expressing genes in the same manner as described above. ru.

[0105] The antibody of the present invention may be multiplied to increase its affinity for the antigen. Even a single antibody can recognize multiple epitopes of the same antigen. Multiple antibodies may be used. One method for increasing antibody volume is to use the IgG CH3 domain and Binding to two scFvs, binding to streptavidin, helic-stern-helic Examples include the introduction of motifs. The antibody of the present invention is a mixture of multiple types of anti-SIRPα antibodies with different amino acid sequences. Polyclonal antibodies are also acceptable. An example of a polyclonal antibody is one with different CDRs. A mixture of multiple types of antibodies can be cited. Such a polyclonal antibody is This involves culturing a mixture of cells that produce different antibodies and using antibodies purified from the culture. It is possible (see WO2004 / 061104).

[0106] Antibodies modified with various molecules such as polyethylene glycol (PEG). You can also use this.

[0107] The antibodies of the present invention are further formed by these antibodies conjugating with other drugs. An immunoconjugate may also be used. An example of such an antibody is one in which the antibody releases Examples include substances that are combined with projectile substances or compounds with pharmacological effects (Natur e Biotechnology (2005) 23, p. 1137-1146).

[0108] Furthermore, the full-length sequences of the antibody's heavy and light chains are linked using an appropriate linker, and a single-strand immunoglobulin is formed. Those who are obtaining globulin (single-chain immunoglobulin) The law 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-stranded immunoglobulins By dimerizing, antibodies retain a structure and activity similar to those of antibodies, which are originally tetramers. This is possible. Furthermore, the antibody of the present invention has a single heavy chain variable region and does not have a light chain sequence. It is also acceptable for it to be a single-domain antibody. Such antibodies are single-domain antibodies. in antibody (sdAb) or nanobody Furthermore, it has been observed in camels or llamas that the antigen-binding ability is retained. (Muyldemans S.et.al., Protein Eng.(1994) 7(9),1129-35,Hamers-Casterman C.et.al.,N ature(1993)363(6428)446-8). The above antibody is used in the present invention. 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 DN encoding a light chain variable region. A is inserted into an expression vector, and host cells for expression are transformed using the vector. By culturing the cells, recombinant antibodies can be produced in the cells.

[0110] The DNA encoding the antibody consists of DNA encoding the heavy chain variable region and DNA encoding the heavy chain constant region. By ligating the DNA, DNA encoding the heavy chain is obtained, and furthermore, the light chain variable region By linking the DNA encoding the light chain constant region with the DNA encoding the light chain constant region, the light chain is coded You will obtain the DNA you want to use.

[0111] The anti-SIRPα antibody of the present invention comprises the DNA encoding the heavy chain and the D encoding the light chain. NA is inserted into an expression vector, host cells are transformed using the vector, and the host cells It can be produced by culturing. In this process, the DNA encoding the heavy chain and the light chain mentioned above are used. The DNA to be expressed is introduced into the same expression vector, and the host cells are transformed using the vector. Alternatively, the DNA encoding the heavy chain and the DNA encoding the light chain can be inserted into separate vectors. Alternatively, the host cells may be transformed using two vectors. In this case, the heavy chain constant region may be used. A vector into which the DNA encoding the light chain constant region and the DNA encoding the light chain constant region have been pre-introduced is then used to create a heavy chain variable region. DNA encoding the region and the light chain variable region may be introduced into the vector. It may also contain DNA encoding a signal peptide that promotes antibody secretion from cells. In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are inverted. They are linked together with a linkage. After the antibody is produced, the signal peptide is removed, thus preventing the antibody The body can be obtained as mature protein.

[0112] In this case, DNA encoding the heavy chain variable region, DNA encoding the light chain variable region, heavy chain variable DNA formed by linking DNA encoding a variable region and DNA encoding a heavy chain constant region, light chain DNA that ligates DNA encoding a variable region and DNA encoding a light chain constant region is used in programming. It can be functionally linked with elements such as motors, enhancers, and polyadenylated signals. Here, functional connection means that elements are connected in a way that allows them to perform their intended function. .

[0113] Expression vectors are not particularly limited as long as they can replicate in a host such as animal cells, bacteria, or yeast. Examples include known plasmids and phages, which are used in the construction of expression vectors. Examples of vectors that can be used include pcDNA (trademark) (ThermoFisher). SCIENTIFIC), Flexi® Vector (Promega Corporation), pUC19 pUEX2 (manufactured by Amersham), pGEX-4T, pKK233-2 (manufactured by Pharmacia) Examples include pMAM-neo (manufactured by Clontech), etc. Host cells include the colon. Both prokaryotic cells such as bacteria and Bacillus subtilis, and eukaryotic cells such as yeast and animal cells can be used, but eukaryotic cells It is preferable to use cells. For example, as animal cells, the human embryonic kidney cell line HEK2 93 cells, Chinese hamster ovary (CHO) cells, etc. can be used. Expression vector The transformer can be introduced into host cells using a known method, and the host cells can be transformed. For example, electro Troporation method, calcium phosphate precipitation method, DEAE-dextran transfect Examples include the suction method. The produced antibodies are separated using the same methods as for regular proteins. It can be purified using purification methods, such as affinity chromatography. Other chromatography, filtering, ultrafiltration, salting out, dialysis, etc. are selected as appropriate and combined. Just put them together.

[0114] Antitumor agents The present invention encompasses an antitumor agent containing the anti-SIRPα antibody of the present invention as an active ingredient. Furthermore, the heavy chain constant region of the anti-SIRPα antibody of the present invention is the heavy chain constant region of the IgG4 subclass. It is a heavy chain constant region IgG4proFALA that has Pro mutations and FALA mutations. It does not have an effects function, and inhibits the binding of SIRPα and CD47. It only has the function of inhibiting the “Don't-eat-me” signaling pathway. Therefore, the anti-SIRPα antibody of the present invention alone cannot sufficiently damage tumor cells. Thus, the present invention has an effector function and can attack and damage tumor cells, and other antitumor agents and tumors. It is used in combination with other antitumor agents that inhibit immune checkpoints on immune cells caused by tumor cells. Other antitumor agents used in this context bind to tumor cells and interact with phagocytic cells such as macrophages. It can be brought into contact with the CD47 of the tumor cells. At that time, the anti-SIRPα antibody of the present invention can be brought into contact with the CD47 of the tumor cells. By inhibiting the binding of phagocytic cells to SIRPα, the phagocytic ability of phagocytic cells to tumor cells is impaired. Because it enhances the tumor, tumor cells are damaged. That is, the anti-SIRPα antibody of the present invention and other anti-tumor antibodies By using anti-tumor agents in combination, a synergistic anti-tumor effect can be achieved.

[0115] As an antitumor agent to be used in combination with the anti-SIRPα antibody of the present invention, an immune checkpoint inhibitor, Examples include antibody drugs that specifically bind to cancer antigens and possess ADCC and / or ADCP activity. Examples of immune checkpoint inhibitors include PD-1 and its ligand, PD-L Examples include inhibitors that bind to 1, 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 ( Examples include ipilimumab and tremelimumab. In addition, cancer antigens Antibody drugs that specifically react to and possess ADCC and / or ADCP activity include anti-CD2 0 antibody (rituximab), anti-HER2 antibody (trastuzumab), anti-EGF Examples include R antibodies (cetuximab) and anti-CD52 antibodies (alemutuzumab). It is possible.

[0116] ADCCs are nonspecific cytotoxic cells that express the Fcγ receptor (e.g., NK cells). Neutrophils and macrophages (etc.) recognize antibodies bound to target cells, and then... This refers to a cell-mediated reaction that causes the lysis of GET cells. These are primary cells responsible for ADCC. In NK cells, FcγRIIC and FcγRIIIA are expressed, while in monocytes, FcγRI It expresses FcγRIIA, FcγRIIC, and FcγRIIIA. On the other hand, ADC P is targeted by phagocytic cells that express the Fc receptor (e.g., macrophages, neutrophils, etc.). Cell-mediated phagocytosis involves recognizing antibodies bound to cells and subsequently engulfing target cells into the cell. This refers to the reaction. In monocytes, which are primary cells responsible for ADCP, FcγRI and FcγRII A, FcγRIIC, and FcγRIIIA are expressed.

[0117] The present invention is an antitumor agent comprising an anti-SIRPα antibody as an active ingredient, and is an antitumor agent for other antitumors as described above. This includes antitumor agents used in combination with anti-inflammatory drugs.

[0118] Furthermore, the present invention includes an antitumor agent containing an anti-SIRPα antibody as an active ingredient and the above-mentioned other antitumor agents. Includes an antitumor agent or kit containing both agents.

[0119] The antitumor agent containing the anti-SIRPα antibody of the present invention as an active ingredient and the above-mentioned other antitumor agents can be used simultaneously. It may be administered sequentially or in batches. Furthermore, the order of administration is not limited to the anti-SIRP of the present invention. It is also possible to administer other antitumor agents after administering an antitumor agent containing an α antibody as an active ingredient. After administering the antitumor agent, the 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 against carcinoma, sarcoma, lymphoma, leukemia, myeloma, germ cell tumor, brain tumor, For one or more types selected from carcinoid, neuroblastoma, retinoblastoma, and nephroblastoma. It can be used. Specifically, in cancers, it can be used for 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, large intestine cancer, rectal cancer, appendiceal cancer, anal cancer Liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, bladder cancer, prostate cancer, uterine cancer Vaginal cancer is one example, and among sarcomas, liposarcoma, angiosarcoma, chondrosarcoma, rhabdomyosarcoma, and euphorbia Ing's sarcoma, osteosarcoma, undifferentiated pleomorphic sarcoma, myxoid fibrosarcoma, malignant peripheral schwannoma, retroperitoneal sarcoma Examples include tumors, synovial sarcomas, uterine sarcomas, gastrointestinal stromal tumors, leiomyosarcomas, epithelioid sarcomas, etc. Examples of lymphomas include B-cell lymphoma, T- and NK-cell lymphoma, and Hodgkin lymphoma. Leukemia includes myeloid leukemia, lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndrome, etc. Examples include multiple myeloma, and germ cell tumors such as testicular cancer and ovarian cancer. Cancer is one example, and 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 cellular immune enhancers containing anti-SIRPα antibodies as active ingredients. In cellular immune enhancers, the enhancement of natural killer cells and / or T cells is associated with It enhances cellular immunity.

[0122] The antitumor agent of the present invention comprises an effective amount of anti-SIRPα antibody for treatment, a pharmaceutically acceptable carrier, and a dilution agent. This may include agents, solubilizers, emulsifiers, preservatives, and auxiliary agents. The term "body," etc., can be appropriately selected from a wide range depending on the type of disease and the form of drug administration. The method of administering the antitumor agent of the present invention can be appropriately selected, for example, by injection. It can be administered via local injection, intraperitoneal administration, selective intravenous infusion, intravenous injection, subcutaneous injection, or organ perfusion. Liquid injection can be employed. Furthermore, the solution for injection can be a salt solution, a glucose solution, or This involves formulation using a carrier consisting of a mixture of saline solution and glucose solution, various buffers, etc. This can be done. Alternatively, the formulation can be prepared in powder form and mixed with the liquid carrier at the time of use to prepare the injection solution. You may do so.

[0123] Other administration methods can be selected as appropriate in conjunction with the development of the formulation. For example, oral administration In such cases, oral liquid preparations, powders, pills, capsules, and tablets may be used. In the case of oral liquid preparations, the oral liquid preparations such as suspensions and syrups include water, syrup, etc. Sugars such as chlorous acid, sorbitol, and fructose, and glycosides such as polyethylene glycol. Oils such as sesame oil and soybean oil, preservatives such as alkyl p-hydroxybenzoate, It can be manufactured using flavors such as strawberry and peppermint. Powders, pills, capsules, and tablets contain lactose, glucose, sucrose, Excipients such as mannitol, starch, disintegrants such as sodium alginate, magnesium stearate Lubricants such as arrate and talc, polyvinyl alcohol, hydroxypropyl cellulose, Using binders such as gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin It can be formulated into a pharmaceutical product. Tablets and capsules are convenient in that they are easy to administer. This is a preferred unit dosage form in the composition of this invention. When manufacturing tablets or capsules. Solid manufacturing supports are used for this purpose.

[0124] The amount of antibody effective for treatment varies depending on the nature of the disease being treated, the patient's age, and their condition. Ultimately, the doctor should make the decision. For example, 0.0001 mg per kg of body weight per dose. The dose is ~100 mg. The prescribed dose may be administered once every 1 to 180 days, or per day The drug may be administered in two, three, four, or more divided doses at appropriate intervals. [Examples]

[0125] The present invention will be specifically described by the following embodiments, but the present invention will be described by these embodiments It is not limited to that.

[0126] Example 1. Preparation of rat anti-SIRPA antibody 1)-1 Preparation of expression constructs 1)-1-1 Construction of the SIRPA_V1_ECD expression vector Human SIRPA_V1 (NCBI protein database ACCESSION number: NP) Add HHHHHHH to the C-terminal side of the amino acid sequence _001035111) from position 1 to 373. DNA encoding linked polypeptides and polypeptides digested with restriction enzymes XbaI and PmeI pcDNA3.3-TOPO / LaxZ(ThermoFisher SCIENT IFIC) to In‐Fusion HD Cloning Kit(CLONTECH) By using SI to bind, a SIRPA_V1_ECD expression vector was constructed. The amino acid sequence of RPA_V1_ECD is entered as sequence number 45 in the sequence listing, SIRPA_V1_E The nucleotide sequence encoding CD is shown as sequence number 44 in the sequence listing.

[0127] 1)-1-2 Construction of SIRPA_V1_IgV expression vector SIRPA_V1 (Accession number NP_00 in the NCBI protein database) HHHHHH is ligated to the C-terminus of the amino acid sequence 1035111) from position 1 to 149. Using the DNA encoding the polypeptide, SIRPA_V was generated in the same manner as in 1)-1-1. A 1_IgV expression vector was constructed. The amino acid sequence of SIRPA_V1_IgV is listed in the sequence listing. Sequence ID 47 contains the nucleotide sequence encoding SIRPA_V1_IgV in the sequence listing. This is shown in sequence number 46.

[0128] 1)-1-3 Construction of the SIRPA_V2_ECD expression vector SIRPA_V2(V1 sequence, JBC Vol.289, No.14, 10024(20 Based on (14), HHHHHHH is ligated to the C-terminus of the amino acid sequence from position 1 to 372. Using the DNA encoding the polypeptide, SIRPA_ A V2_ECD expression vector was constructed. The amino acid sequence of SIRPA_V2_ECD was used as the sequence. The sequence number 49 in the table contains the nucleotide sequence encoding SIRPA_V2_ECD. This is shown in sequence number 48.

[0129] 1)-1-4 Construction of SIRPA_V2_IgV expression vector HHHHHHH was ligated to the C-terminus of amino acid sequences 1 through 148 of SIRPA_V2. Using the DNA encoding the polypeptide, SIRPA_ A V2_IgV expression vector was constructed. The amino acid sequence of SIRPA_V2_IgV was used as the sequence. The sequence number 51 in the table contains the nucleotide sequence encoding SIRPA_V2_IgV. This is shown in sequence number 50.

[0130] 1)-1-5 Construction of the cSIRPA_ECD expression vector cSIRPA (NCBI protein database ACCESSION number NP_0017) 68) A polyparticle in which HHHHHHH is ligated to the C-terminus of the amino acid sequence from position 1 to 372. Using the DNA encoding ptydos, cSIRPA_ECD was generated in the same manner as in 1)-1-1. The current vector was constructed. The amino acid sequence of cSIRPA_ECD was entered into sequence number 53 of the sequence listing. The nucleotide sequence encoding cSIRPA_ECD is shown as sequence number 52 in the sequence listing.

[0131] 1)-1-6 Construction of CD47-Fc expression vector Human CD47 (NCBI protein database ACCESSION number NP_0012) Using the DNA encoding the polypeptide of 71679), C in the same manner as in 1)-1-1 A D47-Fc expression vector was constructed. The amino acid sequence of CD47-Fc was entered into the sequence listing. In sequence 55, the nucleotide sequence encoding SIRPA_V1_ECD is entered in the sequence number field of the sequence listing. This is shown in 54.

[0132] 1)-2 Preparation of recombinant proteins 1)-2-1 Preparation of SIRPA_V1_ECD 1) The SIRPA_V1_ECD expression vector prepared in 1-1-1 is expressed in FreeStyle Transplantation into 293F cells (ThermoFisher SCIENTIFIC) Transient expression was achieved by sfection. The culture supernatant was equilibrated with 3×PBS. After adding to sTrap excel (GE Healthcare Japan), use 3x PBS. The column was washed. Next, 3×PBS, 500 mM Imidazole, pH 7.5 It was eluted. From the recovered SIRPA_V1_ECD fraction, HiLoad 26 / 600 Using Superdex 75 pg (GE Healthcare Japan) with SIRPA_V 1_ECD was purified.

[0133] 1)-2-2 Preparation of SIRPA_V1_IgV 1) The SIRPA_V1_IgV expression vector prepared in 1-1-2 is processed in FreeStyle Transplantation into 293F cells (ThermoFisher SCIENTIFIC) Transient expression was achieved by sfection. The culture supernatant was equilibrated with 3×PBS. After adding to sTrap excel (GE Healthcare Japan), use 3x PBS. The column was washed. Next, 3×PBS, 500 mM Imidazole, pH 7.5 It was eluted. From the recovered SIRPA_V1_IgV fraction, HiLoad 26 / 600 Using Superdex 75 pg (GE Healthcare Japan) with SIRPA_V IgV was purified.

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

[0135] 1)-2-4 Preparation of SIRPA_V2_IgV Using the SIRPA_V2_ECD expression vector prepared in 1)-1-4, 1)-2-2 SIRPA_V2_ECD was purified using the same method as before.

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

[0137] 1)-2-6 Preparation of CD47-Fc CD47-Fc expression vector in FreeStyle 293F cells (Ther Transfection with moFisher SCIENTIFIC results in transient The expression was achieved. The culture supernatant was equilibrated with PBS. MabSelectSuRe(GE Healthcare) After adding everything to the A. Japan (company) container, the column was washed with PBS. Next, 2M arginine The fraction containing CD47-Fc was collected by eluting with a hydrochloride solution (pH 4.0). CD47-Fc fraction, HiLoad 26 / 600 Superdex 200 pg CD47-Fc was purified using (GE Healthcare Japan).

[0138] 1)-3 Immunity Female WKY / Izm rats (SLC Japan) were used for immunization. 1)-2 Prepared antigen proteins: SIRPA_V1_ECD, SIRPA_V1_IgV, SIRPA_V 2_ECD, SIRPA_V2_IgV and Freund's Complete Lymph nodes of rats that were administered a mixture of Adjuvant (Wako Pure Chemical Industries, Ltd.) to the tail ridge. The spleen was also collected and used to create hybridomas.

[0139] 1)-4 Hybridoma Construction Lymph node cells or spleen cells and mouse myeloma SP2 / 0-ag14 cells (ATC) C:CRL-1581) to LF301-Cell Fusion Unit (BEX Corporation) Using electrocellular fusion, Clona Cell‐HY Selection Medi The cells were diluted and cultured in umD (Stem Cell Technologies). Monoclone hybridomas were created by recovering the hybridoma colonies. Each recovered hybridoma colony was cultured, and the resulting hybridoma culture supernatant was used Screening was then performed for anti-SIRPA antibody-producing hybridomas.

[0140] 1)-5 Construction of expression vectors for antigen-binding antibody screening 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( [Modified from 2014] cDNA encoding pcDNA3.2 V5-DEST vector The vectors pcDNA3.2 V5-DEST were cloned into a vector and expressed accordingly. -SIRPA_V1_ECD, V2_ECD (or pcDNA3.2 V5-DEST-) SIRPA_V1 and V2 were constructed. The amino acid sequence of the human SIRPA_V1 protein was used. The amino acid sequence of the human SIRPA_V2 protein is entered in sequence number 56 of the sequence listing. These are shown in 57.

[0141] 1)-5-2 Monkey SIRPA and mouse SIRPA expression vector (pcDNA3.2 V5-DEST-SALSIRPA, pFLAG V5-DEST-SALSIRPA or p FLAG V5-DEST-Mouse SIRPA) Construction Monkey SIRPA protein (NP_001271679) or mouse SIRPA protein (C 57BL6:NP_031573, BALB / c:BAA20376, 129:P977 Based on 97, NOD SCID: Immunology, 143, 61-67, 2014. cDNA encoding (modified) into a pcDNA3.2 V5-DEST vector, or pFL The AG V5-DEST vector was cloned to express each protein. pcDNA3.2 V5-DEST-Sal SIRPA, pFLAG V5-DEST-Sal SIRPA and pFLAG V5-DEST-Mouse SIRPA (C57BL6, BA We constructed the LB / c, 129, NOD) amino acid sequence of monkey SIRPA. Number 58 is the amino acid sequence of mouse SIRPA_C57BL / 6, as shown in sequence number 59 of the sequence listing. The amino acid sequence of mouse SIRPA_BALB / c is entered as sequence number 60 in the sequence listing. The amino acid sequence of SIRPA_129 is entered as sequence number 61 in the sequence listing, mouse SIRPA_NO The amino acid sequences of D are shown in sequence number 62 of 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) (A stable expression cell line derived from this cell line) was placed in 7.5 × 10 units of DMEM medium containing 10% FBS. 5 cells / mL It was prepared to achieve this. In contrast, Lipofectamine 2000 (Therm Following the translocation procedure using Fisher Scientific, Inc., pcDNA 3.2 V5-DEST-SIRPA_V1 or pcDNA3.2 V5-DEST - Use SIRPA_V2, or pcDNA3.2 V5-DEST as a control. Then, 50 μL is added to a 96-Half area well plate (Corning). Dispense into individual portions, or into 100 μL portions in a 96-well plate (Corning). Note: In 10% FBS-containing DMEM medium, under conditions of 37°C and 5% CO2, 24 to 2 The cells were cultured for 7 hours. The resulting transduced cells were used in Cell-ELISA while still attached to the cells. .

[0143] 1)-6-2 Evaluation of binding affinity to human SIRPA (Cell-ELISA) After removing the culture supernatant of the 293α cells into which the expression vector was introduced in Example 1)-6-1, p cDNA3.2 V5‐DEST‐SIRPA_V1, V2 or pcDNA3.2 V5 - Hybridoma culture supernatant was added to each of the DEST-transformed 293α cells, and at 4°C The cells were allowed to stand for 1 hour. After washing the cells in the wells twice with PBS containing 5% FBS, 5% FBS Anti-Rat IgG-Peroxidase a diluted 500-fold with PBS containing Adding "ntibody produced in rabbit" (SIGMA Corporation), 4 The cells were allowed to stand at °C for 1 hour. After washing the cells in the well twice with PBS containing 5% FBS, O PD colorant (OPD dissolving solution (0.05 M trisodium citrate, 0.1 M aqueous phosphoric acid) Disodium ionopropyl alcohol (12-hydrate, pH 4.5) with o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) (Dissolve H2O2 in 0.4 mg / mL and 0.6% (v / v) solutions respectively) and 5 The solution was added at 0 μL / well. The color reaction was carried out with occasional stirring, and 50 μL of 1 M HCl was added. After adding the colorant in L / well to stop the color reaction, use a plate reader (ENVISION Absorbance at 490 nm was measured using a PerkinElmer instrument. To select a hybridoma that produces an antibody that specifically binds to SIRPA, Compared to Rolle's pcDNA3.2 V5-DEST-transformed 293α cells, pcDNA3. 2 V5-DEST-SIRPA_V1 and SIRPA_V2 expression vectors introduced into 293α Hybridomas that produce culture supernatant with higher absorbance in the cell side are identified by anti-SIRPA antibodies. It was selected as a production-positive result.

[0144] 1)-6-3 Evaluation of SIRPA-CD47 binding inhibitory activity After removing the culture supernatant of the 293α cells into which the expression vector was introduced in Example 1)-6-1, p cDNA3.2 V5‐DEST‐SIRPA_V1, SIRPA_V2 or pcDNA 3.2 Hybridoma culture supernatant was added to each of the V5-DEST-transformed 293α cells. Add the solution and immediately prepare it with PBS containing 5% FBS to a final concentration of 10,000 ng / mL. Add 50 μL / well of peroxidase-labeled CD47-Fc, The cells were allowed to stand at 4°C for 1 hour. After washing the cells in the wells twice with PBS containing 5% FBS, O PD colorant (OPD dissolving solution (0.05 M trisodium citrate, 0.1 M aqueous phosphoric acid) Disodium ionopropyl alcohol (12-hydrate, pH 4.5) with o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) (Prepared by) Dissolve H2O2 in 0.4 mg / mL and 0.6% (v / v) solutions respectively. The solution was added at a rate of 100 μL / well. The color reaction was carried out with occasional stirring, and 1M HCl was added in 10 minutes. After adding 0 μL / well to stop the color reaction, use a plate reader (Spectra The absorbance at 490 nm was measured using max:Molecular devices. It produces antibodies that specifically inhibit the binding of SIRPA, expressed on the surface of the vesicle membrane, to CD47-Fc. To select the hybridomas, we compared them with the control group with added culture medium and pcDNA3 2. Introduction of V5-DEST-SIRPA_V1 or SIRPA_V2 expression vector 29 In 3α cells, hybridomas that produce culture supernatant with lower absorbance are used in Rigan It was selected as a candidate that produced an anti-SIRPA antibody with binding inhibitory activity.

[0145] 1)-6-4 Evaluation of species cross-reactivity in mouse and monkey SIRPA pcDNA3.2 V5-DEST-Sal SIRPA prepared in Example 1)-5-2, Alternatively, mouse SIRPA expression vector-transformed 293α cells, and pcDNA3.2 V5-D After removing the culture supernatant of EST-introduced 293α cells, the same method as for evaluating human SIRPA binding activity was used. The binding affinity to monkey or mouse SIRPA was evaluated. The above-mentioned binding affinity to humans and animal species was evaluated. Based on binding activity and SIRPA-CD47 binding inhibitory activity, D13, F42, F44 A total of seven clones were selected, consisting of antibodies F47, F60, F63, and F86.

[0146] 1)-7 Determination of antibody isotype From among the obtained rat anti-SIRPA antibody-producing hybridomas, human SI was strongly and specifically selected. Binds to RPA_V1 and SIRPA_V2, as well as monkey SIRPA, and has high SIRPA-C D13, F42, F44, F47, and F6 were suggested to possess D47 binding inhibitory activity. Hybridomas producing 0, F63, and F86 antibodies were selected, and the antibody isotypes were the same. Determined. The isotype is Rat Immunoglobulin Isotyping The determination was made using an ELISA kit (BD Pharmingen). As a result, Rat anti-SIRPA monoclonal antibodies D13, F42, F60, and F86 isotypes The IPU is IgG1 / κ chain, the isotypes F44 and F47 are IgG2a / κ chain, and the isotype F63 is IgG2a / 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 types of rat anti-SIRPA monoclonal antibodies were purified from hybridoma culture supernatant. First, each antibody-producing hybridoma was selected using ClonaCell-HY Selection. Increase to a sufficient amount with Medium E (StemCell Technologies). After propagation, Ultra Low IgG FBS (Thermo Fisher Sc 20% of gentamicin (Thermo) (from ientific) was added to 5 μg / mL of gentamicin (Thermo Fisher Scientific) containing Hybridoma SFM (Ther The culture medium was changed to (Fire Scientific) and incubated for 7 days. The supernatant was collected and sterilized by passing it through a 0.22 μm filter (Corning).

[0148] 1)-8-2 Purification of antibodies From the culture supernatant of the hybridoma prepared in Example 1)-8-1, the antibody was extracted from Protein Purified by G affinity chromatography. Protein G column (GE H The antibody was adsorbed onto the ealthcare Bioscience column, and the column was washed with PBS. After purification, the sample was eluted with a 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7). A 1 M Tr solution was added to the eluate. After adding is-HCl (pH 9.0) to adjust the pH to 7.0-7.5, Centri fugal UF Filter Device VIVASPIN20 (Fractional Molecular Weight U The buffer was replaced with PBS using F30K (Sartorius), and the antibody was also used. The antibody was concentrated to a concentration of 2 mg / mL or higher. Finally, Minisart-Plu The sample was purified by filtering with an S filter (Sartorius).

[0149] Example 2. In vitro evaluation of seven rat anti-human SIRPA antibodies. 2)-1 Construction of expression vectors for antigen-binding antibody screening 2)-1-1 FLAG-Human SIRPA Expression Vector (pFLAG V5-DEST- Construction of SIRPA_V1-V10) 10 types of human SIRPA variant proteins (Nature Immunology) cDNA encoding (excerpted from 8,1313-1323,2007) is used in pFLAG V5 - A vector into which each variant protein is cloned and expressed. pFLAG V5-DEST-SIRPA_V1-V10 was constructed. The amino acid sequence of human SIRPA_V3 is entered as sequence number 63 in the sequence listing, human SIRPA_V The amino acid sequence of 4 is assigned to sequence number 64 in the sequence listing, and the amino acid sequence of human SIRPA_V5 is assigned to it. In sequence number 65 of the sequence listing, enter the amino acid sequence of human SIRPA_V6 into sequence number 66 of the sequence listing. The amino acid sequence of human SIRPA_V7 is entered as sequence number 67 in the sequence listing, human SIRPA_V The amino acid sequence of 8 is assigned to sequence number 68 in the sequence listing, and the amino acid sequence of human SIRPA_V9 is assigned to it. In the sequence list, under sequence number 69, the amino acid sequence of human SIRPA_V10 is shown under sequence number 70. These are shown below.

[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 acids (1 to 504) of human SIRPA_V2, and amino acids 165 to 371 Defects in the region (hereinafter referred to as "IgV bodies"), defects in regions 225 to 371 (hereinafter referred to as " The cDNA encoding the "IgV_IgC body" is used in the pFLAG V5-DEST vector. We cloned the proteins and constructed vectors to express each mutant protein.

[0151] The amino acid sequence of the human SIRPA_V2_IgV is listed as sequence number 71 in the sequence listing, human SI The amino acid sequence of RPA_V2_IgV_IgC1 is shown in sequence number 72 of the sequence listing.

[0152] 2)-1-2-2 hmSIRPA_Δ0, Δ1, and Δ2_mouse SIRPA expression vector Construction of the ter (pFLAG V5-DEST-hmSIRPA_Δ0, Δ1, and Δ2) S consists of amino acid residues 81 to 85 of mouse SIRPA as described in Sequence ID No. 60. The FTGE sequence (sequence number 78) is replaced with the NQKEG sequence (sequence number 76), and 126 to The RGSSE sequence (SEQ ID NO: 79), consisting of the 130th amino acid residue, is replaced with the KGS sequence. The resulting SIRPA mutant was named hmSIRPA_Δ0. (See mouse code 60) The SFTGE sequence (SEQ ID NO: 78) consisting of amino acid residues 81 to 85 of SIRPA A SIRPA mutant in which the NQKEE sequence (sequence number 77) is substituted is called hmSIRPA_Δ1. It was named. Furthermore, amino acids 81 to 85 of mouse SIRPA described in Sequence ID No. 60 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 encoding cDNA was cloned into the pFLAG V5-DEST vector, and each We constructed a vector expressing the SIRPA variant. The amino acid sequence of hmSIRPA_Δ0 is entered as sequence number 73 in the sequence listing, hmSIRPA_Δ The amino acid 1 is assigned to sequence number 74 in the sequence listing, and the amino acid of hmSIRPA_Δ2 is assigned to sequence number 74 in the sequence listing. These are shown in column 75.

[0153] 2)-2 Evaluation of binding compatibility to human SIRPA variants V1-V10 Example 2) Introduction of expression vectors for 10 variant proteins prepared in Example 1-1 293 After removing the culture supernatant of α cells, pFLAG V5‐DEST‐SIRPA_V1~V10 or Each of the pFLAG V5-DEST-introduced 293α cells was treated with a final concentration of 10,000 nucleotides. Rat anti-human SIRPA purified antibody diluted in PBS containing 5% FBS to a concentration of g / mL. 50 μL / well was added and allowed to stand at 4°C for 1 hour. Furthermore, the expression of each SIRPA variant was assessed. The detection wells contain PBS with 5% FBS to achieve a final concentration of 10,000 ng / mL. Add 50 μL / well of diluted anti-FLAG M2 antibody (SIGMA Corporation) and ferment at 4°C. It was allowed to stand for 1 hour. The following was done using the same method as the binding activity table value of human SIRPA shown in 1)-6-2. The binding affinity to 10 different human SIRPA variants was evaluated. The binding affinity of the rat anti-human SIRPA antibody was standardized based on the expression of the FLAG tag. As shown in Table 1, all clones showed binding affinity to all variants.

[0154] [Table 1]

[0155] 2)-3 Evaluation of species cross-reactivity in mouse and monkey SIRPA pFLAG V5-DEST-SalSIRPA prepared in Example 1)-5-2, or p FLAG V5-DEST-mouse SIRPA expression vector-transformed 293α cells, and pF After removing the culture supernatant of LAG V5-DEST-transformed 293α cells, the binding activity of human SIRPA was detected. Binding affinity to monkey or mouse SIRPA was evaluated using the same method as for sex evaluation. As shown in Table 2, all rat anti-human SIRPA antibodies do not bind to monkey SIRPA. Although it was shown to bind, it did not show binding to mouse SIRPA.

[0156] [Table 2]

[0157] 2)-4 Epitope Analysis 2)-4-1-1 Epitope analysis by Cell-ELISA method (1) 2) The pFLAG V5-DEST-ECD and IgV forms prepared in 2-1-2-1, and IgV_IgC1 expression vector-introduced 293α cells, and pFLAG V5-DEST After removing the culture supernatant of the introduced 293α cells, each cell was treated with a final concentration of 10,000 ng / Rat anti-human SIRPA purified antibody diluted in PBS containing 5% FBS to a total volume of 5 mL. 0 μL / well was added and allowed to stand at 4°C for 1 hour. Furthermore, each SIRPA construct expression was performed. The detection wells contain PBS with 5% FBS to achieve a final concentration of 10,000 ng / mL. Add 50 μL / well of diluted anti-FLAG M2 antibody (SIGMA Corporation) and ferment at 4°C. The sample was allowed to stand for 1 hour. The following is a list of 7 clones of purified antibodies, using the same method as for evaluating the binding activity of human SIRPA. The binding affinity to each domain was evaluated. Rat anti-human SIRP against each construct. The binding affinity of antibody A was standardized based on the expression of the FLAG tag. As shown in Figure 1A, the rat anti-human SIRPA antibody was found to be effective against all constructs. The matching result suggests that it recognizes the IgV domain.

[0158] 2)-4-1-2 Epitope analysis by Cell-ELISA method (2) 2) pFLAG V5-DEST-hmSIRPA_Δ0, Δ prepared in 2)-1-2-2 1, and Δ2 expression vector-transformed 293α cells, and pFLAG V5-DEST-transformed 2 After removing the culture supernatant of 93α cells, each cell group was treated to a final concentration of 10,000 ng / mL. Four types of D13 humanized anti-human SIRPA antibodies diluted in PBS containing 5% FBS, and Add 50 μL / well of each chimeric anti-human SIRPA antibody and allow to stand at 4°C for 1 hour. In addition, each well for detecting SIRPA construct expression contained 10,000 units at a final concentration. Anti-FLAG M2 antibody (SIG) diluted in PBS containing 5% FBS to a concentration of ng / mL. 50 μL / well of (manufactured by MA Corporation) was added and allowed to stand at 4°C for 1 hour. The following is a study of human SIRPA. The binding affinity to each construct was evaluated using the same method as the binding activity evaluation. The binding affinity of the anti-human SIRPA antibody was standardized based on the expression of the FLAG tag. As shown in Figure 1B, hD13_H1L3, hD13_H1L4h, hD13_H2L2 hD13_H2L3, or cD13, is paired with hmSIRPA_Δ0 which has an NQKEG sequence. It showed a binding affinity dependent on the concentration of the added antibody, but for Δ1 and Δ2, it did not. No bonding activity was observed at this concentration. From the above, it can be concluded that the NQKEG sequence is necessary for the binding of hD13 and cD13. This was suggested.

[0159] 2)-4-2 Epitope analysis by X-ray crystal structure analysis 2)-4-2-1 Crystallization of the complex Full-length cD13 antibody is administered under weakly acidic conditions to Lysyl Endopeptidase (Wak o) is used to selectively cleave the sample, and a BioAssist S cation exchange column (Tosoh) is used. The Fab fragment of the cD13 antibody was then isolated. SIRPA_V2 obtained in Example 1)-2 IgV and cD13 Fab fragments were mixed in a molar ratio of 1:1, and Superdex 75,1 The complex fraction was separated using a 0 / 300GL gel filtration column (GE Healthcare). Then, replace the buffer with 10 mM Tris HCl (pH 8.2) by ultrafiltration, and 3 The solution was concentrated to g / L. The complex solution was crystallized by vapor diffusion. Protein solution 0.5 μg In L, a precipitating agent solution (0.2M Potassium phosphate dibasic , 20%(w / v) Polyethylene Glycol 3350, pH 9. Add an equal volume of 2) to the solution and place it in a sealed container containing 0.05 mL of precipitant solution until both solutions are in contact. It was carefully placed and left to stand at 25°C. After one week, 0.2mm x 0.2mm x 0.05m A rod-shaped crystal of m was obtained. The obtained crystal was diluted approximately 1.4 times with Glycol. The sample was immersed in a solution and then frozen with liquid nitrogen. (Photon Factory, Tsukuba) X-ray diffraction data was collected at beamline PF BL-17A. From the obtained diffraction patterns... Software XDS (Max Plank Institute for Medica) The diffraction intensity was quantified using (L Research) and the crystal structure factor was determined. The crystal was six The crystal is in the gonal system, the space group is R32, and the unit cell of the crystal is a=b=149.61 Å, c=155.61 Å. The values ​​were Å, alpha=beta=90°, and gamma=120°.

[0160] 2)-4-2-2 Structural analysis of the complex Homology models of the obtained structure factors and Fab fragments and IgV domains of human SIRPA Using the three-dimensional structural coordinates of the known structure (PDBID:2JJS), the molecular substitution method was performed, and the position The phase was determined. The calculation was performed using the software phaser (CCP4:Collaboration). The Computational Project No. 4) was used. The crystals are non The symmetrical unit contained one complex. Software Refmac5 (CCP4:Col Using Laborative Computational Project No. 4) We refined the structure and modified the model using the software COOT. The process was repeated, and a final R value of 22% and a free R value of 25% were obtained at a resolution of 2.4 Å. The final model consists of L-chain amino acid residues 1-213 and H-chain amino acid residues of the Fab fragment of cD13. It contains amino acid residues 1-225 and 33-143 of human SIRPA variant 2.

[0161] 2)-4-2-3 Structural analysis of the complex and identification of the D13 epitope. amino acid residues of human SIRPA within 4 Å of the cD13 Fab fragment (each amino acid) The residue position (corresponding to sequence number 57 in the sequence listing) is as follows: Gly64 , Pro65, Leu78, Gln82, Lys83, Glu84, Gly85, His 86, Phe87, Thr91, Thr92, Glu95, Thr97, Lys98, L ys126. Figure 2 shows the ribbon model and surface of the entire complex, and Figure 3 shows the bet of human SIRPA. The interaction between the regions before a5 (A) and after beta5 (B) and cD13 was shown. 3 is the beta4-5 loop, which has low sequence diversity in human SIRPA, i.e., residue number 8. Strong recognition of 2-87 enables strong coupling with various variants. This was suggested (Figure 4). On the other hand, the beta5-6 loop, i.e., residue numbers 92-105 The interaction in this region is weak. For example, if glutamate is replaced with aspartic acid... Glu95, which contains the variant, is located near Fab, but the electron density of its side chain is observed. This suggests that it is not measured and does not significantly contribute to the interaction. Furthermore, in the sequence shown in Figure 4... The "·" indicates the same amino acid residue as SIRPA_V1, and the amino acid residue is listed. The locations indicate different amino acid residues.

[0162] 2)-5 Evaluation of human SIRPA-CD47 binding inhibitory activity 1) Remove the culture supernatant of human SIRPA expression vector-transformed 293α cells prepared in 5-1. After that, pcDNA3.2 V5-DEST-SIRPA_V1, V2 or pcDNA3. For each of the 293α cells introduced with V5-DEST, a final concentration of 0 to 10,000 ng was administered. Rat anti-human SIRPA purified antibody diluted in 5% FBS-containing PBS to a concentration of / mL 50 μL / well was added. Immediately afterward, 5% FB was added to achieve a final concentration of 10,000 ng / mL. Peroxidase-labeled CD47-Fc prepared in S-containing PBS was used for 50 minutes. The solution was added in μL / well and allowed to stand at 4°C for 1 hour. The following steps were performed using the same method as in 1)-6-3 to inhibit binding. The harmful activity was evaluated. As shown in Table 3, all rat anti-human SIRPA antibodies are human SIRPA-CD47 It showed binding inhibitory activity against [the target substance].

[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 Human gastric cancer cell line AGS cells are used in TrypLE Express (Life Techno (manufactured by Logie Inc.) was added, reacted at 37°C for 5 minutes, and then peeled off. 10% FBS-containing RPM After adding I1640 medium (Life Technology) and washing twice, PBS After two washes, the number of viable cells was measured using a trypan blue exclusion test. 4 × 10 7 Cells After extraction and centrifugation, use PKH26 Red Fluorescent Cell Linker. Kit for General Cell Membrane Labeling(S) Cells were suspended in 2 mL of Dilluent C (provided by IGMA). A labeling solution was used. After diluting mM PKH26 Linker to 10 μM with Dilluent C, immediately... The cell suspension was mixed with an equal volume of PKH26 Linker solution and allowed to stand at room temperature for 5 minutes. 25 RPMI1640 medium containing 10% FBS per mL (manufactured by Life Technology Corporation) ) Add ) and wash twice, then 2 x 10 6 The cells were resuspended to a concentration of cells / mL and used as the target cells. I used it.

[0165] 2)-6-2 Preparation of PBMC cells 25 mL of healthy donor blood is placed in a 20 mL Ficoll Paque Plus (manufactured by GE). After slow stratification, the plasma was centrifuged at room temperature at 1500 rpm for 30 minutes. The cell layer located in the middle of the ue Plus was collected with a dropper and placed in 20 mL of 10% FBS. It was suspended in RPMI1640 medium (manufactured by Life Technology). 150 Centrifuge at 0 rpm for 5 minutes, remove supernatant, and add 20 mL RPMI1640 medium containing 10% FBS. Add soil and wash twice. Add 1 mL of Robosep buffer (STEMCELL After suspension in (company), the number of viable cells was measured using a trypan blue dye exclusion test. It was used as a cell.

[0166] 2)-6-3 Preparation of effector cells The PBMC cells prepared in Example 2)-6-2 were used in RoboSep buffer (STE (Made by MCELL) 5x10 7 Prepared to a concentration of 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. Added. After reacting at 4°C for 10 minutes, EasySep Magnetic Particulate 50 μL of es was added per 1 mL of PBMC cell suspension. After reacting at 4°C for 5 minutes, 2.5 Add RoboSep buffer (manufactured by STEMCELL) to make a total of mL, E The asySep Magnet was set up. After 2 minutes and 30 seconds, the supernatant was collected and then 1200r was used. The monocyte fraction was separated after centrifuging for 5 minutes at 5pm. RPMI1 containing 10% FBS. After adding 640 medium (Life Technology) and washing once, the concentration is 10 ng / mL. RPMI1640 medium containing 10% FBS and M-CSF (PEPROTEC). (manufactured by Life Technology) was added, and the suspension was seeded into a 225 cm flask (manufactured by Sumitomo Bakelite). The cells were cultured for 10 days under the conditions of 37 °C and 5% CO2. After removing the culture supernatant, RPMI1640 medium containing 10% FBS (manufactured by Life Technology) containing 10 ng / mL IL-10 and 10 ng / mL M-CSF (manufactured by PeproTech) was added, and the cells were further cultured for 2 days. After 12 days, TrypLE Express (manufactured by Life Technology) was added to the differentiated macrophages, and after reacting at 37 °C for 40 minutes, the cells were detached. After adding RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and washing twice, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a concentration of 5×10 cells / mL and used as effector cells. 2 flask (Sumitomo Bakelite). After removing the culture supernatant, RPMI1640 medium containing 10% FBS (manufactured by Life Technology) containing 10 ng / mL IL-10 and 10 ng / mL M-CSF (manufactured by Pepro Tech) was added, and the cells were further cultured for 2 days. After 12 days, TrypLE Express (manufactured by Life Technology) was added to the differentiated macrophages, and after reacting at 37 °C for 40 minutes, the cells were detached. After adding RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and washing twice, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a concentration of 5×10 cells / mL and used as effector cells. After 12 days, TrypLE Express (manufactured by Life Technology) was added to the differentiated macrophages, and after reacting at 37 °C for 40 minutes, the cells were detached. After adding RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and washing twice, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a concentration of 5×10 cells / mL and used as effector cells. macrophages, TrypLE Express (manufactured by Life Technology) was added, and after reacting at 37 °C for 40 minutes, the cells were detached. After adding RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and washing twice, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a concentration of 5×10 cells / mL and used as effector cells. After adding RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and washing twice, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a concentration of 5×10 cells / mL and used as effector cells. After adding RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and washing twice, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a concentration of 5×10 cells / mL and used as effector cells. 5 cells / mL, the cells were resuspended in RPMI1640 medium containing 10% FBS (manufactured by Life Technology) and used as effector cells. (manufactured by Life Technology) and used as effector cells.

[0167] 2) Evaluation of 2-6-4 ADCP activity 50 μL / well of the target cells prepared by the method of Example 2-6-1 was added to a 96-well U-bottom microplate with a low-adhesion surface (manufactured by Sumitomo Bakelite). Then, 7 clones of rat anti-human SIRPA antibody, Hu5F9 G4 (anti-human CD47 antibody: prepared based on PloS ONE 10[9]:e0137345, US20 15183874), TTI-621 (human SIRPA-Fc: prepared based on WO2014 / 094122), or various control IgG were diluted with RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a final concentration of 0 to 10 000 ng / mL and added at 50 μL / well. 15183874), TTI-621 (human SIRPA-Fc: prepared based on WO2014 / 094122), or various control IgG were diluted with RPMI1640 medium containing 10% FBS (manufactured by Life Technology) to a final concentration of 0 to 10 In the monotherapy group, RPMI1640 medium containing 10% FBS (Life Technology (Manufactured by [Company Name]) was added at a rate of 50 μL / well, and in the combined group, the final concentration was adjusted to 250 ng / mL. Dilute with % FBS-containing RPMI1640 medium (Life Technology). Trastuzumab (Roche) was added at a concentration of 50 μL / well. (Example 2) Prepared in -6-3, 1 × 10 6 Cells / mL, with 50 μL / well effector cells added. After addition, the mixture was left to stand for 16 hours under conditions of 37°C and 5% CO2. (4°C, 1200 rpm x 5 minutes) After intercentrifugation and removal of supernatant, the cells were washed with 200 μL / well of PBS containing 5% FBS. 5 μL / well of PBS containing 5% FBS, 5 μL / well of APC human CD1 1b (Becton Dickison) was added, and the mixture was allowed to stand at 4°C for 15 minutes. 20 Washed twice with 0 μL / well of PBS containing 5% FBS. 100 μL / well of 1×BD Stabilizing Fixative (manufactured by Becton Dickison) The solution was suspended and left to stand overnight at 4°C. The following day, flow cytometry (FACS CantoII) was performed. Measurements were taken using a Becton (manufactured by Dickison). Data analysis was performed using Flowjo. (TreeStar Corporation) was used. Exploration was performed using FSC (forward scatter) / SSC (side scatter). Afterward, the number of cells that were PE-positive (A) and those that were positive for both APC and PE (B) was calculated. AP Cells that tested positive for both C and PE (B) were phagocytosed by macrophages into target cells. The rate of cell phagocytosis due to ADCP activity was calculated using the following formula. Cell phagocytosis rate (%)=B / (A+B)×100 As shown in Figure 5, against the CD47-positive human gastric cancer cell line AGS, rat anti-human SIRPA Antibody monotherapy options include Hu5F9G4 (anti-human CD47 antibody) and TTI-621 (human SI Compared to RPA-Fc), it showed lower ADCP activity (Figure 5A). On the other hand, Trastuz When used in combination with umab, ADCP activity equivalent to that of Hu5F9-G4 and TTI-621 should be maintained. As shown (Figure 5B). Therefore, the binding of SIRPA-CD47 is inhibited by the anti-SIRPA antibody. This suggests that it enhances the phagocytic ability of macrophages.

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

[0169] 3)-1-2 Nucleation of cDNA in the light chain variable region of D13 by 5'-RACE PCR Otidone sequence analysis and amino acid sequence determination Amplification of the cDNA encoding the light chain variable region was performed using the tota prepared in Example 3)-1-1. Approximately 1 μg of l RNA and SMARTer RACE 5' / 3' Kit (Clonte The procedure was performed using (ch company). The cDNA encoding the variable region of the D13 light chain gene was processed using PC As a primer for amplification in R, UPM (Universal Primer) A Mix: (Included in the SMARTer RACE 5' / 3' Kit), and known racks Primers designed from the sequence of the constant region of the light chain were used. cDNA encoding the variable region of the light chain, amplified by 5'-RACE PCR, is used as a plasmid. The nucleotide sequence of the cDNA encoding the variable region of the light chain was then sequenced. Quens analysis was performed. The variable region of the D13 light chain encoded in the determined cDNA nucleotide sequence The no-acid sequence is an amino acid sequence consisting of amino acid residues 21-126 of sequence number 23 in the sequence listing. This corresponds to a column. The amino acid sequences of CDRL1, CDRL2, and CDRL3 of D13 are listed in the sequence list. These are shown in Sequence IDs 1 to 3. The amino acids of these CDRs are also shown in Figure 28. The amino chain sequence of each CDR is defined as AbM (Martin, ACR, Cheetham, JC and Rees, AR (1989) P roc. Natl Acad. Sci. USA, 86,9268-9272) It is described based on the following.

[0170] 3)-1-3 Nucleation of cDNA in the heavy chain variable region of D13 by 5'-RACE PCR Otidone sequence analysis and amino acid sequence determination Amplification of the cDNA encoding the heavy chain variable region was performed using the tota prepared in Example 3)-1-1. Approximately 1 μg of l RNA and SMARTer RACE 5' / 3' Kit (Clonte The procedure was performed using (ch company). The cDNA encoding the variable region of the heavy chain gene D13 was processed using PC As a primer for amplification in R, UPM (Universal Primer A Mix: (Included in the SMARTer RACE 5' / 3' Kit), and known rats We used primers designed from the arrangement of the constant region of the heavy chain. cDNA encoding the variable region of the heavy chain, amplified by 5'-RACE PCR, is placed in a plasmid. The nucleotide sequence of the cDNA encoding the variable region of the heavy chain was then sequenced. Quens analysis was performed. The variable region of the D13 heavy chain encoded in the determined cDNA nucleotide sequence The no-acid sequence is an amino acid sequence consisting of amino acid residues 20-139 of sequence number 25 in the sequence listing. This corresponds to a column. The amino acid sequences of CDRH1, CDRH2, and CDRH3 of D13 are listed in the sequence list. These are shown in sequence numbers 4 to 6. The amino acid sequences of these CDRs are also shown in Figure 28.

[0171] 3)-2 Nucleotide sequence analysis and amino acid sequence determination of cDNA in the variable region of F44 The procedure was carried out in the same manner as in Example 3)-1. The determined cDNA nucleotide sequence was then converted to The amino acid sequence of the variable region of the light chain of F44 is from 21 to 12 of sequence number 27 in the sequence listing. This corresponds to the amino acid sequence consisting of the 7th amino acid residue. Also, the determined cDNA The amino acid sequence of the variable region of the heavy chain of F44 encoded by the creotide sequence is given by the sequence number in the sequence listing. This corresponds to the amino acid sequence consisting of amino acid residues 20-138 of number 29. C of F44 Amino acid combination of DRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 The columns are shown in sequence numbers 7 through 12 of the sequence listing. The amino acid sequences of these CDRs are also shown in Figure 29. It is shown.

[0172] 3)-3 Nucleotide sequence analysis and amino acid sequence determination of the cDNA of the variable region of F63 The procedure was carried out in the same manner as in Example 3)-1. The determined cDNA nucleotide sequence was then converted to The amino acid sequence of the variable region of the light chain of F63 is from 21 to 13 of sequence number 31 in the sequence listing. This corresponds to the amino acid sequence starting from the 0th amino acid residue. Also, the determined cDNA The amino acid sequence of the variable region of the heavy chain of F63 encoded by the creotide sequence is the same as in SEQ ID NO: 33. This corresponds to an amino acid sequence consisting of amino acid residues 20 to 143. F63 CDRL1 The amino acid sequences of CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 are distributed These are shown in sequence numbers 13 to 18 of the table. The amino acid sequences of these CDRs are also shown in Figure 30. It is.

[0173] Example 4. Preparation of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) 4)-1 Human chimerization and construction of humanized κ-type light chain expression vector pCMA-LK Restriction of plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) A fragment of approximately 5.4kb obtained by digestion with enzymes XbaI and PmeI, and the sequence number. D, which includes the DNA sequence encoding the human light chain signal sequence and the human κ chain constant region shown in 19. NA fragments are extracted using the In-Fusion HD PCR Cloning Kit (Clontech). pcDNA3.3 / LK was produced by binding using (). By removing the neomycin expression unit from pcDNA3.3 / LK, pCMA - LK was constructed.

[0174] 4)-2 Construction of human chimera and humanized λ-type light chain expression vector pCMA-LL pCMA-LK was digested with XbaI and PmeI to obtain the light chain signal sequence and human κ chain constant state. DNA fragment with region removed, and the human light chain signal sequence shown in Sequence ID No. 20 and human DNA fragments containing DNA sequences encoding the λ-chain constant region are processed using In-Fusion HD P The pCMA-LL was formed by binding using a CR cloning kit (Clontech). It was built.

[0175] 4)-3 Human chimeric and humanized IgG4proFALA type heavy chain expression vector pC Construction of MA-G4PFALA The human heavy chain signal sequence and human IgG4PFALA constant region shown in Sequence ID No. 21 Using a DNA fragment containing a DNA sequence encoding an amino acid, the same method as in Example 4)-2 was used. pCMA-G4proFALA was constructed according to the law.

[0176] 4)-4 Construction of the cD13 expression vector 4)-4-1 Construction of an IgG4proFALA type heavy chain expression vector for cD13 Example 3) The cDNA encoding the variable region of the D13 heavy chain obtained in Example 1 was used as a template. Therefore, we will perform PCR using primers designed for infusion cloning. DNA fragments containing cDNA encoding a heavier variable region were amplified using pCMA-G4. In-Fusion HD PC The amplified DNA fragment is inserted using the R cloning kit (Clontech). By doing so, a cD13 heavy chain expression vector was constructed. Nucleotids encoding the cD13 heavy chain The sequence is shown as sequence number 24 in the sequence listing. The nucleotide sequence consists of a signal sequence and a nucleotide sequence consisting of nucleotides 58 to 417. The variable region is the nucleotide sequence consisting of nucleotides 418 to 1398, which is the constant region. Each region is coded. The amino acid sequence of the cD13 heavy chain is shown in sequence number 25 of the sequence listing. As shown, the amino acid sequence consisting of amino acid residues 1-19 is the signal sequence, and 20-1 The amino acid sequence consisting of the 39th amino acid residue has a variable region, with amino acids 140-466. The amino acid sequence consisting of no acid residues corresponds to the constant region. Sequence ID No. 24 and sequence The sequence number 25 is also shown in Figure 18.

[0177] 4)-4-2 Construction of a cD13 light chain expression vector Example 3) The cDNA encoding the variable region of the D13 light chain obtained in Example 1 was used as a template. Therefore, we will perform PCR using primers designed for infusion cloning. DNA fragments containing cDNA encoding the variable region of the light chain were amplified. pCMA-LK In-Fusion HD PCR clonin was added to the site where the restriction enzyme BsiWI was used to cleave the molecule. By inserting amplified DNA fragments using a kit (Clontech), c A D13 light chain expression vector was constructed. The nucleotide sequence encoding the cD13 light chain was arranged As shown in sequence number 22 of the table, the nucleotide sequence consisting of nucleotides 1 to 60 is The nucleotide sequence consisting of nucleotides 61-378 is a variable region. The nucleotide sequence consisting of nucleotides 379 to 699 constitutes the constant region, It codes for the cD13 light chain. The amino acid sequence of the cD13 light chain is shown in sequence number 23 of the sequence listing. 1-2 The amino acid sequence starting from the 0th amino acid residue is the signal sequence, and the amino acids from 21 to 126th amino acids The amino acid sequence consisting of no acid residues has a variable region, starting from amino acid residues 127 to 233. The amino acid sequences correspond to the constant region, respectively. Sequences of SEQ ID NOs. 22 and 23. This is also shown in Figure 17.

[0178] 4)-5 Construction of the cF44 expression vector 4)-5-1 Construction of an IgG4proFALA-type heavy chain expression vector for cF44 Example 3) The cDNA encoding the variable region of the F44 heavy chain obtained in Example 2 was used as a template. As a result, a cF44 heavy chain expression vector was constructed using the same method as in Example 4)-4-1. The nucleotide sequence encoding the 44-chain is shown in sequence number 28 of the sequence listing. Nucleotides 1-57 The nucleotide sequence consisting of these nucleotides is the signal sequence, and the nucleotides from position 58 to 414 The nucleotide sequence consisting of ocides has a variable region, from nucleotides 415 to 1395. The following nucleotide sequences each encode a constant region. cF44 heavy chain amino The acid sequence is shown in sequence number 29 of the sequence listing. It consists of amino acids from amino acid residues 1 to 19. The sequence is the signal sequence, and the amino acid sequence consisting of amino acid residues 20-138 is the variable region. In the region, the amino acid sequence consisting of amino acid residues 139 to 465 is in the constant region, They correspond. The sequences of sequence numbers 28 and 29 are also shown in Figure 20.

[0179] 4)-5-2 Construction of a cF44 light chain expression vector Example 3) The cDNA encoding the variable region of the F44 light chain obtained in Example 2 was used as a template. As a result, a cF44 light chain expression vector was constructed using the same method as in Example 4)-4-2. The nucleotide sequence encoding the 44 light chain is shown in sequence number 26 of the sequence listing. Nucleotides 1-60 The nucleotide sequence consisting of these nucleotides is the signal sequence, and the nucleotides from position 61 to 381 The nucleotide sequence consisting of ocids has a variable region starting from nucleotides 382 to 702. The nucleotide sequences shown here code for the constant region, respectively. The amino acids of the cF44 light chain. The sequence is shown as sequence number 27 in the sequence listing. The amino acid sequence consists of amino acid residues 1 to 20. The column represents the signal sequence, and the amino acid sequence consisting of amino acid residues 21-127 represents the variable region. In the amino acid sequence consisting of amino acid residues 128 to 234, each is in a phase within the constant region. This is correct. The sequences of sequence numbers 26 and 27 are also shown in Figure 19.

[0180] 4)-6 Construction of the cF63 expression vector 4)-6-1 Construction of an IgG4proFALA-type heavy chain expression vector for cF63 Example 3) The cDNA encoding the variable region of the F63 heavy chain obtained in Example 3 was used as a template. As a result, a cF63 heavy chain expression vector was constructed using the same method as in Example 4)-4-1. The nucleotide sequence encoding the 63-chain is shown in Sequence ID No. 32 of the sequence listing. Nucleotides 1-57 The nucleotide sequence consisting of these nucleotides is the signal sequence, and the nucleotides from position 58 to 429 The nucleotide sequence consisting of ocides has a variable region, from nucleotides 430 to 1410. The following nucleotide sequences each encode a constant region. cF63 heavy chain amino The acid sequence is shown in sequence number 33 of the sequence listing. It consists of amino acids from amino acid residues 1 to 19. The sequence is the signal sequence, and the amino acid sequence consisting of amino acid residues 20-143 is the variable region. In the region, the amino acid sequence consisting of amino acid residues 144 to 470 is in the constant region, They correspond. The sequences of sequence numbers 32 and 33 are also shown in Figure 22.

[0181] 4)-6-2 Construction of a cF63 light chain expression vector Example 3) The cDNA encoding the variable region of the F63 light chain obtained in Example 3 was used as a template. Therefore, we will perform PCR using primers designed for infusion cloning. DNA fragments containing cDNA encoding the variable region of the light chain were amplified. pCMA-LL In-Fusion HD PCR was performed on the sites where the restriction enzymes BsiWI and HpaI were cleaved. Using a cloning kit (Clontech), the amplified DNA fragment is inserted. A cF63 light chain expression vector was constructed using the following method. Nucleotides encoding the cF63 light chain The sequence is shown as sequence number 30 in the sequence listing. Nucleotides consisting of nucleotides 1 to 60 The do sequence is the signal sequence, and the nucleotide sequence consisting of nucleotides 61-390 is The variable region is the nucleotide sequence consisting of nucleotides 391 to 708, while the constant region is the nucleotide sequence. They each encode. The amino acid sequence of the cF63 light chain is shown in sequence number 31 of the sequence listing. The amino acid sequence consisting of amino acid residues 1-20 is the signal sequence, and 21-130 The amino acid sequence consisting of the nth amino acid residue has a variable region, with amino acids 131-236. The amino acid sequences consisting of residues correspond to the constant region. (Sequence ID 30 and Sequence ID 30) The sequence of 31 is also shown in Figure 21.

[0182] 4)-7 Preparation of cD13, cF44, and cF63 4)-7-1 Production of cD13, cF44, and cF63 FreeStyle 293F cells (Invitrogen) are prepared according to the manual. Subculturing and culture were performed. 1.2 × 10⁻⁶ during the logarithmic growth phase. 9 293 FreeStyle pages F cells (Invitrogen) 3L Fernbach Erlenmeyer Sow seeds in a Flask (CORNING), then use FreeStyle 293 express. Dilute with sion medium (Invitrogen) to 2.0 x 10 6 cells / m Prepared to L. In 40 mL of Opti-Pro SFM medium (Invitrogen) 0.24 mg heavy chain expression vector, 0.36 mg light chain expression vector, and 1.8 mg Po Adding lyethyleneimine (Polyscience #24765) makes it milder. After being gently stirred and left to stand for another 5 minutes, it was added to FreeStyle 293F cells. After culturing in a 37°C, 8% CO2 incubator for 4 hours with shaking at 90 rpm, the culture was transferred to 600m³. EX-CELL VPRO medium (SAFC Biosciences), 18 mL GlutaMAX I (GIBCO) and 30 mL of Yeastolate Ul Add trafiltrate (GIBCO) and incubate at 37°C in an 8% CO2 incubator. - Cultured for 7 days with shaking at 90 rpm, the culture supernatant obtained was disposed of as C Filtered with an apsule filter (Advantec #CCS-045-E1H) Ta.

[0183] 4)-7-2 Purification of cD13, cF44, and cF63 The antibody obtained from the culture supernatant in Example 4)-7-1 was analyzed for rProtein A affinity. - Purified in a single step of chromatography. MabS obtained by equilibrating the culture supernatant with PBS. Column packed with electSuRe (GE Healthcare Bioscie After applying the sample to a column (manufactured by nce), the column was washed with PBS at a volume more than twice the column's volume. Next, the antibody-containing fraction was collected by eluting with a 2M arginine hydrochloride solution (pH 4.0). The fraction was then dialyzed (Thermo Scientific, Slide-A-Lyz). Buffer replacement to PBS(-) using a Dialysis Cassette I went. Centrifugal UF Filter Device VIVASPI The antibody was concentrated using N20 (fractionated molecular weight UF10K, Sartorius), and the IgG concentration was adjusted. Prepared to a concentration of 10 mg / mL or higher. Finally, Minisart-Plus filter (S The sample was filtered using Artorius and purified.

[0184] Example 5. In vitro of human chimeric anti-SIRPA antibodies (cD13, cF44, cF63) iTR evaluation 5)-1 Evaluation of binding compatibility to human SIRPA 5)-1-1 Evaluation of binding affinity to human SIRPA (Cell-ELISA) 293α cells [as described in Example 1)-6] were placed in 5x10 units of DMEM medium containing 10% FBS. 5 It was prepared to a cell / mL ratio. In contrast, Lipofectamine LTX ( Using pFLAG V5-DEST-SIRPA_V1 (manufactured by Invitrogen Inc.), , V2, or pFLAG V5-DEST is introduced, and a 96-well plate ( Dispense 100 μL each into Corning (FBS) and then into 10% FBS-containing DMEM medium. The cells were cultured overnight under conditions of 37°C and 5% CO2. The resulting transduced cells were kept in an adherent state. It was used in Cell-ELISA. After removing the culture supernatant, pFLAG V5-DEST- For each of the SIRPA_V1, V2, or pFLAG V5-DEST-transformed cells Furthermore, the cD13(IgG2, IgG4pf) and cF44( prepared in Examples 3 and 4) IgG1, IgG2, IgG4p, IgG4pf), cF63(IgG2, IgG4pf) Add the antibody at a final concentration of 0 to 10000 ng / mL, 50 μL / well, and incubate at 4°C for 1 hour. The mixture was allowed to stand. Additionally, each well for detecting SIRPA construct expression contained a final concentration of 100. Anti-FLAG M2 antibody (S) diluted in PBS containing 5% FBS to a concentration of 00 ng / mL 50 μL / well of IGMA (manufactured by IGMA) was added and allowed to stand at 4°C for 1 hour. 5 cells in the well After washing once with PBS containing % FBS, P was diluted 1000 times with PBS containing 5% FBS. eroxidase AffiniPure F(ab')2Fragment Goat Anti-Human IgG, Fcγ Fragment Specific(J (Added from ackson ImmunoResearch) and allowed to stand at 4°C for 1 hour. After washing the cells in the wells five times with PBS containing 5% FBS, the cells were treated with OPD chromogenic solution (OPD dissolution). Solution (0.05M trisodium citrate, 0.1M disodium hydrogen phosphate, dodecahydrate) (pH 4.5) o-phenylenediamine dihydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) and H2O2 are added separately Add 100 μL / well of a solution (dissolved to 0.4 mg / mL, 0.6% (v / v)). The color reaction was carried out while stirring occasionally, and 100 μL / well of 1 M HCl was added. After stopping the color reaction, use a plate reader (ARVO: PerkinElmer) Absorbance at 490 nm was measured. Human chimeric anti-human SIRP for each construct. The binding affinity of antibody A was standardized based on the expression of the FLAG tag. As shown in Figure 6, the cD13, cF44, and cF63 antibodies are SIRPA_V1 and SIRPA _V2 bound to both (Figures 6A, B), and the binding affinity was nearly equivalent between each isotype (Figure 6A, B). 6C, D).

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

[0186] [Table 4]

[0187] 5)-2 Evaluation of species cross-reactivity in monkey SIRPA 5x10 293α cells in DMEM medium containing 10% FBS 5 Adjust to achieve a cell / mL ratio. It was manufactured. In contrast, Lipofectamine LTX (manufactured by Invitrogen) ) Using pFLAG V5-DEST-SALSIRPA, or pFLAG V5 - DEST is introduced, and 100 μL is placed in a 96-well plate (Corning). After dispensing into individual portions, the mixture was placed in DMEM medium containing 10% FBS at 37°C under conditions of 5% CO2. The cells were cultured overnight. The resulting transduced cells were used in Cell-ELISA while still attached to the cells. After removing the culture supernatant, the binding activity to monkey SIRPA was measured in the same manner as for human SIRPA. The binding activity was evaluated. As shown in Figure 7, the cD13, cF44, and cF63 antibodies showed binding affinity to monkey SIRPA. did.

[0188] 5)-3 Evaluation of SIRPA-CD47 binding inhibitory activity in humans or monkeys Human SIRPA or monkey SIRPA expression vector prepared in Examples 5)-1 and 5)-2 - After removing the culture supernatant of introduced 293α cells, pcDNA3.2 V5-DEST-SIRP A_V1, pcDNA3.2 V5-DEST-Sal SIRPA or pcDNA3.2 For each of the V5-DEST-introduced 293α cells, the final concentration was 0 to 10,000 ng / m³. cD13 (IgG2, IgG4pf) diluted with 5% FBS-containing PBS to L, cF44 (four constant regions of IgG1, IgG2, IgG4p, and IgG4pf), and cF 63 (IgG2, IgG4pf) was added at 50 μL / well. Immediately afterward, the final concentration was 1000. Peroxidase la prepared in PBS containing 5% FBS to a concentration of 0 ng / mL 50 μL / well of beled CD47-Fc was added and allowed to stand at 4°C for 1 hour. SIRPA-CD47 binding inhibitory activity was evaluated using the same method as in 1)-6-3. As shown in Figure 8, cF44, cF63, and cD13 are human and monkey SIRPA-CD4 It exhibits 7-binding inhibitory activity (Figure 8A(i), (ii), (iii)), and its activity is approximately the same across each isotype. They were equivalent (Figure 8B(i), (ii), (iii)).

[0189] 5)-4 ADCP activity of human chimeric anti-human SIRPA antibodies against cancer cell lines 5)-4-1 Preparation of target cells CD47-positive human Burkitt's lymphoma cell line, Raji cells, were collected. After washing twice with PBS, the number of viable cells was measured using a trypan blue dye exclusion test. The following is 2 Target cells were prepared using the same method as in -6-1.

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

[0191] 5)-4-3 Preparation of effector cells Effector cells were prepared using the same method 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 were placed in 96 wells of an ultra-low adhesion surface. It was added to a U-bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.). The final concentration was 0 to 10 RPMI1640 medium containing 10% FBS (Life Te) to achieve a concentration of 000 ng / mL. cD13, cF44, cF63, Hu5F9G4, T (diluted by Chemology Corporation) TI-621 or various control Human IgG were added at 50 μL / well. The formulation group used RPMI1640 medium containing 10% FBS (Life Technology Corporation). Add 50 μL / well of (manufactured by) and in the combination group, adjust to a final concentration of 400 ng / mL using 10% Diluted with FBS-containing RPMI1640 medium (Life Technology). Rituximab (manufactured by Zenyaku Kogyo Co., Ltd.) was added at a rate of 50 μL / well. The following is 2-6-4 and ADCP activity was evaluated using a similar method. As shown in Figure 9, cD13, cF44, and cF63 are CD47-positive human Burkitt In the 's lymphoma cell line Raji cells, the drug alone did not show ADCP activity (Figure) 9A) When used in combination with Rituximab, Hu5F9G4 and TTI-621 are the same It showed a certain degree of ADCP activity (Figure 9B).

[0193] 5)-5 Toxicity of human chimeric anti-human SIRPA antibodies against PBMCs and macrophages Sexual evaluation 5)-5-1 Preparation of PBMCs as target cells and macrophages 2)-6-2 (PBMC) and 2)-6-3 (macrophage) by the same method Target cells were prepared. Each harvested cell was fluorescently labeled in the same manner as in 2)-6-1, and the target cells were identified. It was used as a cell.

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

[0195] 5)-5-3 Evaluation of ADCP activity 50 μL / well of PBMCs or macrophages prepared by the method of Example 5)-4-2 It was added to an ultra-low adhesion surface 96-well U-bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.). RPMI1 containing 10% FBS to achieve a final concentration of 0.64 to 10000 ng / mL cD13 (IgG4pf) diluted with 640 medium (Life Technology). ), cF44 (four constant regions of IgG1, IgG2, IgG4p, and IgG4pf), cF 63(IgG4pf), Hu5F9G4, TTI-621, or various control Humas 50 μL / well of IgG was added. RPMI1640 medium containing 10% FBS (L 50 μL / well was added (manufactured by ife Technology). The following is 2-6-4 and ADCP activity was evaluated using a similar method. Furthermore, the ADCP activity against macrophages was... The ratio is the number of macrophages when each antibody is added compared to the number of macrophages when the control antibody is added. It was calculated by dividing by a number. As shown in Figure 10, the ADCP activity of cD13, cF44, and cF63 toward PBMCs is It was almost equivalent to the control IgG (Figure 10A). Also, cF44, which has a different constant region When comparing antibodies, ADCP was found to be concentration-dependent for IgG1 and IgG4p types. While ADCP activity was observed in IgG2 and IgG4pf types, ADCP activity was not shown (Figure). 10B). On the other hand, regarding the ratio of macrophages 16 hours after antibody addition, cF with different constant regions Comparing the 44 antibodies, the IgG4pf type showed the lowest rate of macrophage reduction. The antibody addition has shown to have the lowest toxicity to SIRPA-positive cells. (Figure 10C).

[0196] Example 6: Design of a humanized anti-SIRPA antibody 6)-1 Molecular modeling of the variable region of the chimeric antibody cD13 Molecular modeling of the variable region of cD13 is performed using homology modeling, a known method [M [ethods in Enzymology, 203, 121-153 (1991)] It was used. Prote has high sequence identity for the variable regions of the heavy and light chains of cD13. in Data Bank[Nuc.Acid Res.35,D301‐D303(2 Using the structure registered in (007) (PDB ID: 3CSY) as a template, a commercially available protein We used the BioLuminate (Schrodinger) program for analyzing the three-dimensional structure of materials. He went.

[0197] 6)-2 Design of humanized amino acid sequences cD13 is CDR grafting [Proc.Natl.Acad.Sci.USA] Humanization was achieved by 86, 10029-10033 (1989). IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION Human gem registered in SYSTEM (http: / / www.imgt.org) rmline sequences IGHV3-30*13 and IGHJ3*01, and IGKV1-6* 01 and IGKJ2*01, and Kabat et al. [Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service National Institute [As defined in *Lites of Health*, Bethesda, MD. (1991)] The consensus sequence of human kappa chain subgroup 4 is in the framework region of cD13. It was selected as an acceptor due to its high degree of identity with the domain. The donor residue to be transplanted is Queen et al. [Proc.Natl.Aca [d.Sci.USA 86,10029-10033(1989)] The selection was made by analyzing a three-dimensional model based on criteria and other factors.

[0198] 6)-3 Humanization of the cD13 heavy chain The two designed heavy chains were named hH1 and hH2. The full-length amino acid sequence of the hH1 heavy chain. This is listed as sequence number 41 in the sequence listing. The nucleus encoding the amino acid sequence of sequence number 41. The Otid sequence is listed as sequence number 40 in the sequence listing. The full-length heavy chain amino acid sequence of hH2 is listed. The nucleotide encoding the amino acid sequence of SEQ ID NO: 43 is listed in the table. The sequence is listed as sequence number 42 in the sequence listing. Sequence numbers 41 and 43 are numbered 1 to 19. The amino acid sequence consisting of amino acid residues in the eye is the signal sequence, and amino acids 20-139 The amino acid sequence consisting of residues has a variable region, with the amino acid residues from position 140 to 466 being a The mino acid sequences correspond to the constant region, respectively. Also, in sequence numbers 40 and 42, 1 The nucleotide sequence consisting of nucleotides ~57 is the signal sequence, and nucleotides 58~417 The nucleotide sequence consisting of the nucleotides of the eye has a variable region, from nucleotide 418 to 1398. The nucleotide sequences consisting of rheosides each encode a constant region. Sequence ID 4 The sequences of 0 and sequence number 41 are also shown in Figure 26, and the sequences of sequence numbers 42 and 43 are shown in Figure 27. This is also shown. cD13_H is the heavy chain of the human chimeric anti-SIRPA antibody cD13, and the humanized antibody heavy chain h Figure 11 shows a comparison of the amino acid sequences of H1 and hH2. The "·" indicates the same amino acid residue as c013_H, and the amino acid residue is listed in the section. The 'location' indicates the substituted amino acid residue.

[0199] 6)-4 Humanization of the cD13 light chain The three light chains designed were named hL2, hL3, and hL4. The total length of the hL2 light chain is The amino acid sequence is listed in sequence number 35 of the sequence listing. The amino acid sequence of sequence number 34 is coded. The nucleotide sequence is described in sequence number 34 of the sequence listing. The full-length light chain amino acids of hL3. The sequence is listed as sequence number 37 in the sequence listing. The amino acid sequence of sequence number 37 is encoded as follows: The creotide sequence is described in Sequence ID No. 36. The full-length light chain amino acid sequence of hL4 is shown in the sequence listing. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 39 is described below. This is described in Sequence ID No. 38. In Sequence ID Nos. 35, 37 and 39, the 1st to 20th ami The amino acid sequence consisting of no acid residues is the signal sequence, starting from amino acid residues 21-127. The resulting amino acid sequence has a variable region containing amino acid residues from positions 128 to 234. Each column corresponds to a steady-state region. Also, in sequence numbers 34, 36, and 38, 1~ The nucleotide sequence starting from the 60th nucleotide is the signal sequence, and from the 61st to the 381st nucleotide The nucleotide sequence consisting of these nucleotides has a variable region, and the nucleos from 382 to 702nd nucleotide The nucleotide sequence consisting of nucleotides codes for the constant region. Sequence ID 34 and The sequence of sequence number 35 is also shown in Figure 23, and the sequences of sequence numbers 36 and 37 are also shown in Figure 24. The sequences of sequence numbers 38 and 39 are also shown in Figure 25, respectively. cD13_L is the light chain of the human chimeric anti-SIRPA antibody cD13, and the humanized antibody light chain h Figure 12 shows a comparison of the amino acid sequences of L2, hL3, and hL4. In the L4 sequence, "·" indicates the same amino acid residue as cD13_L, and amino acid residue The indicated sections show the substituted amino acid residues.

[0200] 6)-5 Design of humanized antibodies using heavy and light chain combinations An antibody consisting of hH1 and hL3 is called "H1L3 antibody" or "H1L3". An antibody consisting of hH2 and hL4 is called "H1L4 antibody" or "H1L4". An antibody consisting of two components is called an "H2L2 antibody" or "H2L2". The antibody is called "H2L3 antibody" or "H2L3".

[0201] Example 7. Preparation of humanized anti-SIRPA antibody 7)-1 Construction of heavy chain expression vectors for humanized antibodies 7)-1-1 Construction of hH1 expression vector Nucleotide numbers 36 to 4 of the nucleotide sequence of hH1 shown in sequence number 40 of the sequence listing. The DNA fragment shown in 34 was synthesized (GENEART). In-Fusion HD Using a PCR cloning kit (Clontech), pCMA-G4proF By inserting a synthesized DNA fragment into the site where ALA was cut with the restriction enzyme BlbI, h 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 sequence number 42 of the sequence listing. The DNA fragment shown in 34 was synthesized (GENEART). Same as Example 7)-1-1. The hH2 expression vector was constructed using the following method.

[0203] 7)-2 Construction of light chain expression vectors for humanized antibodies 7)-2-1 Construction of hL2 expression vector Nucleotide numbers 37 to 4 of the hL2 nucleotide sequence shown in sequence number 34 of the sequence listing. The DNA fragment shown in 02 was synthesized (GENEART). In-Fusion HD Using a PCR cloning kit (Clontech), pCMA-LK was restricted in yeast. By inserting a synthesized DNA fragment into the site cut by plain BsiWI, the hL2 expression vector is created. I built a tar.

[0204] 7)-2-2 Construction of an hL3 expression vector Nucleotide numbers 37 to 4 of the hL3 nucleotide sequence shown in sequence number 36 of the sequence listing. The DNA fragment shown in 02 was synthesized (GENEART). (Same as Example 7)-2-1 The hL3 expression vector was constructed using the following method.

[0205] 7)-2-3 Construction of hL4 expression vector Nucleotide numbers 37 to 4 of the hL4 nucleotide sequence shown in sequence number 38 of the sequence listing. The DNA fragment shown in 02 was synthesized (GENEART). (Same as Example 7)-2-1 The hL4 expression vector was constructed using the following method.

[0206] 7)-3 Preparation of humanized antibodies 7)-3-1 Production of humanized antibodies The same method as in Example 4)-7-1 was used for production. The combination of H chain and L chain shown in Example 6)-5 By combining H chain expression vectors and L chain expression vectors that correspond to specific pairings, various humanized antibodies can be produced. I acquired a 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 using a two-step process involving tography and ceramic hydroxyapatite. A column packed with MabSelectSuRe equilibrated in PBS (GE Health After applying to hcare Bioscience, more than twice the column volume The column was washed with PBS. Next, the antibody was dissolved in 2 M arginine hydrochloride solution (pH 4.0). The antibody-containing fraction was dialysis (Thermo Scientific, Slid). Buffer to PBS using eA-Lyzer Dialysis Cassette - Substitution was performed, and the solution was then prepared using a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0. After diluting 5 times, use 5mM NaPi / 50mM MES / 30mM NaCl / pH7 Ceramic hydroxyapatite column equilibrated with 0 buffer (Nippon BioLa) Bio-Scale CHT Type-1 Hydroxyapatite C The sample was applied to olumn. Linear concentration gradient elution with sodium chloride was performed, and the antibody was detected. The fractions containing [the specified substance] were collected. These fractions were then subjected to dialysis (Thermo Scientific, S HBSor (via lide-A-Lyzer Dialysis Cassette) The buffer was replaced with 25 mM histidine / 5% sorbitol (pH 6.0). . Centrifugal UF Filter Device VIVASPIN20 The antibody was concentrated using (Fractional molecular weight UF10K, Sartorius), and the IgG concentration was increased to 50%. Prepared in mg / mL. Finally, Minisart-Plus filter (Sarto The sample was filtered using a RIUS filter to obtain a purified sample.

[0208] Example 8. Humanized anti-SIRPA antibodies (hD13_H1L3, hD13_H1L4h, hD In vitro evaluation of 13_H2L2 and 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 [as described in Example 1-6] were placed in 10% FBS-containing DMEM medium in 5x10 5 It was prepared to a cell / mL ratio. In contrast, Lipofectamine LTX ( Using pFLAG V5-DEST-SIRPA_V1 (manufactured by Invitrogen Inc.), ‐V10, pFLAG V5‐DEST‐Monkey SIRPA, pFLAG V5‐DEST - Introduce mouse SIRPA or pFLAG V5-DEST and 96-well p Dispense 100 μL into late (Corning) bottles, then add 10% FBS-containing D The cells were cultured overnight in MEM medium at 37°C under 5% CO2 conditions. The resulting transduced cells were then adhered to the cells. The cells were used in Cell-ELISA as is. After removing the culture supernatant, various SIRPA genes were extracted. For each of the introduced cells, hD13_H1L3 prepared in Example 6 and Example 7, h D13_H1L4h, hD13_H2L2, hD13_H2L3, or cD13 and con Add 50 μL / well of trol antibody at a final concentration of 0 to 10000 ng / mL and incubate at 4°C for 1 The samples were allowed to stand for a set time. The binding affinity to human SIRPA was then evaluated using the same method as in Example 5-1. Ta. As shown in Figures 13A-C, hD13_H1L3, hD13_H1L4h, hD13_H 2L2, hD13_H2L3 antibodies are SIRPA variants (V1-V10), monkey SIR It showed binding affinity to PA equivalent to or better than that of the cD13 antibody. On the other hand, as shown in Figures 14A and B Neither the humanized antibody nor the human chimeric antibody showed binding affinity to mouse SIRPA. Ta.

[0209] 8)-1-2 Evaluation of the binding affinity of humanized antibodies to SIRPA hD13_H1L3, hD13_H1L4, hD13_H2L2 and the materials prepared in Example 7 The human SIRPA_V1_IgV and monkey S prepared in Example 1 of hD13_H2L3 Dissociation constant measurement for IRPA_ECD is performed using Biacore T200 (GE Heal Human Antibody (manufactured by thcare Bioscience) Capture Kit (manufactured by GE Healthcare Bioscience) Humanized antibodies were used to immobilize Anti-Human IgG(Fc) antibody. The procedure was performed using a capture method, in which the ligand is captured and the antigen is measured as an analyte. HBS-EP+ (GE Healthcare Biosc) as a running buffer (Manufactured by ience), CM5 (GE Healthcare Bio) as the sensor chip. A device manufactured by Science Inc. was used. 1 μg / mL of humanized antibody was applied to the chip at a rate of 10 μL / min. After adding for 60 seconds, a diluted series of human SIRPA protein solutions (0.5-8 μg) is used as the antigen. ( / mL) or a diluted series solution of monkey SIRPA protein (1-16 μg / mL) at a flow rate of 30 μg / mL The solution was added at a rate of L / min for 120 seconds, and the dissociation phase was monitored for 600 seconds thereafter. 3M magnesium chloride(GE Healthcare Bi (OScience Co., Ltd.) was added at a flow rate of 20 μL / min for 30 seconds. For data analysis, 1: Using a single-bond model, the bond rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD;KD) are used. The formula (=kd / ka) was calculated. The results are shown in Table 5.

[0210] [Table 5]

[0211] 8)-2 Human or monkey SIRPA-CD47 binding inhibitory activity of humanized anti-SIRPA antibodies evaluation Example 8) Human SIRPA prepared in 1, or monkey SIRPA expression vector introduced 29 After removing the culture supernatant of 3α cells, pcDNA3.2 V5-DEST-SIRPA_V1, V2, pcDNA3.2 V5-DEST-Sal SIRPA or pcDNA3.2 V5 - For each of the DEST-introduced 293α cells, the final concentration was between 0 and 10000 ng / mL. hD13_H1L3, hD13_H1L4h diluted in PBS containing 5% FBS. Add 50 μL / well of hD13_H2L2 and hD13_H2L3, and immediately afterwards 5 Peroxidase labeled, prepared at 1 μg / mL in PBS containing % FBS. CD47-Fc was added and allowed to stand at 4°C for 1 hour. The following procedure was carried out in the same manner as in 1)-6-3. SIRPA-CD47 binding inhibitory activity was evaluated. As shown in Figure 15, hD13_H1L3, hD13_H1L4h, hD13_H2L2 The hD13_H2L3 antibodies are SIRPA_V1 (Figure 15A) and SIRPA_V2 (Figure 15A). 5B) It showed binding inhibitory activity equivalent to or greater than that of the cD13 antibody against monkey SIRPA (Figure 15C). did.

[0212] 8)-3 ADCP activity of humanized anti-human SIRPA antibodies 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 number of viable cells was tested using trypan blue exclusion. Measured as follows: 4 x 10 7 After separating the cells and centrifuging them, CellVue Claret Far Red Fluorescent Cell Linker Kit (manufactured by Sigma) Cells were suspended in 2 mL of the included Dilluent C. 1 mM Cell was used as the labeling solution. After diluting Vue Claret Dye to 10 μM with Dilluent C, immediately, Mix the cell suspension with an equal volume of CellVue Claret Dye solution and let stand at room temperature for 15 minutes. Placed in 25 mL of RPMI1640 medium containing 10% FBS (Life Techno). After adding (manufactured by Logy Inc.) and washing twice, 2 x 10 6 Resuspend the cells / mL These cells were used as target cells. The target cells were prepared using the same method as in 2)-6-1.

[0213] 8)-3-2 Preparation of PBMC cells 2) PBMC cells were prepared using the same method as in 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 1× 10 6 The cells were resuspended in PBS to a concentration of cells / mL. 1 μL / 10 was used as the labeling solution. 6 cell Add CFSE solution (ThermoFisher) at a concentration of / mL and let stand at room temperature for 10 minutes. 20 ml of RPMI1640 medium containing 10% FBS (Life Technolo After adding (manufactured by GY Co.) and washing twice, 1 x 10 6 Resuspend the cells / mL in the F They were used as dendritic cells.

[0215] 8)-3-4 Evaluation of ADCP activity 50 μL / well of target cells prepared by the method of Example 8)-3-1 were placed in 96 wells of an ultra-low adhesion surface. It was added to a U-bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.). The final concentration was 0 to 100. RPMI1640 medium containing 10% FBS (Life Te) to achieve a concentration of 00 ng / mL diluted hD13_H1L3, hD13_H1L4h, hD1 (manufactured by Chemology Inc.) 3_H2L2, hD13_H2L3, or cD13 antibody, Hu5F9G4, TTI-62 1 or various control Human IgGs were added at 50 μL / well. In the monotherapy group, 1 50 RPMI1640 medium (Life Technology) containing 0% FBS μL / well was added, and in the combination group, 10% FBS was added to achieve a final concentration of 400 ng / mL. Ritux diluted with RPMI1640 medium (Life Technology Corporation) 50 μL / well of imab (manufactured by Zenyaku Kogyo Co., Ltd.) was added. The solution prepared in Example 8-3-3 was used. 1 x 10 6 After adding 50 μL / well of effector cells at 37°C, 5% The mixture was left to stand for 16 hours under CO2 conditions. After centrifugation at 4°C and 1200 rpm for 5 minutes, and removal of the supernatant, Washed with 200 μL / well of PBS containing 5% FBS. Washed with 100 μL / well of 1×BD. Stabilizing Fixative (manufactured by Becton Dickison) The solution was suspended and left to stand overnight at 4°C. The following day, flow cytometry (FACS CantoII) was performed. Measurements were taken using a Becton (manufactured by Dickison). Data analysis was performed using Flowjo. (TreeStar Corporation) was used. Exploration was performed using FSC (forward scatter) / SSC (side scatter). Afterward, the number of cells that were APC-positive (A) and those that were positive for both APC and FITC (B) was calculated. Cells that are positive for both APC and FITC (B) are phagocytosed by target cells by macrophages. It was assumed that this was the case. The rate of cell phagocytosis 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 group supplemented with hD13_H2L3 or cD13 antibody was CD47-positive human Burkitt's In lymphoma cell line Raji and Ramos cells, the single agent stimulated ADCP activity. This was not shown (Figure 16A, C), and when used in combination with Rituximab, the concentration of the added antibody was dependent. It showed typical ADCP activity (Figure 16B, D). Furthermore, the humanized antibody clone was human chimeric. It showed ADCP activity equivalent to or greater than that of the antibody clone.

[0216] Example 9. In vitro evaluation of various anti-SIRPA antibodies 9)-1 Evaluation of the binding affinity of various anti-SIRPA antibodies to SIRPA The hD13_H1L3 antibody prepared in Example 7, and OSE-172 (International Publication No. WO1 (Prepared in reference to issue 7 / 178653), KWAR23 (International Public Post No. WO18 / 02660) (Prepared according to No. 0), or ADU-1805 (International Public Relations No. WO18 / 190719) Human SIRPA_V1_IgV and human SIRP prepared in Example 1 (as referenced), The dissociation constant for A_V2_IgV was measured. Note that the heavy chain amino acids of OSE-172 were also measured. The sequence is shown in sequence number 81 of the sequence listing, and the amino acid sequence of the light chain of OSE-172 is shown in sequence number 82. The amino acid sequence of the heavy chain of KWAR23 is shown in SEQ ID NO: 83, and the amino acid sequence of the light chain of KWAR23 is shown in SEQ ID NO: 83. The sequence is in SEQ ID NO: 84, and the amino acid sequence of the heavy chain of ADU-1805 is in SEQ ID NO: 85, AD The amino acid sequences of the light chain of U-1805 are shown in SEQ ID NO: 86. Dissociation constant For measurement, use the Biacore T200 (GE Healthcare Bioscie Using a Human Antibody Capture Kit (GE) manufactured by nce Anti-H (manufactured by Healthcare Bioscience) immobilized Each antibody is captured as a ligand in the human IgG(Fc) antibody, and the antigen is... The measurement was performed using a capture method with nalite. HBS was used as the running buffer. -EP+ (manufactured by GE Healthcare Bioscience), sensor chip CM5 (manufactured by GE Healthcare Bioscience) was used as the chip. After adding various antibodies at a rate of 10 μL / min for 30 seconds to a 2 μg / mL sample, human antigens were used. Diluted series solutions of SIRPA protein (0.25-16 nM) were administered at a flow rate of 30 μL / min for 120 seconds. The solution was added intermittently, and the dissociation phase was monitored for 600 seconds. 3M Mag was used as the regeneration solution. nesium chloride(GE Healthcare Bioscience The product (manufactured by the company) was added at a flow rate of 20 μL / min for 30 seconds. A 1:1 binding model was used for data analysis. Furthermore, the binding rate constant ka, the dissociation rate constant kd, and the dissociation constant (KD; KD = kd / ka) are used. The calculation was performed. The results are shown in Table 6.

[0217] [Table 6]

[0218] 9)-2 Evaluation of the human SIRPA-CD47 binding inhibitory activity of various anti-human SIRPA antibodies 293α cells imported into human SIRPA_V1 or V2 expression vector prepared in Example 9)-1 After removing the culture supernatant from the cells, pcDNA3.2 V5-DEST-SIRPA_V1, V2 derivatives were used. Each of the incoming 293α cells was treated with 5% to achieve a final concentration of 0 to 10,000 ng / mL. Various anti-SIRPA antibodies or various control human antibodies diluted in FBS-containing PBS. Add 50 μL / well of IgG and immediately adjust to 1 μg / mL with PBS containing 5% FBS. Add 50 μL / well of the prepared Peroxidase-labeled CD47-Fc. Then, it was left to stand at 4°C for 1 hour. The following is the same method as in 1)-6-3 for SIRPA-CD47 bonding. The 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. On the other hand, As shown in Figure 31B, hD13_H1L3, K WAR23 and ADU-1805 showed binding affinity, but OSE-172 showed binding inhibitory activity. It did not show that. Also, as shown in Figure 31C, hD13_H1L3 was at the lowest concentration. The binding was inhibited.

[0219] 9)-3 Evaluation of the human SIRPB and human SIRPG binding activity of various anti-human SIRPA antibodies price SIRPβ1(signal regulatory protein β1:Ref The amino acid sequence is publicly available under Seq accession number NP_006056. and SIRPγ (signal regulatory protein γ:Ref The amino acid sequence (published as Seq accession number NP_061026) is It is a family molecule of SIRPA. In this invention, "SIRPα" is referred to as "SIRP A, SIRPβ1 is referred to as SIRPB1, and SIRPγ is referred to as SIRPG. In some cases, CHO-K1 cells are placed in Ham's F-12K medium containing 10% FBS. 3x10 5 The solution was prepared to the desired cell / mL concentration and incubated overnight at 37°C under 5% CO2 conditions. In contrast, Lipofectamine LTX (manufactured by Invitrogen) is used. pFLAG V5-DEST-Human SIRPB, pFLAG V5-DEST-Human Implement SIRPG or pFLAG V5-DEST, and add 10% FBS-containing Ha The cells were incubated in m's F-12K medium at 37°C and 5% CO2 for 24 hours. The introduced cells were collected and seeded in a 96-well plate. After removing the culture supernatant, various... Each of the gene-transformed cells was treated with various anti-human SIRPA antibodies or various control H2O2 antibodies. Uman IgG was added at a final concentration of 0 to 10000 ng / mL, 100 μL / well, and 4 The mixture was allowed 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, remove the supernatant and extract PE Mouse anti-Human IgG antibody (Bio Add 50 μL / well of a 1 / 400 diluted solution (manufactured by Legend) and allow to stand at 4°C for 25 minutes. The sample was placed in a cool, dry place. After centrifugation, the supernatant was removed and the sample was washed twice with PBS containing 5% FBS. After centrifugation, remove the supernatant and add 1×BD Stabilizing F to 100 μL / well. The sample was suspended in ixative (Becton Dickison) and flow cytometry was performed. Measurements were taken using FACS CantoII (manufactured by Becton Dickison). Flowjo (TreeStar) was used for data analysis. FSC (Forward Scattered Light) After development using SSC (side-scattered light), the average fluorescence intensity of PE was measured. Secondary antibody only. By standardizing with the average fluorescence intensity of the reacted samples, family differentiation of various antibodies can be achieved. The compatibility with offspring was calculated. As shown in Figures 32A and B, various anti-human SIRPA antibodies are human SIRPB and human S While it showed concentration-dependent binding affinity to IRPG, OSE-172 did not bind to human SIRPG. It did not show any bonding properties.

[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 collected. After washing twice with PBS, the number of viable cells was measured using a trypan blue exclusion test. 1 × 10 6 The cells were resuspended in PBS to a concentration of cells / mL. 1 μL / 10 was used as the labeling solution. 6 cells / m Add Cell Trace Far Red solution (manufactured by ThermoFisher) to L. Add and let stand at room temperature for 10 minutes. 25 mL of RPMI1640 medium containing 10% FBS (L After adding (ife Technology) and washing twice, 2 x 10 6 cells / mL The resuspended cells were used as target cells. The following target cells were targeted using the same method as in 2)-6-1. Cells were prepared.

[0221] 9)-4-2 Preparation of PBMC cells 2) PBMC cells were prepared using the same method as in 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 1× 10 6 The cells were resuspended in PBS to a concentration of cells / mL. 1 μL / 10 was used as the labeling solution. 6 cell Add CFSE solution (ThermoFisher) at a concentration of / mL and let stand at room temperature for 10 minutes. 20 ml of RPMI1640 medium containing 10% FBS (Life Technolo After adding (manufactured by GY Co.) and washing twice, 1 x 10 6 Resuspend the cells / mL in the F They were used as dendritic cells.

[0223] 9)-4-4 Evaluation of ADCP activity 50 μL / well of target cells prepared by the method in Example 8-3-1 were placed in an ultra-low adhesion surface with 96 wells U It was added to a bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.). The final concentration was 0 to 1000. RPMI1640 medium containing 10% FBS (Life Tec) to achieve a concentration of 0 ng / mL Various anti-SIRPA antibodies diluted (manufactured by Hnology), or various control Huma 50 μL / well of IgG was added. The final concentration in the combined group was 1000 ng / mL. RPMI1640 medium containing 10% FBS (manufactured by Life Technology Corporation) 50 μL / well of rituximab (manufactured by Zenyaku Kogyo Co., Ltd.), diluted with ), was added. Example Prepared with 8-3-3, 1 × 10 6 Cells / mL, effector cells in 50 μL / well After addition, the mixture was allowed to stand for 2 to 16 hours under conditions of 37°C and 5% CO2. (4°C, 1200 rpm) After centrifugation for 5 minutes and removal of the supernatant, the samples were washed with 200 μL / well of PBS containing 5% FBS. 0 μL / well 1×BD Stabilizing Fixative (Becton Suspension using Dickison (FACS CantoII), and flow cytometry (FACS CantoII) Measurements were taken using a Becton (manufactured by Dickison). Data analysis was performed using Flowjo. (TreeStar Corporation) was used. Exploration was performed using FSC (forward scatter) / SSC (side scatter). Afterward, the number of cells that were APC-positive (A) and those that were positive for both APC and FITC (B) was calculated. Cells that are positive for both APC and FITC (B) are phagocytosed by target cells by macrophages. It was assumed that this was the case. The rate of cell phagocytosis 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 group treated with ADU-1805 antibody showed CD47-positive human Burkitt's lymphoma. ADCP activity was observed in the α cell line Raji when combined with rituximab. The strength of ADCP-enhancing activity after 2 hours of reaction was observed for 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 presumed to be at the saturation point of phagocytic activity. Concentration dependence and ADCP after 2 hours of reaction. Regarding the activity comparison, hD13_H1L3 showed the highest activity even at the lowest concentration, and ADU-1 805 and KWAR23 were almost equivalent, followed by OSE-172. Based on these results, hD1 3_H1L3 enhanced ADCP activity from the lowest concentration in a short time.

[0224] 9)-4-5 Phagocytic activity of 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 were placed on an ultra-low adhesion surface. It was added to a 96-well U-bottom microplate (manufactured by Sumitomo Bakelite Co., Ltd.). The final concentration was 0 to RPMI1640 medium containing 10% FBS to achieve a concentration of 5000 ng / mL (Life Various anti-SIRPA antibodies diluted (manufactured by Technology Inc.), or various control H2. 50 μL / well of Uman IgG was added. 10% FBS-containing RPMI 1640 cultures. 100 μL / well of soil (manufactured by Life Technology) was added. 37°C, 5 The samples were allowed to stand for 16-20 hours under % CO2 conditions. Centrifuged at 4°C at 1200 rpm for 5 minutes. After removal, the samples were washed with 200 μL / well of PBS containing 5% FBS. (100 μL / well) 1 x BD Stabilizing Fixative (Becton Dickis The solution is suspended using (manufactured by o-on) and flow cytometry (FACS CantoII: Becton) Measurements were taken using a Dickison (manufactured by Dickison). Data analysis was performed using Flowjo (TreeSta). (Manufactured by company r) was used. After being unfolded using FSC (forward scatter) / SSC (side scatter), each The number of FITC-positive cells in the well was calculated (A). F in the well without antibody addition By standardizing with the count of ITC-positive cells (B), the decrease in each sample is calculated using macrophage. This was attributed to phagocytosis between phages. 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 Adding U-1805 antibody causes macrophages, which are effector cells, to... - ADCP activity was observed. The highest rate of decrease was observed in OSE-172, KWAR23, and ADU-18. The order was 05, then hD13_H1L3. The high rate of reduction was due to Sel by anti-SIRPA antibody. This indicates high f-ADCP activity. This phenomenon is due to the administration of each anti-SIRPA antibody. This suggests the possibility of a decrease or depletion of SIRPA-positive cells such as macrophages and dendritic cells. This serves as 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, human SIRPA antibodies To evaluate the antitumor effect, studies in mice expressing human SIRPA are necessary. [Ring et al. PNAS, 2017 (114) 49, E10578‐ E10585]. On the other hand, in order to evaluate the contribution of the immune system to the antitumor effect, immunoassay It is important to use immunocompetent mice, not all mice. [Yanagita e [t al. JCI, 2017 (2) 1, 1-15]. Human SIRPA alone, or This refers to genetically modified mice in which both human SIRPA and human CD47 have been introduced into immunonormal mice. Then, a mouse cancer cell line genetically modified with human CD47 was transplanted, and the tumor volume was 100 mm. 3 At this stage, the mice are divided into groups. These mice are then subjected to various anti-SIRPA antibodies and anti- CD47 antibodies or anti-CD47 biologics such as SIRPA-Fc fusion proteins, or negative. As a control group, PBS, etc., will be administered 1 to 3 times a week for 1 to 3 weeks. Antitumor treatment with concomitant drugs. When considering adding efficacy, chemotherapeutic agents, antibody drugs, and molecular targets may be applied to each of these groups. The medications are administered in combination. The tumor diameter (long diameter / short diameter) in each treatment group is measured every 2-3 days using an electronic cymbal. The tumor volume is calculated by measuring it using a meter, etc. The tumor growth rate is calculated from the tumor volumes of the antibody-administered group and the negative control group. By calculating the reproductive inhibition rate, it is possible to compare and evaluate the in vivo efficacy of each drug. It will come. Furthermore, the tumor volume and tumor growth inhibition rate are shown by the following formulas. Tumor volume (mm 3 ) = (major axis × minor axis × minor axis) / 2 Tumor growth suppression rate (%) = (1 - Tumor volume in each treatment group / Tumor volume in the negative control group) × 100 [Industrial applicability]

[0226] The anti-SIRPα antibody of the present invention is used with other antibody drugs and immunotherapy drugs that have other effector functions. It can be used as an antibody drug for use in combination with other antibody drugs that have block point inhibitory activity. It is possible. [Sequence Listing Free Text]

[0227] SEQ ID NO: 1:D13 CDR-L1 amino acid sequence SEQ ID NO: D13 CDR-L2 amino acid sequence SEQ ID NO: 3:D13 CDR-L3 amino acid sequence SEQ ID NO: 4:D13 CDR-H1 amino acid sequence SEQ ID NO: D13 CDR-H2 amino acid sequence SEQ ID NO: 6: D13 CDR-H3 amino acid sequence Amino acid sequence of SEQ ID NO: F44 CDR-L1 SEQ ID NO: 8:F44 CDR-L2 amino acid sequence SEQ ID NO: 9:F44 CDR-L3 amino acid sequence SEQ ID NO: 10:F44 CDR-H1 amino acid sequence Amino acid sequence of SEQ ID NO: 11:F44 CDR-H2 Amino acid sequence of SEQ ID NO: 12:F44 CDR-H3 SEQ ID NO: 13:F63 CDR-L1 amino acid sequence Amino acid sequence of SEQ ID NO: 14:F63 CDR-L2 Amino acid sequence of SEQ ID NO: 15:F63 CDR-L3 Amino acid sequence of SEQ ID NO: 16:F63 CDR-H1 Amino acid sequence of SEQ ID NO: 17:F63 CDR-H2 Amino acid sequence of SEQ ID NO: 18:F63 CDR-H3 Sequence ID 19: Nucleotide encoding the human light chain signal sequence and the constant region of the human κ light chain. DNA fragments containing a sequence Sequence ID 20: Nucleotide encoding the human light chain signal sequence and the constant region of the human λ light chain. DNA fragments containing a sequence Sequence ID 21: Human heavy chain signal sequence and human IgG4ProFALA heavy chain constant region DNA fragment containing a nucleotide sequence Sequence ID 22: Nucleotide sequence encoding the light chain of human chimeric antibody D13 Sequence ID 23: Amino acid sequence of the light chain of human chimeric antibody D13 Sequence ID 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 Sequence ID 26: Nucleotide sequence encoding the light chain of human chimeric antibody F44 Sequence ID 27: Amino acid sequence of the light chain of human chimeric antibody F44 Sequence ID 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 Sequence ID 30: Nucleotide sequence encoding the light chain of human chimeric antibody F63 SEQ ID NO: 31: Amino acid sequence of the light chain of human chimeric antibody F63 Sequence ID 32: Nucleotide sequence encoding the heavy chain of human chimeric antibody F63 SEQ ID NO: 33: Amino acid sequence of the heavy chain of human chimeric antibody F63 Sequence ID 34: Nucleotide sequence encoding the hL2 light chain of humanized D13 Sequence ID 35: Amino acid sequence of the hL2 light chain of humanized D13 Sequence ID 36: Nucleotide sequence encoding the hL3 light chain of humanized D13 Sequence ID 37: Amino acid sequence of the hL3 light chain of humanized D13 Sequence ID 38: Nucleotide sequence encoding the hL4 light chain of humanized D13 Sequence ID 39: Amino acid sequence of the hL4 light chain of humanized D13 Sequence ID 40: Nucleotide sequence encoding the hH1 heavy chain of humanized D13 Sequence ID 41: Amino acid sequence of the hH1 heavy chain of humanized D13 Sequence ID 42: Nucleotide sequence encoding the hH2 heavy chain of humanized D13 Sequence ID 43: Amino acid sequence of the hH2 double chain of humanized D13 Sequence ID 44: Nucleotide sequence encoding the ECD of human SIRPA variant 1 Sequence ID 45: Amino acid sequence of ECD of human SIRPA variant 1 Sequence ID 46: Nucleotide sequence encoding IgV of human SIRPA variant 1 Sequence ID 47: Amino acid sequence of human SIRPA variant 1 IgV Sequence ID 48: Nucleotide sequence encoding the ECD of human SIRPA variant 2 Sequence ID 49: Amino acid sequence of ECD of human SIRPA variant 2 Sequence ID 50: Nucleotide sequence encoding human SIRPA variant 2 IgV Sequence ID 51: Amino acid sequence of human SIRPA variant 2 IgV Sequence ID 52: Nucleotide sequence encoding the ECD of monkey SIRPA Sequence ID 53: Amino acid sequence of ECD in monkey SIRPA Sequence ID 54: Nucleotide sequence encoding human CD47-Fc Sequence ID 55: Amino acid sequence of human CD47-Fc IgV Sequence ID 56: Amino acid sequence of human SIRPA variant 1 Sequence ID 57: Amino acid sequence of human SIRPA variant 2 Sequence ID 58: Amino acid sequence of monkey SIRPA SEQ ID NO: 59: Amino acid sequence of C57 BL / 6 mouse SIRPA Sequence ID 60: Amino acid sequence of BALB / C mouse SIRPA SEQ ID NO: 61:129 Amino acid sequence of mouse SIRPA Sequence ID 62: Amino acid sequence of NOD mouse SIRPA Sequence ID 63: Amino acid sequence of human SIRPA variant 3 Sequence ID 64: Amino acid sequence of human SIRPA variant 4 Sequence ID 65: Amino acid sequence of human SIRPA variant 5 Sequence ID 66: Amino acid sequence of human SIRPA variant 6 Sequence ID 67: Amino acid sequence of human SIRPA variant 7 Sequence ID 68: Amino acid sequence of human SIRPA variant 8 Sequence ID 69: Amino acid sequence of human SIRPA variant 9 Sequence ID 70: Amino acid sequence of human SIRPA variant 10 Sequence ID 71: Amino acid sequence of human SIRPA_V2_IgV Sequence ID 72: Amino acid sequence of human SIRPA_V2_IgV_IgC1 Sequence ID 73: Amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ0 Sequence ID 74: Amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ1 Sequence ID 75: Amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ2 Sequence ID 76: Amino acid number 81 in the amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ0 Amino acid sequence from the 8th to the 85th Sequence ID 77: Amino acid number 81 in the amino acid sequence of mouse SIRPA mutant hmSIRPA_Δ1 Amino acid sequence from the 8th to the 85th Sequence ID 78: Amino acid sequences of BALB / C mouse SIRPA, from position 81 to 85 amino acid sequence Sequence ID 79: Amino acid sequences of BALB / C mouse SIRPA, positions 126-130 Amino acid sequence of the eye Sequence ID 80: Amino acid number 81 in the amino acid sequence of the mouse SIRPA mutant hmSIRPA_Δ2 Amino acid sequence from the 8th to the 85th Sequence ID 81: Amino acid distribution of OSE-172 antibody heavy chain (OSE-172_hG4Pro) column Sequence ID 82: Amino acid sequence of the OSE-172 antibody light chain (OSE-172_hK) Sequence ID 83: Amino acid sequence of KWAR23 antibody heavy chain (KWAR23_hG4Pro) Sequence ID 84: Amino acid sequence of the KWAR23 antibody light chain (KWAR23_hK) SEQ ID NO: 85: Amino acid sequence of ADU-1805 antibody heavy chain (ADU-1805_hG2) SEQ ID NO: 86: Amino acid sequence of the ADU-1805 antibody light chain (ADU-1805_hK)

[0228] All publications, patents, and patent applications cited herein are provided herein by direct reference. It will be incorporated.

Claims

[Claim 1] The invention described in the specification or drawings.

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