Pharmaceutical composition containing cells expressing chimeric receptors

Abstract

This invention provides a method for creating general-purpose chimeric antigen receptor-expressing cells and a therapeutic method using them. [Solution] The present invention provides a pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with the administration of an antigen-binding molecule, wherein the chimeric receptor comprises an extracellular domain, the extracellular domain comprising an extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof, and the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor.

Description

[Technical Field]

[0001] The present invention relates to a chimeric receptor, a cell that expresses the chimeric receptor, and a method for treating a disease using said cell, for example, adoptive cell immunotherapy, particularly adoptive cell immunotherapy using T cells that express the chimeric receptor. [Background Art]

[0002] A chimeric antigen receptor (hereinafter also referred to as "CAR") is an artificially fused chimeric protein of an antigen-binding molecule that recognizes a cell surface antigen and a signal transduction domain that induces activation of immune cells. By introducing a gene encoding a CAR into an immune cell, an immune cell that expresses the CAR is produced. CAR-expressing cells exert cytotoxicity against cells that express a target antigen without depending on interaction with major histocompatibility complex (MHC), and are used for the treatment of diseases such as cancer by adoptive cell immunotherapy (Non-Patent Document 1).

[0003] Clinical trials of adoptive cell immunotherapy using CAR-T cells or CAR-NK cells, wherein the CAR gene is introduced into T cells or natural killer cells, are being conducted worldwide. In particular, results demonstrating efficacy against hematopoietic malignancies such as leukemia and lymphoma have been obtained, and Kymriah (registered trademark) (Novartis, tisagenlecleucel) and Yescarta (registered trademark) (Gilead Sciences, axicabtagene ciloleucel), which are CD19-targeting CAR-T products, have been approved as pharmaceuticals.

[0004] Establishing technology for producing immune cells that can universally recognize target cells expressing various antigens has important clinical significance for the generalization of adoptive cell immunotherapy. There have been reports on such studies to date (Patent Documents 1 to 5, and Non-Patent Documents 2 to 5)

[0005] Furthermore, as an alternative treatment for cancer, methods are being developed to activate endogenous immune cells using antigen-binding molecules and thereby stimulate tumor immunity. To date, the therapeutic effects of antibodies that inhibit the immune checkpoint molecules CTLA-4, PD-1, and PD-L1 have been reported (Non-Patent Literature 6-7), and these have been approved as pharmaceuticals. In addition, it has been demonstrated in mouse models that activating costimulatory factors expressed on immune cells with agonist antibodies can produce antitumor effects (Non-Patent Literature 8). Moreover, a treatment method has been devised to enhance the efficacy of drugs by co-administering a CD137 agonist antibody that activates immune cells with CAR-T receptors (Patent Literature 6, Non-Patent Literature 9). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2012 / 082841 [Patent Document 2] WO2016 / 040441 [Patent Document 3] WO2017 / 161333 [Patent Document 4] WO2018 / 002358 [Patent Document 5] WO2018 / 177966 [Patent Document 6] WO2019 / 140425 [Non-patent literature]

[0007] [Non-Patent Document 1] June CH and Sadelain M, N Engl J Med. (2018) 379, 64 [Non-Patent Document 2] Tamada et al, Clin Cancer Res. (2012) 18, 6436 [Non-Patent Document 3] Urbanska K et al, J Transl Med. (2014) 12, 347 [Non-Patent Document 4] Kudo K et al, Cancer Res. (2014) 74, 93 [Non-Patent Document 5] Karches CH et al, Clin Cancer Res. (2019) 25, 5890 [Non-Patent Document 6] Hodi FS et al, N Engl J Med. (2010) 363, 711 [Non-Patent Document 7] Robert C et al, N Engl J Med. (2015) 372, 320 [Non-Patent Document 8] Houot R et al, Blood. (2009) 114, 3431 [Non-Patent Document 9] Mardiana S et al, Cancer Res. (2017) 77, 1296 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] One challenge in cancer treatment using CAR-based adoptive cell immunotherapy is its limited efficacy against solid tumors. This is likely due to the heterogeneity of tumor antigens expressed on individual cancer cells in cancer tissue, preventing CAR-expressing cells, which recognize only a single target antigen, from recognizing all tumor cells, as well as the immunosuppressive mechanisms within the tumor microenvironment. A strategy to address this challenge involves activating not only CAR-expressing cells but also endogenous immune cells to achieve antitumor effects in coordination with these endogenous immune cells. One possible method for achieving this is combination therapy with CD137 agonist antibodies that activate immune cells. However, both clinically and nonclinically, CD137 agonist antibodies have been shown to induce activation of immune cells in non-tumor tissues, including the liver, leading to toxicity. Furthermore, the following are some of the challenges in treating cancer with adoptive cell immunotherapy using CARs. Firstly, since the tumor antigens expressed on the surface of tumor cells differ from one cancer cell to another, the antigen recognition site of the CAR needs to be constructed for each target tumor antigen. Secondly, the tumor antigen targeted by the CAR may experience decreased expression or mutation due to treatment, leading to tumor antigen evasion. Since most current CAR-expressing cells recognize only a single target antigen, tumor antigen evasion reduces the therapeutic effect. Thirdly, the economic cost and labor burden of identifying antigen-binding molecules for various tumor antigens and establishing CAR-expressing cells fused with them are challenges. Therefore, there is a need for the creation of versatile CAR-expressing cells that can universally recognize multiple target antigens and whose recognized tumor antigen can be changed according to the characteristics of the patient's target antigen cells and the stage of treatment, as well as the development of therapeutic methods using such cells. [Means for solving the problem]

[0009] This disclosure provides a therapeutic method that combines a multispecific antigen-binding molecule having a site that exerts agonist activity against an immune receptor and a site that recognizes a target antigen, with immune cells that have been transduced with a chimeric receptor having the immune receptor recognized by the antigen-binding molecule in its extracellular domain. In one embodiment, the antigen-binding molecule in this disclosure accumulates at the lesion site expressing the target antigen, so its agonist activity is selectively expressed at the lesion site, and as a result, it is possible to avoid the expression of toxicity. Furthermore, unlike conventional CARs, the chimeric receptor in this disclosure does not have binding affinity to the target antigen, and therefore does not inhibit the binding of the antigen-binding molecule in this disclosure to the target antigen. Moreover, since the chimeric receptor in this disclosure has binding affinity to the antigen-binding molecule in this disclosure, it is possible to simultaneously induce the activation of endogenous immune cells and the activation of immune cells expressing the chimeric receptor with the antigen-binding molecule alone. In other words, the present invention discloses the following:

[0010] More specifically, in one aspect of this disclosure, the following invention is provided.

[0011] [1] A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with the administration of an antigen-binding molecule, A chimeric receptor includes an extracellular domain, which comprises the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. A pharmaceutical composition in which the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor. [2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of cells expressing a chimeric receptor, The chimeric receptor includes an extracellular domain, which includes the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. A pharmaceutical composition in which the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor. [3] The pharmaceutical composition according to [1] or [2], wherein the immune receptor recognition site recognizes the extracellular domain of a chimeric receptor and an endogenous immune receptor. [4] A pharmaceutical composition according to any one of [1] to [3], comprising an extracellular domain variant of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to the ligand of the endogenous immune receptor. [5] A pharmaceutical composition according to any one of [1] to [4], wherein the immune receptor is a co-stimulatory molecule.

[0012] [6] A pharmaceutical composition according to any one of [1] to [5], wherein the immune receptor is human CD137. [7] The pharmaceutical composition according to any one of [1] to [6], wherein the extracellular domain of the chimeric receptor comprises a human CD137 extracellular domain variant or a fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted. [8] A pharmaceutical composition according to any one of [1] to [7], wherein the chimeric receptor comprises an intracellular signaling domain, and the intracellular signaling domain comprises the intracellular signaling domain of CD3 zeta. [9] A pharmaceutical composition according to any one of [1] to [8], wherein the target antigen is a receptor, tumor antigen, MHC antigen, or differentiation antigen.

[10] A pharmaceutical composition according to any one of [1] to [9], wherein the target antigen is a tumor antigen.

[11] A pharmaceutical composition according to any one of [1] to

[10] , wherein the antigen-binding molecule is a bispecific antibody.

[0013]

[12] A pharmaceutical composition according to any one of [1] to

[11] for use in the treatment or prevention of cancer.

[13] Chimeric receptors comprising an extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or an extracellular domain comprising a fragment thereof.

[14] The chimeric receptor according to

[13] , wherein the extracellular domain of the chimeric receptor is a modified extracellular domain of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to the ligand of an endogenous immune receptor.

[15] A chimeric receptor as described in

[13] or

[14] , wherein the immune receptor is a costimulatory molecule.

[16] A chimeric receptor described in any of

[13] to

[15] , wherein the immune receptor is human CD137.

[17] The chimeric receptor according to any one of

[13] to

[16] , wherein the extracellular domain of the chimeric receptor comprises a human CD137 variant or fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted.

[18] The chimeric receptor contains an intracellular signaling domain, and the intracellular signaling domain contains the intracellular signaling domain of CD3 zeta, either

[13] -

[17] The chimeric receptor described above.

[0014] Cells expressing any one of the chimeric receptors described in

[19] ,

[13] , or

[18] . A composition containing cells

[20]

[19] . A nucleic acid encoding a chimeric receptor as described in any of

[21]

[13] ~

[18] . A vector into which the nucleic acids described in

[22]

[21] have been inserted. A method for producing the cells described in

[19] , comprising transfecting or transducing cells with the nucleic acid described in

[23]

[21] or the vector described in

[22] .

[24] A pharmaceutical composition according to any one of [1] to

[12] , or a chimeric receptor according to any one of

[13] to

[18] , further comprising a transmembrane domain.

[0015] [A1] A pharmaceutical composition comprising cells expressing a chimeric receptor for use in combination with the administration of an antigen-binding molecule, A chimeric receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain includes the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. A pharmaceutical composition comprising an antigen-binding molecule having a bispecific antigen-binding site having a target antigen-recognition site and an immune receptor-recognition site that recognizes the immune receptor. [A2] A pharmaceutical composition comprising an antigen-binding molecule for use in combination with the administration of cells expressing a chimeric receptor, A chimeric receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain includes the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. A pharmaceutical composition comprising an antigen-binding molecule having a bispecific antigen-binding site having a target antigen-recognition site and an immune receptor-recognition site that recognizes the immune receptor. [A3] The pharmaceutical composition according to [A1] or [A2], wherein the immune receptor recognition site recognizes the extracellular domain of a chimeric receptor and an endogenous immune receptor. [A4] A pharmaceutical composition according to any one of [A1] to [A3], comprising a modified extracellular domain of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to an endogenous immune ligand. [A5] A pharmaceutical composition according to any one of [A1] to [A4], wherein the immune receptor is a costimulatory molecule.

[0016] [A6] A pharmaceutical composition according to any one of [A1] to [A5], wherein the immune receptor is human CD137. [A7] The pharmaceutical composition according to any one of [A1] to [A6], wherein the extracellular domain of the chimeric receptor comprises an extracellular domain variant or fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted. [A8] A pharmaceutical composition according to any one of [A1] to [A7], wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3 zeta. [A9] A pharmaceutical composition according to any one of [A1] to [A8], wherein the target antigen is a receptor, tumor antigen, MHC antigen, or differentiation antigen. [A10] A pharmaceutical composition according to any one of [A1] to [A9], wherein the target antigen is a tumor antigen. [A11] A pharmaceutical composition according to any one of [A1] to [A10], wherein the antigen-binding molecule is a bispecific antibody.

[0017] [A12] A pharmaceutical composition according to any one of [A1] to [A11] for use in the treatment or prevention of cancer. [A13] A chimeric receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. [A14] The chimeric receptor according to [A13], comprising an extracellular domain variant or fragment of an immune receptor in which the extracellular domain of the chimeric receptor has reduced binding to an endogenous immune ligand. [A15] A chimeric receptor as described in [A13] or [A14], wherein the immune receptor is a costimulatory molecule. [A16] A chimeric receptor described in any of [A13] to [A15], wherein the immune receptor is human CD137. [A17] The chimeric receptor according to any one of [A13] to [A16], wherein the extracellular domain of the chimeric receptor comprises an extracellular domain variant or fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted. [A18] A chimeric receptor described in any of [A13] to [A17], wherein the intracellular signaling domain includes the intracellular signaling domain of CD3 zeta.

[0018] Cells expressing any of the chimeric receptors described in [A19], [A13], or [A18]. A composition containing cells [A20] and [A19]. A nucleic acid encoding a chimeric receptor as described in any of [A21], [A13], or [A17]. A vector into which the nucleic acids described in [A22] and [A21] have been inserted. A method for producing the cells described in [A18], comprising transfecting or transducing cells using the nucleic acids described in [A23] and [A21] or the vector described in [A22].

[0019] [B1] A method of treating a disease, Administering an effective amount of antigen-binding molecule to a target requiring treatment. To administer cells expressing chimeric receptors to the aforementioned subjects, Includes, A chimeric receptor includes an extracellular domain, which comprises the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. The method, wherein the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor. [B2] The method according to [B1], wherein the chimeric receptor further comprises a transmembrane domain and an intracellular signaling domain. [B3] The method according to [B1] or [B2], wherein the immune receptor recognition site recognizes the extracellular domain of the chimeric receptor and the endogenous immune receptor. [B4] The method according to any one of [B1] to [B3], comprising an extracellular domain variant of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to the ligand of the endogenous immune receptor. [B5] The method according to any of [B1] to [B4], wherein the immune receptor is a co-stimulatory molecule.

[0020] [B6] The method according to any of [B1] to [B5], wherein the immune receptor is human CD137. [B7] The method according to any one of [B1] to [B6], wherein the extracellular domain of the chimeric receptor comprises a human CD137 extracellular domain variant or a fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted. [B8] The method according to any one of [B1] to [B7], wherein the chimeric receptor comprises an intracellular signaling domain, and the intracellular signaling domain comprises the intracellular signaling domain of CD3 zeta. [B9] The method according to any of [B1] to [B8], wherein the target antigen is a receptor, tumor antigen, MHC antigen, or differentiation antigen. [B10] The method according to any of [B1] to [9], wherein the target antigen is a tumor antigen. [B11] The method according to any of [B1] to [B10], wherein the antigen-binding molecule is a bispecific antibody.

[0021] [B12] The disease is cancer, and the method described in any of [B1] to [B11]. [B13] The method according to [B12], wherein the cancer is selected from primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, colorectal cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.

[0022] [C1] A method of treating a disease, Administering an effective amount of antigen-binding molecule to a target requiring treatment. To administer cells expressing chimeric receptors to the aforementioned subjects, Includes, A chimeric receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain includes the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. The method wherein the antigen-binding molecule comprises a bispecific antigen-binding site having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor. [C2] The method according to [C1], wherein the chimeric receptor further comprises a transmembrane domain and an intracellular signaling domain. [C3] The method according to [C1] or [C2], wherein the immune receptor recognition site recognizes the extracellular domain of the chimeric receptor and the endogenous immune receptor. [C4] The method according to any one of [C1] to [C3], comprising a modified extracellular domain of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to an endogenous immune ligand. [C5] The method according to any of [C1] to [C4], wherein the immune receptor is a co-stimulatory molecule.

[0023] [C6] The method according to any of [C1] to [C5], wherein the immune receptor is human CD137. [C7] The method according to any one of [C1] to [C6], wherein the extracellular domain of the chimeric receptor comprises an extracellular domain variant or fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted. [C8] The method according to any of [C1] to [C7], wherein the intracellular signaling domain comprises the intracellular signaling domain of CD3 zeta. [C9] The method according to any of [C1] to [C8], wherein the target antigen is a receptor, tumor antigen, MHC antigen, or differentiation antigen. [C10] The method described in any of [C1] to [C9], wherein the target antigen is a tumor antigen. [C11] The method according to any of [C1] to [C10], wherein the antigen-binding molecule is a bispecific antibody.

[0024] [C12] Any method according to any of [C1] to [C11] for use in the treatment or prevention of cancer. [C13] The method according to [C12], wherein the cancer is selected from primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, colorectal cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 is a conceptual diagram of a therapeutic method that combines an antigen-binding molecule that exhibits agonist activity against an immune receptor (for example, a bispecific antibody) with immune cells that have been transduced to a chimeric receptor having the immune receptor recognized by the antigen-binding molecule in its extracellular domain. The diagram illustrates the features of the present invention, which involves mobilizing endogenous immune cells in addition to chimeric receptor-expressing cells. [Figure 2] Figure 2 is a schematic diagram showing the chimeric receptor CD137-CR2, which has I64R and V71R mutations in the extracellular domain of CD137, compared to the chimeric receptor CD137-CD8-CD28-CD137-CD3 zeta (CD137-CR1), trCD137-CD8-CD28-CD137-CD3 zeta (trCD137-CR), and CD137-CR1. [Figure 3] Figure 3 is a schematic diagram of the lentiviral vector construct expressing pCDH-CD137-CR1 described in Example 3-1 and the sequence order of the frame-unit components from the 5' end to the 3' end. [Figure 4]Figure 4 shows the results of evaluating the activation ability of CD137-CR1 Jurkat cells when cells expressing human GPC3 as the target antigen were co-cultured with a bispecific antibody consisting of anti-GPC3 antibody and anti-CD137 antibody, as described in the reporter assay of Examples 3-4. The horizontal axis represents the concentration of the bispecific antibody (H0000-F760nN17 / GL4-k0a / / hCD137VH-F760mnP17 / hCD137VL-k0), and the vertical axis represents the luciferase luminescence intensity. [Figure 5] Figure 5 shows the results of evaluating the activation ability of CD137-CR1-copGFPJurkat cells when cells expressing human GPRC5D as the target antigen were co-cultured with a bispecific antibody consisting of anti-GPRC5D antibody and anti-CD137 antibody, as described in the reporter assays of Examples 3-5. Two bispecific antibodies, namely GPA0018H-F760mnN17 / GPA0018L-k0C / / hCD137VH-F760mnP17 / hCD137VL-k0 (GPA0018 / CD137) and GPA0039H-F760mnN17 / GPA0039L-k0C / / hCD137VH-F760mnP17 / hCD137VL-k0 (GPA0039 / CD137), were used. The horizontal axis indicates their presence or absence, and the vertical axis indicates the luminescence intensity of luciferase. [Figure 6] Figure 6 shows the results of evaluating the activation ability of NFAT-RE-luc2 Jurkat cells (copGFP) and CD137-CR1-copGFP Jurkat cells (CD137-CR1) that express only copGFP and do not express the chimeric receptor, when cells expressing human IL-6R as the target antigen are co-cultured with a bispecific antibody consisting of anti-IL-6R antibody and anti-CD137 antibody, as described in the reporter assays of Examples 3-6. The horizontal axis shows the presence or absence of the bispecific antibody (MRAH.v1-F760mnN17 / MRAL.v1-k0.v1 / / hCD137VH-F760mnP17 / hCD137VL-k0), and the vertical axis shows the luciferase luminescence intensity. [Figure 7]Figure 7 is a schematic diagram of the retroviral vector construct expressing pMSGV1-CD137-CR1 described in Example 4-1 and the sequence order of the frame-unit components from the 5' end to the 3' end. [Figure 8A] Figure 8A is a graph showing the results of evaluating the cytotoxic activity of CD137-CR1-eGFP-expressing T cells, using the number of residual tumor cells as an indicator, when co-cultured human hepatocellular carcinoma cell line (SK-Hep-1) and SK-pca60 cells expressing human GPC3 as the target antigen, with three types of bispecific antibodies (consisting of anti-GPC3 antibody and anti-KLH antibody, anti-KLH antibody and anti-CD137 antibody, and anti-GPC3 antibody and anti-CD137 antibody), as described in Example 4-4. The vertical axis shows the number of residual tumor cells, and the horizontal axis shows the presence or absence of the three types of bispecific antibodies. [Figure 8B] Figure 8B is a graph showing the results of evaluating the cytotoxic activity of CD137-CR1-eGFP-expressing T cells using the xCELLigence system when co-cultured with the human hepatocellular carcinoma cell line (SK-Hep-1) and SK-pca60 cells expressing human GPC3 as the target antigen, as described in Example 4-5, and a bispecific antibody consisting of anti-GPC3 antibody and anti-CD137 antibody. The evaluation was performed by varying the ratio of effector cells to target cells (E:T) and the presence or absence of the bispecific antibody (indicated as "with antibody" and "without antibody" in Figure 8B). The vertical axis shows cell proliferation activity (%), and the horizontal axis shows the number of days since the addition of effector cells and antibody. [Figure 9] Figure 9 is a schematic diagram illustrating the target antigen-independent activation mechanism of chimeric receptor-expressing cells through binding to ligand-expressing cells. [Figure 10]Figure 10 is a graph showing the results of evaluating the activation ability of trCD137-CR-copGFP Jurkat cells (trCD137-CR) when cells expressing human GPC3 as the target antigen were co-cultured with a bispecific antibody consisting of anti-GPC3 antibody and anti-CD137 antibody, as described in Example 6-4. The horizontal axis represents the concentration of the bispecific antibody (H0000-F760nN17 / GL4-k0a / / hCD137VH-F760mnP17 / hCD137VL-k0), and the vertical axis represents the luciferase luminescence intensity. [Figure 11] Figure 11 is a graph showing the results of evaluating the activation ability of CD137-CR2-copGFP Jurkat cells (CD137-CR2) when cells expressing human GPC3 as the target antigen were co-cultured with a bispecific antibody consisting of anti-GPC3 antibody and anti-CD137 antibody, as described in Example 7-4. The horizontal axis represents the concentration of the bispecific antibody (H0000-F760nN17 / GL4-k0a / / hCD137VH-F760mnP17 / hCD137VL-k0), and the vertical axis represents the luciferase luminescence intensity. [Figure 12A] Figure 12A is a graph showing the results of evaluating the activation ability of chimeric receptor-expressing cells in CD137-CR1-copGFP Jurkat cells (CD137-CR1) prepared in Example 3-2, both in the presence and absence of ligand (human CD137L)-expressing cells (Raji) described in Example 8. The vertical axis represents the luminescence intensity of luciferase. [Figure 12B] Figure 12B is a graph showing the results of evaluating the activation ability of chimeric receptor-expressing cells in trCD137-CR-copGFP Jurkat cells (trCD137-CR) prepared in Example 6-2, both in the presence and absence of ligand (human CD137L)-expressing cells (Raji) described in Example 8. The vertical axis represents the luminescence intensity of luciferase. [Figure 12C]Figure 12C is a graph showing the results of evaluating the activation ability of chimeric receptor-expressing cells in CD137-CR2-copGFP Jurkat cells (CD137-CR2) prepared in Example 7-2, both in the presence and absence of ligand (human CD137L)-expressing cells (Raji) described in Example 8. The vertical axis represents the luminescence intensity of luciferase. [Figure 13] Figure 13 is a graph showing the results of measuring the binding activity of BB0000 and BB0077 to the in vivo ligand (human CD137L), as described in Example 9-3-1. The horizontal axis represents the concentration of CD137L, and the vertical axis represents the amount of CD137L bound relative to the amount of B0000 and BB0077 absorbed. [Figure 14] Figure 14 is a graph showing the results of the ECM binding evaluation of BB0000 and BB0077 described in Example 9-3-2. The vertical axis shows the emission intensity indicating ECM binding. [Figure 15] Figure 15 is a graph showing the results of measuring the binding activity of BB0124 to BB0138 to the endogenous ligand (human CD137L) as described in Example 9-3-4. The horizontal axis represents the evaluated CD137 variants, and the vertical axis represents the amount of CD137L bound to each variant relative to the amount absorbed. [Figure 16A] Figure 16A is a graph showing the results of the ECM binding evaluation of CD137 modifiers BB0124 to BB0138 described in Example 9-3-5. The vertical axis shows the luminescence intensity indicating ECM binding. [Figure 16B] Figure 16B is a graph showing the results of the ECM binding evaluation of the CD137 variant BB0139 described in Example 9-3-3. The vertical axis shows the luminescence intensity indicating ECM binding. [Figure 17]Figure 17 is a graph showing the results of evaluating the activation ability of a bispecific antibody (indicated as Ab in Figure 17) consisting of an anti-GPC3 antibody and an anti-CD137 antibody in the presence of target cells using the reporter assay described in Example 10-4. The horizontal axis shows Jurkat cells expressing a chimeric receptor in which eight types of CD137 variants (BB0000, BB0077, BB0127, BB0128, BB0131, BB0133, BB0134, BB0139) are used as extracellular domains and linked to the cytoplasmic domains of CD8-CD28-CD137-CD3 zeta, as well as the parental strain (Mock) of unmodified NFAT-RE-luc2 Jurkat cells. The vertical axis shows the luminescence intensity of luciferase. [Figure 18] Figure 18 is a graph showing the results of evaluating the activation ability of Jurkat cells with a modified CD137 extracellular domain in the presence of ligand-expressing cells, as described in Example 10-5. The axes represent Jurkat cells expressing a chimeric receptor in which the cytoplasmic regions of CD8-CD28-CD137-CD3 zeta are linked to eight types of extracellular domains: BB0000, BB0077, BB0127, BB0128, BB0131, BB0133, BB0134, and BB0139, as well as the parental strain (Mock) of unmodified NFAT-RE-luc2 Jurkat cells. The vertical axis represents the luminescence intensity of luciferase. [Figure 19] Figure 19 is a schematic diagram showing the chimeric receptor CD137-CD8-CD3 zeta encoded by the lentiviral vector described in Example 11-1. [Figure 20]Figure 20 is a graph showing the results of evaluating the CD137 activation ability of three types of Jurkat cells in the presence of target cells using a reporter assay described in Example 11-4, which is dependent on bispecific antibodies consisting of anti-GPC3 antibody and anti-CD137 antibody. The horizontal axis shows the presence or absence of the bispecific antibodies and the three types of Jurkat cells prepared in Example 11-2, namely CD137-CR1 Jurkat cells (CD137-CR1), CD137-CR3-copGFP Jurkat cells (CD137-CR3), and NF-κB-Luc2 / 4-1BB Jurkat cells (copGFP) that do not express the chimeric receptor and express only copGFP. The vertical axis shows the luminescence intensity of luciferase. [Figure 21] Figure 21 is a schematic diagram showing the constructs (CD28-CR1) and (CD28-CR2) that use human CD8 in the transmembrane domain encoded by the lentiviral vector described in Example 12-1. [Figure 22] Figure 22 is a graph showing the results of evaluating the activation ability of CD28-CR1 Jurkat cells and CD28-CR2 Jurkat cells described in Example 12-4 in the presence of target cells. The horizontal axis shows the presence or absence of three types of Jurkat cells: NFAT-RE-luc2 Jurkat cells (copGFP) that express only copGFP without expressing the chimeric receptor, Jurkat cells expressing CD28-CR1-copGFP prepared in Example 12-2 (CD28-CR1-copGFP), and Jurkat cells expressing CD28-CR2-copGFP (CD28-CR2-copGFP), as well as three types of bispecific antibodies: a bispecific antibody consisting of anti-GPC3 antibody and anti-CD28 antibody (GPC3 / CD28), a bispecific antibody consisting of anti-KLH antibody and anti-CD28 antibody (KLH / CD28), and a bispecific antibody consisting of anti-GPC3 antibody and anti-KLH antibody (GPC3 / KLH). The vertical axis shows the luminescence intensity of luciferase. [Modes for carrying out the invention]

[0026] Other features and advantages of this disclosure will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of this disclosure will become obvious to those skilled in the art from this detailed description, the detailed description and specific examples illustrating preferred embodiments of this disclosure should be understood to be provided for illustrative purposes only. The embodiments of this disclosure will be described below with reference to the drawings.

[0027] In the present invention, polypeptides typically refer to peptides and proteins having a length of about 4 amino acids or more. While polypeptides in the present invention are typically polypeptides consisting of artificially designed sequences, they are not particularly limited and may, for example, be polypeptides of biological origin. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.

[0028] In this specification, amino acids are represented by single-letter codes, three-letter codes, or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V. When representing an amino acid at a specific position, a representation that includes both a number indicating the specific position and the single-letter or three-letter code of the amino acid may be used as appropriate. For example, the amino acid 37V, which is contained in a single-domain antibody, represents Val, which is located at position 37 in Kabat numbering.

[0029] For modifying amino acids in the amino acid sequences of polypeptides such as antibodies, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. Furthermore, several known methods for modifying amino acids by substituting them with non-natural amino acids can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is suitably used. In this specification, substitution is mentioned as a modification, but is not limited to this.

[0030] In this specification, the term "and / or" used to describe the modification sites of amino acids includes any combination of "and" and "or" as appropriate. Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following variations of amino acid modification sites: (a) 37, (b) 45, (c) 47, (d) 37 and 45, (e) 37 and 47, (f) 45 and 47, (g) 37, 45 and 47.

[0031] In this specification, expressions that include a number representing a specific position followed by the one-letter or three-letter code of the amino acid before and after the modification may be used as appropriate to represent amino acid modifications. For example, the modification F37V or Phe37Val, used when making amino acid substitutions in the antibody variable region or single-domain antibody, represents the substitution of Phe at position 37 in Kabat numbering to Val. That is, the number represents the position of the amino acid in Kabat numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala, used when making amino acid substitutions in the Fc region included in the antibody constant region, represents the substitution of Pro at position 238 in EU numbering to Ala. That is, the number represents the position of the amino acid in EU numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution. The one-letter or three-letter code for an amino acid represents the substituted amino acid.

[0032] In this specification, the term “antibody” is used in its broadest sense and is not limited to any antibody that exhibits the desired antigen-binding activity, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, and antibody fragments.

[0033] In the present invention, a "multispecific antigen-binding molecule" refers to a molecule that can specifically bind to multiple different antigens, specifically to multiple epitopes contained in multiple antigens. In other words, a multispecific antigen-binding molecule is a molecule that has specificity to at least two different epitopes, and includes molecules that bind to different antigens as well as molecules that bind to different epitopes on the same antigen. The concept of a multispecific antigen-binding molecule encompasses a bispecific antigen-binding molecule, and examples include multispecific antibodies and bispecific antibodies. A bispecific antibody is a molecule that binds to two antigens, while a multispecific antibody may have specificity to two or more (e.g., three types) antigens. Bispecific antibodies and multispecific antibodies can be prepared as full-length antibodies or as molecules containing antibody fragments.

[0034] An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0035] The term "full-length antibody" refers to an antibody that has a structure substantially similar to that of a naturally occurring antibody, or an antibody that has a heavy chain containing an Fc region as defined herein.

[0036] In this specification, the term "homoantibody" refers to an antibody that binds to a single antigen and has a quadruple-chain structure consisting of two light chains and two heavy chain polypeptide chains.

[0037] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH or VL domain provides antigen-binding specificity.

[0038] As used herein, the terms “complementarity-determining region” or “CDR” refer to the regions of the variable domain of an antibody that are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”) and / or antigen contact residues (“antigen contacts”). Typically, an antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative CDRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b(H1), 49-65(H2), 93-102(H3), and 94-102(H3).

[0039] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0040] The "framework" or "FR" refers to variable domain residues other than complementarity-determining region (CDR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the CDR and FR usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0041] In this specification, the term “constant region” or “constant domain” refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing the CH1 domain, hinge region, CH2 domain, and CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of native antibodies may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0042] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the native sequence and the mutant Fc region. In one embodiment, in the case of human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows Kabat et al., Sequences of The EU numbering system (also known as the EU Index) is followed, as described in *Proteins of Immunological Interest*, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0043] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0044] In this specification, the "target antigen recognition site" is a site of an antigen-binding molecule that can recognize a target antigen (target antigen) and specifically bind to it, and can also be called a target antigen-binding domain. As long as the target antigen recognition site binds to the target antigen, It may also be a part of the structure. Examples of such regions, though not limited to these, include, for example, the heavy chain variable region (VH) and light chain variable region (VL) of antibodies, single-domain antibodies (sdAb), a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody (International Publication WO1995 / 001937), which uses the IgG-binding domain that constitutes a bundle of three helices of 58 amino acids of Protein A as a scaffold, and DARPins (Designed Examples include Ankyrin Repeat proteins (International Publication WO2002 / 020565), Anticalin (International Publication WO2003 / 029462), which consists of four loop regions supporting one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL), and recessed regions of parallel sheet structures within a horseshoe-shaped structure formed by repeatedly stacked leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) that do not possess the structure of immunoglobulins, as part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854).

[0045] Preferred examples of the target antigen recognition site of the present invention include the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, and particularly preferred examples include the target antigen recognition site in a multispecific antibody that also has an immune receptor recognition site, such as the target antigen recognition site in a bispecific antibody. The site of the antigen-binding molecule of the present invention that can recognize and bind to a specific antigen may be referred to as an antigen-binding domain. For example, a multispecific antibody has multiple antigen-binding domains, of which the antigen-binding domain that can recognize and bind to a target antigen functions as the target antigen-binding site, and the antigen-binding domain that can recognize and bind to an immune receptor functions as the immune receptor-recognition site. Therefore, both the target antigen-binding site and the immune receptor-recognition site of a multispecific antibody correspond to the antigen-binding domain of the multispecific antibody. "Containing a bispecific antigen-binding site" means having two types of antigen-binding domains.

[0046] In this specification, "antigen" means a substance that a molecule such as an antibody recognizes as a target, and in the present invention, "target antigen" means an antigen to which an antigen-binding molecule can bind via a target antigen recognition site. A target antigen includes an epitope to which the target antigen recognition site can bind. In the present invention, a target antigen is an antigen used to treat a disease caused by a target tissue, and preferred examples, but not limited to, include molecules expressed on the surface of target cells (e.g., cancer cells, inflammatory cells), molecules expressed on the surface of other cells in the tissue containing the target cells, molecules expressed on the surface of cells that have an immunological role with respect to the target cells and the tissue containing the target cells, and macromolecules present in the stroma of the tissue containing the target cells. The antigens listed below can be given as examples of target antigens.

[0047] In one embodiment, the target antigen in the present invention can preferably be, for example, a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.

[0048] Target antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin Tibin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adresin, aFGF, ALCAM, ALK, ALK-1, ALK-7, Alpha-1-Antitrypsin, Alpha-V / Beta-1 Antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-Lymphocyte-Stimulating Factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, Osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, β-NGF, BOK, Bombecin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, Complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, Calcitonin, cAMP, Carcinoembryonic antigen (CEA), Cancer-associated antigen, Cathepsin A, Catheter Cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / Ep hB4, EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast-Activating Protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-Stimulating Hormone, Fractalkine FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF -3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-Alpha 1, GFR-Alpha 2, GFR-Alpha 3, GITR, Glucagon, Glut4, Glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GPR20, GRO, Growth Hormone Releasing Factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNO S, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin alpha 2, Integrin alpha 3, Integrin alpha 4, Integrin alpha 4 / beta 1, Integrin alpha 4 / beta 7, Integrin alpha 5 (alpha V), Integrin alpha 5 / beta 1, Integrin alpha 5 / beta 3, Integrin alpha 6, Integrin beta 1, Integrin beta 2, Interferon gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte growth factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, Nerve Growth Factor (NGF), NGFR, NGF-Beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDK-1, P ECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, polynuclear respiratory virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymus Ck-1, Thyroid-stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(WHEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR) TNFRSF17(BCMA) TNFRSF18(GITR). AITR)、TNFRSF19(TROY CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF RI CD120a、p55-60)、TNFRSF1B(TNF RII). CD120b, p75-80, TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF5(CD40 p50), TNFRSF6(Fas). Apo-1, APT1, CD95, TNFRSF6B(DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2). TNFRH2, TNFRST23(DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10(TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK polymer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM ligand LTg, TNFSF15(TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a). Connectin (Connectin), DIF, TNFSF2, TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40). gp34, TXGP1, TNFSF5(CD40, CD154, gp39, H.S IGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-like receptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DDR2, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, polymeric kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor. Examples include Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P, and receptors for hormones and growth factors.

[0049] While the above examples of target antigens include receptors, these receptors can also be used as antigens to which the target antigen recognition site of the present invention binds, even when they exist in a soluble form in biological fluids. One non-limiting embodiment of such a soluble receptor is, for example, a protein that is soluble IL-6R, as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).

[0050] Examples of receptors include those belonging to receptor families such as the hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase receptor family, serine / threonine kinase receptor family, TNF receptor family, G protein-coupled receptor family, GPI-anchored receptor family, tyrosine phosphatase receptor family, adhesion factor family, and hormone receptor family. Regarding receptors belonging to these receptor families and their characteristics, numerous publications have been consulted, such as Cooke BA., King RJB., van der Molen HJ. ed. New Comprehensive Biochemistry Vol.18B Hormones and their Actions Part II pp.1-46 (1988) Elsevier Science Publishers BV., or reviews such as Miyasaka Masayuki (supervisor), Cell Engineering Supplementary Handbook Series "Adhesion Factor Handbook" (1994) (Shujunsha, Tokyo, Japan), as well as Patthy (Cell (1990) 61 (1)), 13-14), Ullrich et al. (Cell (1990) 61 (2), 203-212), Massague (with an acute accent mark on the e) (Cell (1992) 69 (6), 1067-1070), Miyajima et al. (Annu. Rev. Immunol. (1992) 10, 295-331), Taga et al. (FASEB J. (1992) 6, 3387-3396), Fantl et al. (Annu. Rev. Biochem. (1993), 62, 453-481), Smith et al. (Cell (1994) 76 (6) 959-962), Flower DR. Biochim. Biophys. Acta, Flower (Biochim. Biophys. Acta (1999) 1422) (3) As described in 207-234, etc.

[0051] Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277-285), human or mouse granulocyte colony-stimulating factor (G-CSF) receptors (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), and human or This includes mouse thrombopoietin (TPO) receptors (Proc. Natl. Acad. Sci. US A. (1992) 89 (12), 5640-5644, EMBO J. (1993) 12(7), 2645-53), human or mouse insulin receptors (Nature (1985) 313 (6005), 756-761), human or mouse Flt-3 ligand receptors (Proc. Natl. Acad. Sci. USA. (1994) 91 (2), 459-463), human or mouse platelet-derived growth factor (PDGF) receptors (Proc. Natl. Acad. Sci. USA. (1988) 85 (10) 3435-3439), human or mouse interferon (IFN)-alpha and beta receptors (Cell (1990) 60 (2)), Examples of suitable receptors include human or mouse leptin receptors, human or mouse growth hormone (GH) receptors, human or mouse interleukin (IL)-10 receptors, human or mouse insulin-like growth factor (IGF)-I receptors, human or mouse leukemia suppressor (LIF) receptors, human or mouse ciliary neurotrophic factor (CNTF) receptors, etc. (225-234 and Cell (1994) 77 (3), 391-400).

[0052] Examples of target antigens mentioned above include membrane-bound molecules expressed on cell membranes and soluble molecules secreted extracellularly from cells. When the antigen-binding domain of the present invention binds to a soluble molecule secreted from a cell, it is preferable that the antigen-binding domain has neutralizing activity.

[0053] The soluble molecule is not limited to the solution in which it exists; it can be present in any biological fluid, i.e., any fluid that fills the spaces between blood vessels or tissues and cells within the body. In one non-limiting embodiment, the soluble molecule to which the target antigen recognition site of the present invention binds can be present in extracellular fluid. Extracellular fluid refers to the general term for components in bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, cerebrospinal fluid, puncture fluid, or synovial fluid, as well as cellular permeable fluids such as alveolar fluid (bronchial alveolar lavage fluid), ascites, pleural fluid, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor) (fluids in various glandular lumens resulting from the active transport and secretory activity of cells, and fluids in the gastrointestinal tract and other body cavities).

[0054] The term "tumor antigen" refers to an antigen expressed on cancer cells, meaning an antigenic biomolecule whose expression becomes recognized in relation to the malignant transformation of the cell. Tumor antigens include tumor-specific antigens (antigens present only on tumor cells and not on other normal cells) and tumor-associated antigens (antigens present in other organs and tissues or in heterogeneous and allogeneic normal cells, or antigens expressed during development and / or differentiation). Abnormal glycans that appear on the cell surface or on protein molecules when cells become cancerous are also tumor antigens, and are also called cancer glycan antigens. In one embodiment of the present invention, the target antigen is a tumor antigen.

[0055] Examples of tumor antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4), 455-65), which belongs to the GPI-anchored receptor family as a receptor and is expressed in several cancers, including liver cancer, and EpCAM (Proc Natl Acad Sci US A. (1989) 86 (1)), which is expressed in multiple cancers, including lung cancer. 27-31) (The polynucleotide sequence is described in RefSeq registry number NM_002354.2, and the polypeptide sequence is described in RefSeq registry number NP_002345.2.), EGFR, CA19-9, CA15-3, serial SSEA-1 (SLX), Her2, Her3, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), tumor antigen-125 (CA-125), calretinin, MUC-1, MUC-16, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), chromogranin, cytokeratin, desmin, glial fiber acidic protein (GFAP), gross cystic disease fluid protein Protein (GCDFP-15), HMB-45 antigen, Protein Melan-A (Melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, Muscle-specific actin (MSA), Neurofilament, Nerve-specific enolase (NSE), Placental alkaline phosphatase, Synaptophysis, Thyroglobulin, Thyroid transcription factor-1, Dimeric form of pyruvate kinase isoenzyme type M2 (Tumor M2-PK), GD2 (Ganglioside G2), EGFRvIII (Epidermal growth factor variant III), Sperm protein 17 (Sp17), Mesoserine, PAP (Prostatic acid phosphatase), Prostain, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (Six-transmembrane epithelial antigen of prostate 1), TROP-2, Suitable examples include Rhodin 6, RNF43a, abnormal ras protein, or abnormal p53 protein, integrin alpha v beta 3 (CD61), galectin, K-Ras (V-Ki-ras2 kirsten rat sarcoma virus oncogene), or Ral-B.

[0056] Furthermore, examples of tumor antigens include, for example, thyroid-stimulating hormone receptor (TSHR); CD171; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319 and 19A24); type C lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag); T antigen (T Ag); Fms-like tyrosine kinase 3 (FLT3); CD38; CD44v6; B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2); Interleukin-11 receptor alpha (IL-11Ra); Interleukin-2 receptor alpha (IL-2Ra); Prostate stem cell antigen (PSCA); Protease serine 21 (PRSS21); Vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); Neuronal cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); Proteasome (prosome, macropain) subunit, beta type, 9 (LMP2); Ephrin type A receptor 2 (EphA2); Fucosyl GM1; Sialyl Lewis adhesion molecule (sLe); Ganglioside GM3 (aNeu5A c(2-3)bDGalp(1-4)bDGlcp(1-1)Cer;TGS5;High molecular weight melanoma-associated antigen (HMWMAA);o-acetyl-GD2 ganglioside (OAcGD2);folate receptor beta;tumor endothelial marker 1 (TEM1 / CD248);tumor endothelial marker 7-associated (TEM7R);claudin 6 (CLDN6);G protein-coupled receptor class C group 5, member D (GPRC5D);chromosome X open reading pattern Mu61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexasaccharide portion of globoH glycoceramide (GloboH); Mammary gland differentiation antigen (NY-BR-1); Uloplakin 2 (UPK2); Hepatitis A virus cytotoxic receptor 1 (HAVCR1); Adrenergic receptor beta 3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20);Lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma-selective leading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); Melanoma carcinoma testicular antigen-1 (MAD-CT-1); Melanoma carcinoma testicular antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 variant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Apoptotic melanoma inhibitor (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl-transferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell tumor antigen recognized by T cell 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein p32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-section 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin; Examples include Lambda-like polypeptide 1 (IGLL1).

[0057] MHC antigens are gene products of the major histocompatibility complex (MHC), and among them, glycoproteins expressed on the cell membrane are mainly classified into MHC class I antigens and MHC class II antigens. MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, and -H, while MHC class II antigens include HLA-DR, -DQ, and -DP. In addition, tumor antigen-derived peptides presented by these MHC antigens are also included. Tumor antigens such as GP100, MART-1, MAGE-1, MAGE-A4, and NY-ESO-1, as well as complexes with MHC that present mutated regions such as RAS and p53, can also be considered as tumor antigens.

[0058] "Differentiation antigens" are a general term for cell surface molecules that change during the differentiation of macrophages, T cells, B cells, etc., from bone marrow stem cells. Differentiation antigens include CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15s, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD29, CD30, CD32, CD33, CD34, CD35, CD38, CD40, CD41a, CD41b, CD42a, CD42b, and CD4 3, CD44, CD45, CD45RO, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD51, CD54, CD55, CD56, CD57, CD58, CD61, CD62E, CD62L, CD62P, CD64, CD69, CD70, CD71, CD73, CD95, CD99, CD102, CD106, CD117, CD122, CD126, CDw130 may be included.

[0059] The term “tumor” generally refers to a mass of tissue formed by the autonomous and excessive growth of tissues or cells against the control of the body. Tumors are malignant, characterized by autonomous growth, invasion and metastasis, and cachexia, and benign, characterized only by autonomous growth. Malignant tumors, “cancer,” refer to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Various examples of cancer are described in this disclosure, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and related cancers. The terms “tumor” and “cancer” are used synonymously in this disclosure, and for example, both terms encompass solid and liquid tumors, such as diffuse or circulating tumors. When used in this disclosure, the terms “cancer” or “tumor” encompass precancerous, as well as malignant, cancers and tumors.

[0060] The cancers treated with the anticancer drugs disclosed herein and the cancer treatment methods described later include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colorectal cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, and other cancers, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue. Additionally, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, as well as blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreaticoblastoma, pleuropulmonary blastoma, and retinoblastoma, germ cell tumors, lymphoma, and leukemia.

[0061] In one embodiment, in relation to cancer type, the tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In a particular embodiment, the tumor antigen of the Disclosure is derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, colorectal cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinoma, e.g., breast cancer, prostate cancer, ovarian cancer, pancreatic cancer and similar cancers. In one embodiment, the tumor antigen is an antigen common to a particular proliferative disorder. In one embodiment, the cancer-associated antigen is overexpressed in cancer cells compared to normal cells, e.g., 1x expression, 2x overexpression, 3x overexpression compared to normal cells. These are cell surface molecules that are overexpressed or in excess. In some embodiments, cancer-associated antigens are cell surface molecules that are improperly synthesized in cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed in normal cells. In one embodiment, cancer-associated antigens are expressed exclusively on the cell surface of cancer cells, either whole or as fragments (e.g., MHC / peptide), and are neither synthesized nor expressed on the surface of normal cells. Typically, peptides derived from endogenous proteins fill the pocket of a major histocompatibility complex (MHC) class I molecule, and CD8 +It is recognized by the T cell receptor (TCR) on T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting viral or tumor antigen-derived peptides in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described [see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Bood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100]. For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage-presenting libraries.

[0062] Examples of antigens to which the antigen-binding molecules of this disclosure bind include viral antigens, bacterial (particularly infectious bacteria) antigens, parasitic antigens, cell surface markers on target cells associated with specific pathological conditions (e.g., tumor antigens), and surface molecules of immune cells that give rise to autoimmunity.

[0063] In one aspect, this disclosure provides a chimeric receptor that can bind to antigens derived from the Retroviridae family (e.g., human immunodeficiency virus, e.g., HIV-1 and HIV-LP), Picornaviridae family (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, varicella stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae family, Herpesviridae family [e.g., herpes simplex virus type 1 and type 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesvirus], Poxviridae family (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus via antigen-binding molecules.

[0064] In another aspect, this disclosure provides a chimeric receptor that can bind to antigens derived from strains of the genera Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella via antigen-binding molecules. In particular, this disclosure concerns infectious bacteria, such as Helicobacter pylori, Legionella pneumophila, strains of Mycobacterium species (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, or Mycobacterium gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactie) The present invention provides a chimeric receptor that can bind to antigens derived from *Streptococcus agalactiae*, *Streptococcus pneumoniae*, or *Clostridium tetani* via antigen-binding molecules.

[0065] This disclosure also states that, in another aspect, antigen-binding molecules include 5T4, alpha-5beta-1-integrin, 707-AP, AFP, ART-4, B7H4, B7H3, BAGE, beta- catenin / m, Bcr-abl, MN / C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, C T, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, Gpc3, Gpr20, HAGE, HER-2 / ne u, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / melan-A, MART-2 / Ski, MC1R, myosin / m, MUC1, MUC-16, MUM-1, MUM-2, MUM-3, NA88-A, PAP, proteinase-3, p190 The antibody may bind to tumor antigens such as minor bcr-abl, Pml / RAR alpha, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, Survivin, TEL / AML1, TGF beta, TPI / m, Trop-2, TRP-1, TRP-2, TRP-2 / INT2, VEGF, WT1, NY-Eso-1 or NY-Eso-B. In this disclosure, the antibody may also bind to cell surface adhesion molecules, surface molecules of inflammatory cells found in autoimmune diseases, or TCRs that cause autoimmunity.

[0066] In this specification, the "immune receptor recognition site" is a site of an antigen-binding molecule that can recognize a target immune receptor and specifically bind to it, and may also be referred to as an immune receptor-binding domain. The immune receptor recognition site may have any structure as long as it binds to the target immune receptor.Examples of such sites, though not limited to these, include, for example, the heavy chain variable region (VH) and light chain variable region (VL) of antibodies that recognize immune receptors as antigens, single-domain antibodies (sdAb), a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in the body (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody (International Publication WO1995 / 001937), which uses an IgG-binding domain as a scaffold to form a bundle of three helices consisting of 58 amino acids of Protein A, and ankyrin repeats, which have a structure in which a turn containing 33 amino acid residues and two antiparallel helical and loop subunits are repeatedly stacked. Examples include DARPins (Designed Ankyrin Repeat proteins) (International Publication WO2002 / 020565), which are regions exposed on the molecular surface of repeat (AR); Anticalin, etc. (International Publication WO2003 / 029462), which are four loop regions in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) that support one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands; and recessed regions of parallel sheet structures within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules, which do not possess the structure of immunoglobulins, are repeatedly stacked, as part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854).

[0067] Preferred examples of the immune receptor recognition site of the present invention include the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody that recognize an immune receptor as an antigen. Particularly preferred examples include the immune receptor recognition site in a multispecific antibody that also has a target antigen recognition site, such as the immune receptor recognition site in a bispecific antibody. When the site of the antigen-binding molecule of the present invention that can recognize and bind to a specific immune receptor is part of an antibody that recognizes an immune receptor as an antigen, the immune receptor recognition site may be referred to as an antigen-binding domain. For example, a multispecific antibody has multiple antigen-binding domains, of which the antigen-binding domain that can recognize and bind to a target antigen functions as a target antigen-binding site, and the antigen-binding domain that can recognize and bind to an immune receptor functions as an immune receptor recognition site. Therefore, in both cases, the target antigen-binding site and the immune receptor-recognition site of the multispecific antibody correspond to the antigen-binding domain of the multispecific antibody.

[0068] Examples of immune receptors recognized by antigen-binding molecules include immune receptors on T cells and immune receptors on natural killer (NK) cells. Specific examples include TIGIT, PD-1, PD-L1, TIM-3, LAG-3, BTLA, CD268, CD267, CD266, CD226, CD160, CD137, CD96, CD70, CD47, CD40, CD30, CD28, CD27, CD18, NKG2D, VISTA, ICOS, B7-HE, GITR, OX40, KIR, SLAM7, CTLA-4, RANK, osteoprotegerin, BCMA, TNFR1, TNFR2, Fas, DR1, DR2, DR3, DR4, DR5, DR6, HVEM, NGFR, EDA2R, TR6, and TROY.

[0069] In one embodiment, the immune receptor recognition site of the antigen-binding molecule can be an agonist antibody against a costimulatory molecule belonging to the tumor necrosis factor receptor superfamily (TNFRSF) (hereinafter referred to as a TNFRSF agonist antibody) or its antigen-binding fragment. A TNFRSF agonist antibody means an antibody that, when attached to cells, tissues, or organisms expressing the TNF receptor superfamily, activates at least about 5%, more specifically at least about 10%, and more specifically at least about 15%, of the cells expressing the TNF receptor superfamily, when 100% activation is achieved under physiological conditions by equimolar amounts of a binding partner. In various specific examples, the agonist antibody of TNFRSF used in the pharmaceutical composition of the present invention can activate the activity of the cells by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, 1000%, or more. In one embodiment of the present invention, the immune receptor recognition site of the antigen-binding molecule binds to an endogenous immune receptor as an agonist and also binds to the chimeric receptor of the present invention as an agonist.

[0070] The target molecules for TNFRSF agonist antibodies are not particularly limited as long as they are factors that activate cells expressing the TNF receptor superfamily (e.g., T cells and NK cells). Preferred factors include, for example, CD137 and CD40. A more preferred factor is, for example, CD137. Examples of CD137 agonist antibodies include Urelumab (CAS registry number: 934823-49-1) and various other known CD137 agonist antibodies.

[0071] Examples of CD137 agonist antibodies include the following antibodies, such as those listed in the sequence number WO2015 / 156268: [1] An antibody having the amino acid sequence described in SEQ ID NO: 66 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 85 as the light chain variable region; [2] An antibody having the amino acid sequence described in SEQ ID NO: 67 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 86 as the light chain variable region; [3] An antibody having the amino acid sequence described in SEQ ID NO: 70 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 89 as the light chain variable region; [4] An antibody having the amino acid sequence described in SEQ ID NO: 76 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 95 as the light chain variable region; [5] An antibody having the amino acid sequence described in SEQ ID NO: 77 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 96 as the light chain variable region; [6] An antibody having the amino acid sequence described in SEQ ID NO: 78 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 97 as the light chain variable region; [7] An antibody described in any of [1] to [6], having the amino acid sequence described in SEQ ID NO: 99 as the heavy chain constant region, and the amino acid sequence described in SEQ ID NO: 59 or SEQ ID NO: 60 as the light chain constant region; [8] An antibody having activity equivalent to that of any of the antibodies described in [1] to [7]; [9] An antibody that binds to the same epitope as the antibody described in any of [1]~[7].

[0072] In the antibodies described in [8] above, "equivalent activity" means that the agonist activity to CD137 is 70% or more, preferably 80% or more, and more preferably 90% or more, of the binding activity of the antibody described in any of [1] to [7] above.

[0073] The present invention also provides antibodies that bind to the same epitopes as the anti-CD137 antibodies disclosed in the present invention, as described in [9] above. Such antibodies can be obtained, for example, by the following method.

[0074] Preferred examples of antibodies that bind to the same epitope as the antibody described in any of [1] to [7] above include, for example, an antibody that recognizes the region in the CD137 protein having the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (Sequence ID 15 described in WO2015 / 156268). Furthermore, an antibody that recognizes the region in the CD137 protein having the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC (Sequence ID 16 described in WO2015 / 156268) can also be mentioned.

[0075] By modifying the aforementioned anti-human CD137 antibody into a bispecific antibody with a tumor-specific antigen antibody (e.g., anti-human GPC3 antibody) and evaluating its tumor-specific antigen-dependent CD137 agonist activity, it is possible to provide an anti-tumor antigen / anti-human CD137 bispecific antibody that exhibits the desired effect, and this antibody can be used as the antigen-binding molecule of the present invention.

[0076] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on an antigen (target antigen or immune receptor) to which an antigen-binding molecule disclosed herein binds. Therefore, for example, an epitope can be defined by its structure. An epitope can also be defined by the binding activity of an antigen-binding molecule that recognizes it. If the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute it. Furthermore, if the epitope is a sugar chain, it can also be identified by a specific sugar chain structure.

[0077] A linear epitope is an epitope that contains an epitope whose amino acid primary sequence has been recognized. A linear epitope typically contains at least three, and most commonly at least five, amino acids, e.g., about eight to about ten, or six to twenty, in a specific sequence.

[0078] A stereoepitope, in contrast to a linear epitope, is an epitope in which the primary amino acid sequence containing the epitope is not a single defining component of the recognized epitope (for example, an epitope whose primary amino acid sequence is not necessarily recognized by the antibody defining the epitope). A stereoepitope may contain a larger number of amino acids than a linear epitope. With regard to the recognition of stereoepitopes, the target antigen recognition site or immunoreceptor recognition site of an antigen-binding molecule recognizes the three-dimensional structure of the peptide or protein. For example, if a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbone that form the stereoepitope are parallel, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. For example, see Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0079] The structure of the target antigen recognition site or immune receptor recognition site of an antigen-binding molecule that binds to an epitope of a target antigen or immune receptor is called a paratope. The epitope and paratope are stably bound by hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc., acting between them. This binding force between the epitope and paratope is called affinity. The sum of the binding forces when multiple antigens and multiple antigen-binding domains bind is called avidity. When antibodies containing multiple antigen-binding domains (i.e., polyvalent antibodies) bind to multiple epitopes, the binding forces (affinity) work synergistically, resulting in avidity that is higher than affinity.

[0080] In certain embodiments, the antigen-binding molecules provided herein have a range of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8(1E-08)M or less, for example 10 -8 (1E-08)M~10 -13 (1E-13)M, for example 10 -9 (1E-09)M~10 -13 It has a dissociation constant (Kd) of (1E-13)M).

[0081] The following methods for confirming the binding of an antigen-binding molecule to an epitope, including an antigen-binding domain for an antigen, or an antigen-binding domain as a target antigen recognition site or immune receptor recognition site, can be appropriately carried out in accordance with the following examples.

[0082] For example, if an antigen is a receptor expressed on the cell surface, the recognition of a linear epitope present in a certain antigen molecule by the antigen-binding domain of that antigen can be confirmed, for example, as follows: A linear peptide consisting of the amino acid sequence constituting the extracellular domain of a certain antigen is synthesized for the above purpose. This peptide can be synthesized chemically. Alternatively, it can be obtained by genetic engineering using the region of the cDNA of a certain antigen that codes for the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain and the antigen-binding domain for a certain antigen is evaluated. For example, the binding activity of the antigen-binding domain to the peptide can be evaluated by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity to the linear peptide can be determined based on the level of inhibition by the linear peptide in the binding of the antigen-binding domain to a cell expressing a certain antigen. Through these tests, the binding activity of the antigen-binding domain to the linear peptide can be determined.

[0083] Furthermore, the recognition of a stereoepitope by a target antigen recognition site or an immune receptor recognition site can be confirmed as follows: Cells expressing a target antigen or immune receptor are prepared. When the target antigen recognition site or immune receptor recognition site for the target antigen or immune receptor comes into contact with the cell, it binds strongly to the cell, while the recognition site does not substantially bind to a linear peptide consisting of an amino acid sequence constituting a fixed target antigen or immune receptor, or to a linear peptide consisting of an amino acid sequence constituting the extracellular domain of a target antigen or immune receptor that has been denatured using a common denaturing agent such as guanidine. Here, substantially non-binding means that the binding activity of the latter is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less, of the binding activity to the target antigen or immune receptor expressing cell.

[0084] Furthermore, as a method for confirming the binding activity of an antigen-binding molecule to a target antigen or immune receptor, one method is to measure the Kd value by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using an antigen-binding molecule and a target antigen or immune receptor or fragments thereof. For example, the binding affinity of an antigen-binding molecule in solution is measured by equilibrating the antigen-binding domain with a minimum concentration of (125I) labeled antigen in the presence of an increasing series of unlabeled antigens, and then measuring the antigen-binding domain. It is measured by capturing it with a plate coated with the main substance (see, for example, Chen et al., J. Mol. Biol. 293:865-881(1999)).

[0085] In another embodiment, Kd is measured by surface plasmon resonance using BIACORE®. For example, the measurement method using BIACORE®-2000 or BIACORE®-3000 (BIACORE, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8 before being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78 nM to 500 nM) of the antigen-binding molecule (target antigen recognition site or immunoreceptor recognition site) in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20®) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. The binding rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting binding and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. Furthermore, the apparent dissociation constant (Kd) can also be determined using equilibrium analysis. Refer to the protocols included with BIACORE® for these methods. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999) and Methods Enzymol. 2000;323:325-40.Further, in the surface plasmon resonance assay, the amount of the immobilized protein, the amount of the protein used for the reaction, the temperature, and the solution composition can be varied as appropriate by those skilled in the art. When the on-rate exceeds 10 6 M -1 s -1 -1, the on-rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25°C of 20 nM antigen-binding domain in PBS, pH 7.2 in the presence of increasing concentrations of antigen, measured on a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0086] Furthermore, the binding activity of an antigen-binding molecule to a target antigen or an immune receptor can also be measured by known methods for measuring intermolecular interactions such as electrochemiluminescence. In addition, instead of the antigen-binding molecule used in the assay described herein, a fragment corresponding to the target antigen recognition site or the immune receptor recognition site can be used, and as the target antigen recognition site or the immune receptor recognition site, a fragment corresponding to the target antigen recognition site or the immune receptor recognition site, or an antigen-binding molecule can be used.

[0087] Examples of methods for measuring the binding activity of an antigen-binding molecule to cells expressing a target antigen or an immune receptor include the method described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, evaluation can be performed based on the principles of ELISA or FACS (fluorescence activated cell sorting) using cells expressing the target antigen or immune receptor as the antigen.

[0088] In the ELISA format, the binding activity of an antigen-binding molecule is quantitatively evaluated by comparing the signal levels produced by the enzymatic reaction. Specifically, the test antigen-binding molecule is added to an ELISA plate immobilized with cells expressing a particular antigen, and the antigen-binding molecule bound to the cells is detected using an enzyme-labeled antibody that recognizes the antigen-binding domain. Alternatively, in FACS, a dilution series of the test antigen-binding molecule is prepared and compared against cells expressing a particular antigen. By determining the antibody binding titer, the binding activity of a test antigen-binding molecule to cells expressing a particular antigen can be compared.

[0089] The binding of a test antigen-binding molecule to a target antigen or immune receptor expressed on the cell surface suspended in a buffer solution can be detected by a flow cytometer. Examples of known flow cytometers include the following: FACSCanto TM II FACSAria TM FACSArray TM FACSVantage TM SE FACSCalibur TM (All are product names of BD BioSciences) EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are product names of Beckman Coulter).

[0090] One example of a suitable method for measuring the binding activity of an antigen-binding molecule to a target antigen or immune receptor is as follows: First, cells expressing a certain antigen are stained with a FITC-labeled secondary antibody that recognizes the test antigen-binding molecule. The test antigen-binding molecule is then diluted with a suitable buffer to prepare it to the desired concentration for use. For example, it can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, FACSCalibur TM Fluorescence intensity and cell count are measured by BD (Biotechnology). The amount of antigen-binding molecule bound to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining this Geometric Mean value, the binding activity of the test antigen-binding molecule, which is expressed by the amount of the test antigen-binding molecule bound, can be measured.

[0091] The sharing of an epitope between one antigen-binding molecule and another can be confirmed by competition between the two molecules for the same epitope. Competition between antigen-binding molecules can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay.

[0092] Specifically, in a cross-blocking assay, an antigen protein derived from a target antigen or immune receptor, coated on a well of a microtiter plate, is pre-incubated in the presence or absence of a candidate competing antigen-binding molecule, after which the test antigen-binding molecule is added. The amount of the test antigen-binding molecule bound to a particular antigen protein in the well is indirectly correlated with the binding ability of the competing candidate antigen-binding molecule that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding molecule for the same epitope, the lower the binding activity of the test antigen-binding molecule to the well coated with the antigen protein.

[0093] The amount of antigen-binding molecules bound to a well via an antigen protein can be easily measured by pre-labeling the antigen-binding molecules. For example, biotin-labeled antigen-binding molecules can be measured using an avidin peroxidase conjugate and an appropriate substrate. Cross-blocking assays utilizing enzymatic labeling such as peroxidase are specifically called competitive ELISA assays. Antigen-binding molecules can be labeled with other detectable or measurable labeling substances. Specifically, radiolabeling and fluorescent labeling are well known.

[0094] Compared to the binding activity obtained in a control test performed in the absence of candidate competing antigen-binding molecule aggregates, the binding activity of the competing antigen-binding molecule to a certain antigen is compared to that of the candidate competing antigen-binding molecule. If the test antigen-binding molecule can block at least 20%, preferably at least 20-50%, and more preferably at least 50%, then the test antigen-binding molecule is either binding to substantially the same epitope as the competing antigen-binding molecule, or is an antigen-binding molecule that competes for binding to the same epitope.

[0095] If the structure of the epitope to which an antigen-binding molecule binds has been identified, the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by comparing the binding activity of both antigen-binding molecules to peptides or polypeptides into which amino acid mutations have been introduced into the peptide constituting the epitope.

[0096] One method for measuring such binding activity is to compare the binding activity of a test antigen-binding molecule and a control antigen-binding molecule to a linear peptide into which a mutation has been introduced in the aforementioned ELISA format. Alternatively, the binding activity to the mutated peptide bound to a column can be measured by quantitatively determining the antigen-binding molecule eluted into the eluate after the test antigen-binding molecule and the control antigen-binding molecule have been passed through the column. Methods for adsorbing the mutated peptide onto a column as a fusion peptide with, for example, GST, are well known.

[0097] Furthermore, if the identified epitope is a stereoepitope, the sharing of the epitope between the test antigen-binding molecule and the control antigen-binding molecule can be evaluated by the following method. First, cells expressing a certain antigen and cells expressing a certain antigen with a mutation introduced into the epitope are prepared. The test antigen-binding molecule and the control antigen-binding molecule are added to the cell suspension, in which these cells are suspended in an appropriate buffer such as PBS. Next, FITC-labeled antibodies that can recognize the test antigen-binding molecule and the control antigen-binding molecule are added to the cell suspension, which has been washed with an appropriate buffer. The fluorescence intensity and cell number of cells stained with the labeled antibody are evaluated using FACSCalibur. TM (Measured by BD Corporation). The concentrations of the test antigen-binding molecule and the control antigen-binding molecule are adjusted to the desired concentration by appropriately diluting them with a suitable buffer. For example, concentrations between 10 μg / ml and 10 ng / ml are used. The amount of labeled antibody bound to the cells is measured by CELL QUEST TM The fluorescence intensity obtained by analysis using software (BD Corporation) is reflected in the Geometric Mean value. In other words, by obtaining this Geometric Mean value, the binding activity of the test antigen-binding molecule and the control antigen-binding molecule, which is represented by the amount of labeled antibody bound, can be measured.

[0098] Furthermore, to confirm competition between antigen-binding molecules for the same epitope as other antigen-binding molecules, methods other than ELISA and FACS, such as radiolabeled antigen binding assay (RIA), BIACORE® surface plasmon resonance assay, and electrochemiluminescence, can also be used.

[0099] The Geometric Mean comparison value (ΔGeo-Mean value of the mutated antigen) obtained by the analysis, which reflects the amount of binding of the test antigen-binding molecule to cells expressing a certain mutated antigen, is compared with the ΔGeo-Mean comparison value which reflects the amount of binding of the test antigen-binding molecule to cells expressing a certain antigen. In this case, it is particularly preferable that the concentrations of the test antigen-binding molecule used when determining the ΔGeo-Mean comparison values ​​for cells expressing the mutated antigen and cells expressing a certain antigen are prepared to be the same or substantially the same. An antigen-binding molecule that has been confirmed to recognize an epitope in a certain antigen in advance is used as a control antigen-binding molecule.

[0100] If the ΔGeo-Mean comparison value of the test antigen-binding molecule to cells expressing a mutated antigen is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value of the test antigen-binding molecule to cells expressing a certain antigen, it is considered that the molecule "substantially does not bind to cells expressing a mutated antigen." The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). If the comparative values ​​can be considered substantially equivalent, then the epitopes of the test antigen-binding molecule and the control antigen-binding molecule can be evaluated as identical.

[0101] In this specification, the term "transport portion" refers to the portion of an antigen-binding molecule other than the target antigen recognition site and the immune receptor recognition site. The transport portion of the present invention is typically a peptide or polypeptide composed of amino acids, and in one specific embodiment, the transport portion in the antigen-binding molecule is linked to the target antigen recognition site and the immune receptor recognition site. The transport portion of the present invention may be a series of peptides or polypeptides linked by amide bonds, or a complex in which multiple peptides or polypeptides are formed by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0102] When the antigen-binding molecule is an antibody, one embodiment of extending its half-life in the blood is to confer FcRn binding ability to the antigen-binding molecule. To confer FcRn binding ability, one typically involves creating an FcRn binding region within the antigen-binding molecule. An FcRn binding region is a region that has binding ability to FcRn, and any structure can be used as long as it has binding ability to FcRn.

[0103] The presence of an FcRn binding domain allows IgG molecules to be taken up into cells via the FcRn salvage pathway and then returned to the plasma. For example, the relatively long retention time (slow disappearance) of IgG molecules in plasma is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions. IgG molecules that do not bind to FcRn proceed to lysosomes and are degraded there, but IgG molecules that do bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma, returning to the plasma.

[0104] The FcRn binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn binding region is the Fc region of an antibody. However, regions that can bind to polypeptides that have the ability to bind to FcRn, such as albumin and IgG, can indirectly bind to FcRn via albumin or IgG, so the FcRn binding region in the present invention may be a region that binds to such polypeptides that have the ability to bind to FcRn.

[0105] The binding activity of the FcRn binding domain in the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described in the section on binding activity, and the conditions can be appropriately determined by those skilled in the art. The binding activity to human FcRn can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate (kd), or apparent dissociation rate (kd). These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), scatchard plots, flow cytometers, etc., can be used.

[0106] The conditions for measuring the binding activity of the FcRn binding region to FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under conditions of MES buffer and 37°C, as described in WO2009 / 125825. Furthermore, the measurement of the binding activity of the FcRn binding region of the present invention to FcRn can be performed by methods known to those skilled in the art, for example, by using Biacore (GE Healthcare).

[0107] The pH used for measurement conditions may be any pH between pH 4.0 and pH 6.5 to evaluate the binding affinity between the FcRn binding region and FcRn. Preferably, a pH of 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn binding region and human FcRn. The temperature used for measurement conditions may be the binding affinity between the FcRn binding region and FcRn. The affinity may be evaluated at any temperature between 10°C and 50°C. Preferably, temperatures between 15°C and 40°C are used to determine the binding affinity between the FcRn binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn binding region and FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0108] One example of an FcRn binding region, though not limited to this, is the Fc region of an IgG antibody. When using the Fc region of an IgG antibody, the type is not limited, and it is possible to use Fc regions of IgG1, IgG2, IgG3, IgG4, etc.

[0109] Furthermore, not only the Fc region of natural IgG antibodies, but also modified Fc regions with one or more amino acid substitutions can be used, as long as they retain FcRn binding ability. For example, EU numbering in the IgG antibody Fc region: 237th, 238th, 239th, 248th, 250th, 252nd, 254th, 255th, 256th, 257th, 258th, 265th, 270th, 286th, 289th, 297th, 298th, 303rd, 305th, 307th, 308th, 309th, 311th, 312th, 314th It is possible to use a modified Fc region containing an amino acid sequence in which at least one amino acid selected from positions 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0110] More specifically, EU numbering in the Fc region of IgG antibodies Amino acid substitution where Gly at position 237 is replaced with Met. An amino acid substitution where the 238th Pro is replaced with Ala. An amino acid substitution where Ser at position 239 is replaced with Lys. An amino acid substitution where Lys at position 248 is replaced with Ile. Amino acid substitutions that replace the 250th Thr with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace Met at position 252 with Phe, Trp, or Tyr. An amino acid substitution where Ser at position 254 is replaced with Thr. Amino acid substitution where Arg at position 255 is replaced with Glu, Amino acid substitutions that replace the 256th Thr with Asp, Glu, or Gln. Amino acid substitutions that replace the 257th Pro with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val. An amino acid substitution in which Glu at position 258 is replaced with His. An amino acid substitution in which Asp at position 265 is replaced with Ala. An amino acid substitution where Asp at position 270 is replaced with Phe. Amino acid substitutions that replace the 286th Asn with Ala or Glu. Amino acid substitution that replaces the 289th Thr with His, An amino acid substitution in which the 297th Asn is replaced with Ala. An amino acid substitution in which Ser at position 298 is replaced with Gly. An amino acid substitution in which the 303rd Val is replaced with Ala. An amino acid substitution in which the 305th Val is replaced with Ala. Amino acid substitutions that replace the 307th Thr with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace the 308th Val with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr. Amino acid substitutions that replace the 309th Leu or Val with Ala, Asp, Glu, Pro, or Arg. Amino acid substitutions that replace the 311th Gln with Ala, His, or Ile. Amino acid substitutions that replace Asp at position 312 with Ala or His. Amino acid substitutions that replace the 314th Leu with Lys or Arg. Amino acid substitutions that replace the 315th Asn with Ala or His, An amino acid substitution in which Lys at position 317 is replaced with Ala. Amino acid substitution where Asn at position 325 is replaced with Gly. Amino acid substitution where Ile at position 332 is replaced with Val. An amino acid substitution where Lys at position 334 is replaced with Leu. An amino acid substitution where the 360th Lys is replaced with His. An amino acid substitution in which Asp at position 376 is replaced with Ala. An amino acid substitution where Glu at position 380 is replaced with Ala. An amino acid substitution in which Glu at position 382 is replaced with Ala. Amino acid substitutions that replace the 384th Asn or Ser with Ala, Amino acid substitutions that replace the 385th Gly with Asp or His, Amino acid substitution where Gln at position 386 is replaced with Pro. An amino acid substitution where Pro at position 387 is replaced with Glu. Amino acid substitutions that replace the 389th Asn with Ala or Ser. An amino acid substitution in which Ser at position 424 is replaced with Ala. Amino acid substitutions that replace the 428th Met with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr. An amino acid substitution where His at position 433 is replaced with Lys. Amino acid substitutions that replace the 434th Asn with Ala, Phe, His, Ser, Trp, or Tyr, and Amino acid substitutions that replace Tyr or Phe at position 436 with His It is possible to use a modified Fc region that includes at least one amino acid substitution selected from the above.

[0111] From another perspective, EU numbering in the IgG antibody Fc region. Met at amino acid position 237, Ala at amino acid position 238, Lys at the 239th amino acid, Ile at the 248th amino acid, Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at the 250th amino acid, Phe, Trp, or Tyr at the 252nd amino acid, Thr at amino acid 254, Glu at the 255th amino acid, Asp, Glu, or Gln at the 256th amino acid, Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257 His at amino acid position 258, Ala at the 265th amino acid, Phe at amino acid 270, Ala or Glu at the 286th amino acid, His at the 289th amino acid, Ala at the 297th amino acid, Gly at the 298th amino acid, Ala at amino acid position 303, Ala at amino acid position 305, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid position 307, Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid position 308, Ala, Asp, Glu, Pro, or Arg at amino acid position 309, Ala, His, or Ile at the 311th amino acid, Ala or His at the 312th amino acid, Lys or Arg at the 314th amino acid, Ala or His at the 315th amino acid, Ala at amino acid position 317, Gly at amino acid position 325, Val at amino acid position 332, Leu at amino acid position 334, His at amino acid position 360, Ala at the 376th amino acid, Ala at amino acid position 380, Ala at amino acid position 382, Ala at amino acid position 384, Asp or His at the 385th amino acid, Pro at amino acid 386, Glu at amino acid position 387, Ala or Ser at the 389th amino acid, Ala at amino acid position 424, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid position 428, Lys at amino acid position 433, Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and His at amino acid position 436 It is possible to use an Fc region containing at least one amino acid selected from the following.

[0112] Another method to extend the half-life in the blood is to bind an antigen-binding molecule to albumin. Albumin is not excreted by the kidneys and has FcRn-binding properties, resulting in a long half-life of 17-19 days in the blood (J Clin Invest. 1953 Aug; 32(8): 746-768). Therefore, the protein bound to albumin becomes bulkier and can indirectly bind to FcRn, which has been reported to increase the half-life in the blood (Antibodies 2015, 4(3), 141-156).

[0113] Furthermore, one embodiment of extending the half-life in the blood is to PEGylate the antigen-binding molecule. It is thought that PEGylation of a protein increases its bulk and simultaneously suppresses its degradation by proteases in the blood, thereby extending the half-life of the protein in the blood (J Pharm Sci. 2008 Oct;97(10):4167-83.).

[0114] In some embodiments of the present invention, the antigen-binding molecule includes the antibody Fc region. In one specific embodiment, the antigen-binding molecule includes the CH2 and CH3 domains of a human IgG antibody. In another specific embodiment, the antigen-binding molecule includes a portion extending from Cys226 or Pro230 of the human IgG1 antibody heavy chain to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region may or may not be present.

[0115] In some embodiments of the present invention, the antigen-binding molecule includes an antibody constant region. In a more preferred embodiment, the antigen-binding molecule includes an IgG antibody constant region. In a more preferred embodiment, the antigen-binding molecule includes a human IgG antibody constant region. In an even more preferred embodiment, the antigen-binding molecule is a bispecific antibody and includes a human IgG antibody constant region.

[0116] In some further embodiments of the present invention, the antigen-binding molecule comprises a region having a structure substantially similar to the constant region of the antibody heavy chain, and a region substantially similar to the antibody light chain, which is bound to this region by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions. Includes regions having a structure.

[0117] As used herein, the term "IgG antibody-like molecule" is used to define a molecule having a substantially similar structure to an IgG antibody, specifically a portion substantially similar to the structure of a constant domain or constant region, and a portion substantially similar to the structure of an IgG antibody, specifically a variable domain or variable region, and having a substantially similar three-dimensional structure to an IgG antibody. However, as used herein, an "IgG antibody-like molecule" is not limited to exhibiting antigen-binding activity while maintaining a structure similar to an IgG antibody.

[0118] When the antigen-binding molecule is an IgG antibody-like molecule, embodiments in which a target antigen recognition site and an immune receptor recognition site are provided in the portions corresponding to the two variable regions of an IgG antibody, respectively, are included in the present invention.

[0119] In this specification, the term "specificity" refers to the property of an antigen-binding molecule such that its target antigen recognition site or immune receptor recognition site does not substantially bind to molecules other than a specific target antigen or immune receptor. It is also used when an antigen-binding molecule has specificity to an epitope contained in a specific target antigen or immune receptor. Substantially non-binding is determined in accordance with the above-described method for measuring binding activity, and means that the binding activity of the specific-binding molecule to molecules other than the specific target antigen or immune receptor is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to the other molecule.

[0120] The antigen-binding molecule in this invention can usually be produced by loading (inserting) the nucleic acid encoding it onto a suitable vector, introducing it into a host cell, and using conventional methods. The vector is not particularly limited as long as it stably holds the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (manufactured by Stratagene) is preferred as a cloning vector, but various commercially available vectors can be used. When using a vector for the purpose of producing polypeptides used in the implementation of this invention (e.g., chimeric receptors, IgG antibodies, bispecific antibodies, antigen-binding molecules, etc.), an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses polypeptides in vitro, in E. coli, in cultured cells, or in living organisms. However, for example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) is preferred for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) is preferred for living organisms. Insertion of the DNA of the present invention into the vector can be carried out by conventional methods, for example, by a ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0121] There are no particular restrictions on the host cells used, and various host cells can be used depending on the purpose. Examples of cells used to express antigen-binding molecules include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electropulse puncture (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method (GIBCO-BRL), and microinjection.

[0122] To secrete antigen-binding molecules expressed in host cells into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, the nucleus encoding the target antigen-binding molecule generates an appropriate secretion signal. These signals can be incorporated into acids. These signals may be endogenous or heterogeneous for the target polypeptide.

[0123] In the above manufacturing method, if the antigen-binding molecule of the present invention is secreted into the culture medium, the culture medium is recovered. If the antigen-binding molecule of the present invention is produced inside a cell, the cell is first lysed, and then the antigen-binding molecule is recovered.

[0124] To recover and purify the antigen-binding molecules of the present invention from recombinant cell cultures, known methods can be used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography.

[0125] The amino acids included in the amino acid sequence described in this invention may undergo post-translational modifications (for example, modification to pyroglutamic acid by pyroglutamylation of the N-terminal glutamine is a modification well known to those skilled in the art), but even when amino acids are modified post-translation in this way, they are naturally still included in the amino acid sequence described in this invention.

[0126] Methods for producing antibodies with desired binding activity are known to those skilled in the art. In the present invention, antigen-binding molecules can be used in which a molecule expressed on the surface of a target cell (lesion cell) is the antigen (target antigen). When the target cell is a tumor cell or cancer cell, the antigen is exemplified herein as a tumor antigen. The following is an example of a method for producing an antibody that binds to a tumor antigen.

[0127] Antibodies that bind to tumor antigens can be obtained as polyclonal or monoclonal antibodies using known methods. Mammalian-derived monoclonal antibodies are preferably produced as such antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes using genetic engineering techniques.

[0128] Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as follows: Mammals are immunized according to a standard immunization method using a tumor antigen protein as the sensitizing antigen. The resulting immune cells are fused with known parent cells by a standard cell fusion method. Next, hybridomas that produce anti-tumor antigen antibodies can be selected by screening monoclonal antibody-producing cells using a standard screening method.

[0129] Specifically, the production of monoclonal antibodies is carried out as follows: First, a tumor antigen protein can be obtained by expressing a tumor antigen gene, which can then be used as a sensitizing antigen for antibody acquisition. That is, a suitable host cell is transformed by inserting the gene sequence encoding the tumor antigen into a known expression vector. The desired human tumor antigen protein is purified from the host cell or culture supernatant by a known method. To obtain a soluble tumor antigen from the culture supernatant, for example, a protein in which the hydrophobic region of the tumor antigen polypeptide sequence has been deleted can be used. Similarly, purified natural GPC3 protein can also be used as a sensitizing antigen.

[0130] The purified tumor antigen protein can be used as a sensitizing antigen for immunization against mammals. Partial peptides of the tumor antigen can also be used as sensitizing antigens. These partial peptides can be obtained by chemical synthesis from the amino acid sequence of the human tumor antigen. They can also be obtained by incorporating a portion of the tumor antigen gene into an expression vector and expressing it. Although it can also be obtained by degrading tumor antigen proteins using proteolytic enzymes, the region and size of the tumor antigen peptide used as a partial peptide are not particularly limited to any special form. Preferably, the number of amino acids constituting the peptide to be used as a sensitizing antigen is at least 5, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0131] Furthermore, fusion proteins obtained by fusing a desired partial polypeptide or peptide of a tumor antigen protein with a different polypeptide can be used as sensitizing antigens. For example, antibody Fc fragments or peptide tags can be suitably used to produce fusion proteins used as sensitizing antigens. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in-frame, and then inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). As an example, methods for obtaining GPC3 used as a sensitizing antigen and immunization methods using it are specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0132] While the mammals immunized with the sensitizing antigen are not limited to specific animals, it is preferable to select them considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferred.

[0133] The animals described above are immunized with the sensitizing antigen according to known methods. For example, a common method is to administer the sensitizing antigen to mammals by injection intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen, diluted to an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified. After emulsification, the sensitizing antigen is administered to mammals several times every 4 to 21 days. A suitable carrier may also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0134] Furthermore, hybridomas that produce the desired antibody can also be produced using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered, and the sensitized antigen is expressed in the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which protein antigens are administered to immunized animals, DNA immunization is expected to have the following advantages. - The structure of membrane proteins can be maintained, allowing for immune stimulation. - There is no need to purify immune antigens.

[0135] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing a tumor antigen protein is administered to an immunized animal. The DNA encoding the tumor antigen can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector and administered to an immunized animal. Commercial expression vectors such as pcDNA3.1 can be suitably used as the expression vector. Commonly used methods can be used to administer the vector into a living organism. For example, DNA immunization is performed by introducing gold particles to which the expression vector is adsorbed into the cells of an immunized animal using a gene gun. Furthermore, antibodies that recognize tumor antigens can also be produced using the method described in International Publication WO2003 / 104453.

[0136] After the mammal has been immunized in this manner and an increase in antibody titers binding to tumor antigens in the serum has been confirmed, immune cells are collected from the mammal and used for cell fusion. Splenocytes, in particular, may be used as preferred immune cells.

[0137] Mammalian myeloma cells are used as the cells fused with the aforementioned immune cells. It is preferable that the myeloma cells possess appropriate selection markers for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells lacking HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT-selective medium and die, but when fused with normal cells, they can continue DNA synthesis using the normal cell's salvage pathway and thus proliferate even in HAT-selective medium.

[0138] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA will die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker, known as G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0139] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc., can be suitably used.

[0140] Cell fusion between the immune cells and myeloma cells is basically performed according to known methods, such as the method of Köhler and Myrstein et al. (Methods Enzymol. (1981) 73, 3-46).

[0141] More specifically, the cell fusion can be carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and additional adjuvants such as dimethyl sulfoxide may be added as desired to further enhance fusion efficiency.

[0142] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, or other common culture mediums used for this type of cell culture can be used, and serum supplements such as fetal bovine serum (FCS) may be suitably added.

[0143] Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, and then adding a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C, usually at a concentration of 30 to 60% (w / v). The desired fused cells (hybridomas) are formed by the gradual mixing of the mixture. Subsequently, the appropriate culture medium mentioned above is added sequentially. By repeatedly centrifugating and removing the supernatant, cell fusion agents and other substances unfavorable to hybridoma growth can be removed.

[0144] The hybridomas obtained in this manner can be selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing with the HAT culture medium can be continued for a sufficient time (usually several days to several weeks) to kill cells other than the desired hybridoma (non-fusion cells). Subsequently, screening and single cloning of hybridomas that produce the desired antibody is performed using a standard limiting dilution method.

[0145] The hybridomas obtained in this way can be selected by using a selective culture medium corresponding to the selection markers present in the myeloma used for cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that successfully fuse with normal cells can be selectively proliferated in the HAT culture medium. Culturing with the HAT culture medium is continued for a sufficient amount of time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, generally, the desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas that produce the desired antibody can be performed using the usual limiting dilution method.

[0146] Screening and single cloning of desired antibodies can be suitably carried out by known antigen-antibody reaction-based screening methods. For example, a monoclonal antibody that binds to GPC3 can bind to GPC3 expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows for the measurement of antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

[0147] To screen hybridomas that produce the monoclonal antibody of the present invention by FACS, cells expressing GPC3 are first prepared. Preferred cells for screening are mammalian cells that forcibly express the tumor antigen used. By using untransformed mammalian cells as the host cells as a control, the antibody binding activity to the tumor antigen on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to GPC3-forcibly expressing cells, hybridomas that produce tumor antigen monoclonal antibodies can be obtained.

[0148] Alternatively, the binding activity of antibodies against immobilized tumor antigen-expressing cells can be evaluated based on the principles of ELISA. For example, GPC3-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of hybridomas is brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody with antigen-binding ability, selected through these screenings, can be cloned by methods such as limiting dilution.

[0149] The hybridomas producing monoclonal antibodies thus created can be subcultured in a normal culture medium. Furthermore, these hybridomas can be stored for extended periods in liquid nitrogen.

[0150] The hybridoma is cultured according to a standard method, and the desired monoclonal ion is obtained from the culture supernatant. Monoclonal antibodies can be obtained. Alternatively, hybridomas can be administered to compatible mammals to proliferate, and monoclonal antibodies can be obtained from their ascites fluid. The former method is suitable for obtaining high-purity antibodies.

[0151] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably utilized. By incorporating the cloned antibody gene into a suitable vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur.J. Biochem.(1990)192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0152] For example, cDNA encoding the variable region (V region) of an antibody can be obtained from hybridoma cells that produce antibodies that bind to tumor antigens. To do this, total RNA is usually extracted from the hybridoma first. Methods for extracting mRNA from cells include, for example, the following: - Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159).

[0153] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences), or similar kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences). mRNA can be obtained from hybridomas using such kits. From the obtained mRNA, cDNA encoding the antibody V region can be synthesized using reverse transcriptase. The cDNA is then used for AMV cDNA can be synthesized using the Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation), etc. Furthermore, for cDNA synthesis and amplification, the SMART RACE cDNA amplification kit (manufactured by Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) may be used as appropriate. In addition, appropriate restriction enzyme sites, as described later, can be introduced at both ends of the cDNA during the synthesis process.

[0154] The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. A recombinant vector is thus prepared, introduced into E. coli or other organisms, and after colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector possesses the target cDNA base sequence is confirmed by known methods, such as dideoxynucleotide chain intermination.

[0155] To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a convenient approach. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, yielding a 5'-RACE cDNA library. Commercially available kits, such as the SMART RACE cDNA amplification kit, can be used as appropriate for synthesizing the 5'-RACE cDNA library.

[0156] The obtained 5'-RACE cDNA library is used as a template to amplify the antibody gene by PCR. Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

[0157] Specifically, for example, when the goal is to obtain the gene encoding mouse IgG, primers capable of amplifying the genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains, and the κ and λ chains as light chains, can be used. To amplify the variable region genes of IgG, the 3' side is generally used. For the primers used, a primer that anneals to the constant region, which is close to the variable region, is used. On the other hand, for the 5' side primer, the primer included with the 5' RACE cDNA library preparation kit is used.

[0158] Using the thus amplified PCR product, an immunoglobulin composed of a combination of heavy chains and light chains can be reconstituted. A desired antibody can be screened using the antigen-binding activity of the reconstituted immunoglobulin as an indicator. For example, when the purpose is to obtain an antibody against GPC3, it is further preferable that the binding of the antibody to GPC3 is specific. The antibody used in the present invention can be screened, for example, by the following method; (1) a step of contacting an antibody comprising a V region encoded by cDNA obtained from a hybridoma with an antigen-expressing cell; (2) a step of detecting the binding between the antigen-expressing cell and the antibody, and (3) a step of selecting an antibody that binds to the antigen-expressing cell.

[0159] Methods for detecting the binding between an antibody and a tumor antigen-expressing cell are known in the art. Specifically, the binding between an antibody and a tumor antigen-expressing cell can be detected by a technique such as FACS as mentioned above. Fixed specimens of tumor antigen-expressing cells can be appropriately used to evaluate the binding activity of an antibody.

[0160] As a method for screening antibodies using binding activity as an indicator, a panning method using phage is also suitably used. When antibody genes are obtained as a library of heavy chain and light chain subclasses from a polyclonal antibody-expressing cell population, a screening method using phage is advantageous. Genes encoding variable regions of heavy chains and light chains can be linked by an appropriate linker sequence to form a single-chain Fv (scFv). By inserting a gene encoding scFv into a phage vector, phage expressing scFv on the surface can be obtained. After contacting this phage with a desired antigen, by recovering the phage bound to the antigen, DNA encoding scFv having the target binding activity can be recovered. By repeating this operation as necessary, scFv having the desired binding activity can be concentrated.

[0161] After obtaining cDNA encoding the V region of an antibody that binds to the target tumor antigen, the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest base sequences that appear infrequently in the base sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, insertion of a restriction enzyme that provides an adhesive end is preferable. By inserting the cDNA encoding the V region of the anti-GPC3 antibody digested as described above into a suitable expression vector, an antibody expression vector can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the constant region and the variable region originate from different sources. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human allologous chimeric antibodies are also included in the chimeric antibodies of this invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' end of an expression vector containing DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by in-frame fusion of the two, which have been digested with the same combination of restriction enzymes.

[0162] To produce monoclonal antibodies, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression regulatory region. This expression regulatory region for antibody expression includes, for example, enhancers and promoters. Additionally, an appropriate signal sequence may be added to the amino terminus so that the expressed antibody is secreted extracellularly. In the embodiments described later, a signal sequence is added. As the signal sequence, for example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS is used, but other suitable signal sequences may also be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Then, by transforming an appropriate host cell with this expression vector, a recombinant cell that expresses DNA encoding an antibody that binds to a target tumor antigen can be obtained.

[0163] For expression of an antibody gene, DNAs encoding an antibody heavy chain (H chain) and light chain (L chain) are inserted into separate expression vectors, respectively. When the same host cell is co-transfected simultaneously with vectors into which the H chain and L chain have been inserted, an antibody molecule comprising the H chain and L chain can be expressed. Alternatively, a host cell can be transformed by inserting DNAs encoding the H chain and L chain into a single expression vector (see International Publication WO 94 / 11523).

[0164] Many combinations of host cells and expression vectors for producing an antibody by introducing an isolated antibody gene into an appropriate host are known in the art. Any of these expression systems can be applied to isolating a domain comprising the antibody variable region of the present invention. When a eukaryotic cell is used as the host cell, an animal cell, plant cell, or fungal cell can be appropriately used. Specifically, the following cells can be exemplified as animal cells: (1) Mammalian cells: CHO, COS, myeloma, BHK (baby hamster kidney), Hela, Vero, etc. (2) Amphibian cells: Xenopus laevis oocytes, etc. (3) Insect cells: sf9, sf21, Tn5, etc.

[0165] Alternatively, as for plant cells, antibody gene expression systems using cells from the Nicotiana genus, such as Nicotiana tabacum, are known. Callus-cultured cells can be used as appropriate for plant cell transformation.

[0166] Furthermore, the following types of fungal cells can be used: - Yeast: Saccharomyces species such as Saccharomyces serevisiae, and Pichia species such as methanol-utilizing yeast (Pichia pastoris). - Filamentous fungi: Aspergillus species such as Aspergillus niger.

[0167] Furthermore, antibody gene expression systems using prokaryotic cells are also known. For example, when using bacterial cells, bacterial cells such as Escherichia coli (E. coli) and Bacillus subtilis can be used as appropriate. An expression vector containing the target antibody gene is introduced into these cells by transformation. By culturing the transformed cells in vitro, the desired antibody can be obtained from the culture of the transformed cells.

[0168] In addition to the host cells mentioned above, transgenic animals can also be used to produce recombinant antibodies. That is, antibodies can be obtained from animals into which the gene encoding the desired antibody has been introduced. For example, the antibody gene can be constructed as a fusion gene by inserting it in-frame into a gene encoding a protein that is specifically produced in milk. As the protein secreted in milk, for example, goat β-casein can be used. The DNA fragment containing the fusion gene into which the antibody gene has been inserted is injected into a goat embryo, and the injected embryo is introduced into a female goat. From the milk produced by the transgenic goat (or its offspring) born from the goat that received the embryo, the desired antibody can be obtained as a fusion protein with the milk protein. Furthermore, hormones can be administered to the transgenic goat to increase the amount of milk containing the desired antibody produced from the transgenic goat (Bio / Technology (1994), 12 (7), 699-702).

[0169] When the antigen-binding molecules described herein are administered to humans, the domain containing the antibody variable region in the antigen-binding molecule may appropriately be a domain derived from a recombinant antibody that has been artificially modified for purposes such as reducing heterologous antigenicity to humans. Recombinant antibodies include, for example, humanized antibodies. These modified antibodies can be appropriately manufactured using known methods.

[0170] The variable region of an antibody used to create a domain containing the antibody variable region in the antigen-binding molecule described herein typically consists of three complementarity-determining regions (CDRs) flanked by four framework regions (FRs). The CDRs are essentially the regions that determine the antibody's binding specificity. The amino acid sequences of CDRs are highly diverse. On the other hand, the amino acid sequences constituting the FRs often exhibit high identity even among antibodies with different binding specificities. Therefore, it is generally believed that the binding specificity of one antibody can be transferred to another antibody by transplanting the CDRs.

[0171] Humanized antibodies are also called reshaped human antibodies. Specifically, known examples include humanized antibodies obtained by transplanting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody. General genetic recombination methods for obtaining humanized antibodies are also known. Specifically, Overlap Extension PCR is a known method for transplanting the CDR of a mouse antibody into the FR of a human antibody. In Overlap Extension PCR, the nucleotide sequence encoding the mouse antibody CDR to be transplanted is added to the primer for synthesizing the human antibody FR. Primers are prepared for each of the four FRs. Generally, when transplanting mouse CDRs into human FRs, it is considered advantageous to select human FRs with high identity to the mouse FRs in order to maintain the function of the CDRs. That is, it is generally preferable to use human FRs with amino acid sequences that have high identity to the amino acid sequences of the FRs adjacent to the mouse CDR to be transplanted.

[0172] Furthermore, the nucleotide sequences to be linked are designed to connect in-frame. Human FRs are synthesized individually using each primer. As a result, products are obtained in which DNA encoding mouse CDRs is attached to each FR. The nucleotide sequences encoding mouse CDRs in each product are designed to overlap with each other. Subsequently, a complementary chain synthesis reaction takes place by annealing the overlapping CDR regions of products synthesized using the human antibody gene as a template. Through this reaction, human FRs are linked via the mouse CDR sequences.

[0173] The V-region gene, which ultimately consists of three CDRs and four FRs linked together, is amplified to its full length by primers that anneal to its 5' and 3' ends and have appropriate restriction enzyme recognition sequences added. A vector for human antibody expression can be created by inserting the DNA obtained as described above into an expression vector so as to fuse it in-frame with the DNA encoding the human antibody C-region. After introducing this integration vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, thereby producing the humanized antibody in the cultured cell culture (see European Patent Publication EP 239400, International Publication WO1996 / 002576).

[0174] By qualitatively or quantitatively measuring and evaluating the antigen-binding activity of the humanized antibody prepared as described above, a human antibody FR that forms a good antigen-binding site on the CDR when linked via the CDR can be suitably selected. If necessary, amino acid residues of the FR can be substituted so that the reconstituted human antibody CDR forms an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used for transplanting mouse CDRs into human FRs. Specifically, partial nucleotide sequence mutations can be introduced into primers that anneal to the FR. Nucleotide sequence mutations are introduced into the FR synthesized by such primers. The antigen-binding activity of the mutant antibody with substituted amino acids can be measured by the method described above. By measuring and evaluating, mutant FR sequences with desired properties can be selected (Sato, K. et al.). al., Cancer Res, 1993, 53, 851-856).

[0175] Furthermore, transgenic animals possessing the entire repertoire of human antibody genes (see International Publications WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, WO1996 / 033735) can be used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0176] Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the V region of a human antibody is expressed as a single-chain antibody (scFv) on the surface of a phage using phage display. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined. After determining the DNA sequence of the scFv that binds to the antigen, an expression vector can be created by fusing the V region sequence in-frame with the sequence of the desired human antibody C region and then inserting it into a suitable expression vector. The human antibody can be obtained by introducing this expression vector into suitable expression cells as described above and expressing the gene encoding the human antibody. These methods are already publicly known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).

[0177] In this specification, the term "Fv (variable fragment)" refers to the smallest unit of an antibody-derived antigen-binding domain, consisting of a pair of the antibody's light chain variable region (VL) and heavy chain variable region (VH). In 1988, Skerra and Pluckthun found that antibodies could be prepared from the periplasmic fraction of E. coli in a homogeneous and active state by inserting the antibody gene downstream of a bacterial signal sequence and inducing the expression of the gene in E. coli (Science (1988) 240 (4855)). (1038-1041). Fv prepared from the periplasmic fraction showed association of VH and VL in a manner that had binding to the antigen.

[0178] In this specification, Fv refers to, for example, the following antigen-binding molecules; The antigen-binding molecule includes (1) a divalent antigen-binding domain in which a monovalent scFv is linked to one polypeptide constituting an Fc region via a heavy chain Fv fragment constituting a CD3-binding domain, and the other monovalent scFv is linked to another polypeptide constituting an Fc region via a light chain Fv fragment constituting a CD3-binding domain, and the other monovalent scFv is linked to another polypeptide constituting an Fc region via a light chain Fv fragment constituting a CD3-binding domain, and the divalent antigen-binding domain is a divalent scFv, (2) a domain containing an Fc region among the amino acids constituting the Fc region of IgG1, IgG2a, IgG3, or IgG4 that does not have binding activity to the Fc gamma receptor, and (3) at least a monovalent CD3-binding domain, in which a pair of Fv fragments, including a light chain Fv fragment and a heavy chain Fv fragment, associate in a manner that has binding to the antigen CD3 and constitutes a CD3-binding domain.

[0179] As used herein, the terms "scFv", "single-chain antibody", or "sc(Fv)2" refer to antibody fragments that comprise variable regions derived from both a heavy chain and a light chain within a single polypeptide chain, but lack constant regions. Generally, a single-chain antibody further comprises a polypeptide linker between the VH domain and the VL domain that allows the formation of a desired structure that is thought to enable antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, 269-315 (1994). See also International Patent Application Publication WO1988 / 001649 and U.S. Patent Nos. 4,946,778 and 5,260,203. In certain embodiments, a single-chain antibody may also be bispecific and / or humanized.

[0180] scFv is an antigen-binding domain in which VH and VL constituting Fv are linked by a peptide linker (Proc. Natl. Acad. Sci. U.S.A. (1988) 85 (16), 5879-5883). The said peptide VH and VL can be kept in close proximity to each other by the linker.

[0181] sc(Fv)2 is a single-chain antibody in which four variable regions, two VLs and two VHs, are linked by a linker such as a peptide linker to form a single polypeptide chain (J Immunol. Methods (1999) 231 (1-2), 177-189). These two VH and VL may also originate from different monoclonal antibodies. For example, bispecific sc(Fv)2, which recognizes two different epitopes present in the same antigen, is also preferred, as disclosed in Journal of Immunology (1994) 152 (11), 5368-5374. sc(Fv)2 can be prepared by methods known to those skilled in the art. For example, it can be prepared by linking scFv with a linker such as a peptide linker.

[0182] In this specification, the antigen-binding domain configuration of sc(Fv)2 is characterized by two VHs and two VLs arranged in the order VH, VL, VH, VL ([VH]linker[VL]linker[VH]linker[VL]) starting from the N-terminus of the single-chain polypeptide. However, the order of the two VHs and two VLs is not limited to the above configuration and may be arranged in any order. For example, the following configuration can also be given.

[0183] [VL] Linker [VH] Linker [VH] Linker [VL] [VH] Linker [VL] Linker [VL] Linker [VH] [VH] Linker [VH] Linker [VL] Linker [VL] [VL] Linker [VL] Linker [VH] Linker [VH] [VL] Linker [VH] Linker [VL] Linker [VH]

[0184] The molecular morphology of sc(Fv)2 is described in detail in WO2006 / 132352, and those skilled in the art can use these descriptions to appropriately prepare the desired sc(Fv)2 for the preparation of the antigen-binding molecules disclosed herein.

[0185] Furthermore, the antigen-binding molecule of the present invention may be conjugated with carrier polymers such as PEG or organic compounds such as anticancer agents. Additionally, a glycosylation sequence may be inserted, and the glycosylation may be suitably added to achieve a desired effect.

[0186] As the linker for binding the variable region of the antibody, any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996) can be used, but in the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose, but a preferred length is 5 amino acids or more (the upper limit is not particularly limited, but usually 30 amino acids or less, preferably 20 amino acids or less), and particularly preferably 15 amino acids. If sc(Fv)2 contains three peptide linkers, peptide linkers of the same length may be used, or peptide linkers of different lengths may be used.

[0187] For example, in the case of a peptide linker: Ser Gly·Ser Gly·Gly·Ser Ser·Gly·Gly Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly Gly·Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly·Gly Gly·Gly·Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly·Gly·Gly Gly·Gly·Gly·Gly·Gly·Gly·Ser Ser·Gly·Gly·Gly·Gly·Gly·Gly (Gly·Gly·Gly·Gly·Ser) n (Ser·Gly·Gly·Gly·Gly) n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0188] Synthetic chemical linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these crosslinking agents are commercially available.

[0189] When binding four antibody variable regions, typically three linkers are required, but the same linker may be used for all of them, or different linkers may be used.

[0190] "Fab" consists of a light chain and a heavy chain with a CH1 region and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0191] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulins (monoclonal antibodies) with proteolytic enzymes such as pepsin or papain, and digesting them before and after the disulfide bond between the two H chains in the hinge region. For example, by treating IgG with papain, the disulfide bond between the two H chains in the hinge region is cleaved upstream, producing two homologous antibody fragments: an L chain consisting of VL (variable L chain region) and CL (constant L chain region), and an H chain fragment consisting of VH (variable H chain region) and CH gamma 1 (gamma 1 region in the constant H chain region), which are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab'.

[0192] "F(ab')2" comprises two light chains and two heavy chains containing constant regions of the CH1 domain and a portion of the CH2 domain such that interchain disulfide bonds are formed between the two heavy chains. The antigen-binding molecules F(ab')2 disclosed herein can be suitably obtained by partially digesting a full-length monoclonal antibody having a desired antigen-binding domain with a protease such as pepsin, and then removing the Fc fragment by adsorption onto a protein A column. The protease is not particularly limited as long as it can digest a full-length antibody to produce F(ab')2 restrictively by appropriately setting the reaction conditions of the enzyme, such as pH, for example, pepsin and ficin can be cited.

[0193] The Fc region constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting an antibody, such as a monoclonal antibody, with a proteolytic enzyme such as pepsin, adsorbing the fragment onto a protein A column or a protein G column, and then eluting it with an appropriate elution buffer. The proteolytic enzyme is not particularly limited as long as it can digest an antibody, such as a monoclonal antibody, by appropriately setting the reaction conditions of the enzyme, such as pH. Examples include pepsin and ficin.

[0194] The antigen-binding molecules described herein include Fc regions of IgG1, IgG2, IgG3, or IgG4 in which the binding activity to the Fc gamma receptor is reduced.

[0195] The isotype of an antibody is determined by the structure of its constant region. The constant regions of the IgG1, IgG2, IgG3, and IgG4 isotypes are called C-gamma 1, C-gamma 2, C-gamma 3, and C-gamma 4, respectively.

[0196] The Fc region refers to the region excluding F(ab')2, which includes two light chains and two heavy chains that include a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. The Fc region constituting the antigen-binding molecule disclosed herein can be suitably obtained by partially digesting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies, etc., with a proteolytic enzyme such as pepsin, and then re-eluting the fraction adsorbed onto a protein A column. Such a proteolytic enzyme is not particularly limited as long as it can digest the full-length antibody in a restrictive manner to produce F(ab')2 by appropriately setting the reaction conditions of the enzyme, such as pH, for example, pepsin and ficin can be given as examples.

[0197] The term Fcγ receptor refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and essentially means any member of the family of proteins encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, FcγRI(CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR species or FcγR isoforms or allotypes. FcγR may be derived from any organism, including, but is not limited to, humans, mice, rats, rabbits, and monkeys. Mouse FcγR receptors include, but are not limited to, FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIII-2(CD16-2), as well as any undiscovered mouse FcγR receptors, FcγR isoforms, or allotypes. Preferred examples of such Fcγ receptors include human FcγRI(CD64), FcγRIIA(CD32), FcγRIIB(CD32), FcγRIIIA(CD16), and / or FcγRIIIB(CD16).The polynucleotide and amino acid sequences of FcγRI are listed under RefSeq registry numbers NM_000566.3 and NP_000557.1, respectively; the polynucleotide and amino acid sequences of FcγRIIA are listed under RefSeq registry numbers BC020823.1 and 30AAH20823.1, respectively; the polynucleotide and amino acid sequences of FcγRIIB are listed under RefSeq registry numbers BC146678.1 and AAI46679.1, respectively; the polynucleotide and amino acid sequences of FcγRIIIA are listed under RefSeq registry numbers BC033678.1 and AAH33678.1, respectively; and the polynucleotide and amino acid sequences of FcγRIIIB are listed under RefSeq registry numbers BC128562.1 and AAI28563.1, respectively. Whether or not the Fcγ receptor has binding activity to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies can be confirmed by the FACS and ELISA formats described above, as well as by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0198] Furthermore, "Fc ligand" or "effector ligand" means a molecule, preferably a polypeptide, derived from any organism that binds to the Fc region of an antibody to form an Fc / Fc ligand complex. Binding of an Fc ligand to Fc preferably induces one or more effector functions. Fc ligands include, but are not limited to, Fc receptors, FcγR, FcαR, FcεR, FcRn, C1q, C3, mannan-binding lectins, mannose receptors, Staphylococcus protein A, Staphylococcus protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH) (Davis et al., (2002) Immunological Reviews 190, 123-136), which are a family of Fc receptors homologous to FcγR. Fc ligands may also include undiscovered molecules that bind to Fc.

[0199] The reduced binding activity of the Fc region to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB can be confirmed by the FACS and ELISA formats described above, as well as by ALPHA screening (Amplified Luminescent Proximity Homogeneous Assay) and the BIACORE method utilizing surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0200] The ALPHA screen is performed using ALPHA technology, which employs two beads, a donor and an acceptor, based on the following principle: Molecules bound to the donor bead biologically interact with molecules bound to the acceptor bead, and an emission signal is detected only when the two beads are in close proximity. A photosensitiver within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0201] For example, a biotin-labeled antigen-binding molecule is bound to a donor bead, and a glutathione S-transferase (GST)-tagged Fcγ receptor is bound to an acceptor bead. In the absence of competing antigen-binding molecules with mutant Fc regions, antigen-binding molecules with wild-type Fc regions interact with the Fcγ receptor, producing a signal in the 520-620 nm range. Antigen-binding molecules with untagged mutant Fc regions compete with the interaction between antigen-binding molecules with wild-type Fc regions and the Fcγ receptor. The relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from this competition. Biotinylation of antigen-binding molecules such as antibodies using sulfo-NHS-biotin is well known. As a method for tagging the Fcγ receptor with GST, a fusion gene obtained by in-frame fusion of a polynucleotide encoding the Fcγ receptor and a polynucleotide encoding GST is expressed in cells containing such a fusion gene in an expressionable vector, and then purified using a glutathione column can be appropriately employed. The obtained signals can be suitably analyzed by fitting them to a one-site competition model that utilizes nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0202] When one of the substances whose interaction is to be observed (ligand) is fixed onto a gold thin film on a sensor chip, and light is shone from the back of the sensor chip so as to cause total internal reflection at the interface between the gold thin film and glass, a region of reduced reflection intensity (SPR signal) is formed in a part of the reflected light. When the other substance whose interaction is to be observed (analyte) is flowed onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index causes the position of the SPR signal to shift (conversely, when the bond dissociates, the signal position returns to its original position). The Biacore system plots the amount of the above shift, i.e., the change in mass on the sensor chip surface, on the vertical axis and displays the change in mass over time as measurement data (sensorgram). From the sensorgram curve, kinetics: the binding rate constant (ka) and the dissociation rate constant (kd) can be determined, and affinity (KD) can be determined from the ratio of these constants. Inhibition measurement methods are also suitably used in the BIACORE method. Examples of inhibitory assay methods are described in Proc.Natl.Acad.Sci.USA (2006) 103 (11), 4005-4010.

[0203] Several therapeutic antibodies that exhibit antitumor effects inhibit signals necessary for cancer cell proliferation, induce cell death signals, or cause ADCC (Antibody-Dependent Cell-mediated Cytotoxicity). Antitumor effects against cancer cells are exerted through somatic-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). ADCC is the cytotoxicity exerted by effector cells such as NK cells and macrophages when the Fc region of an antibody binds to Fc receptors present on these effector cells, targeting cancer cells to which the antibody has bound. The complement complex binds to the complement binding site present in the antibody structure. CDC is the cytotoxicity that occurs when complement components present in the complex form pores on the cell membrane of the antibody-bound cell, promoting the influx of water and ions into the cell and destroying the cell. Among Fc receptors, the Fcγ receptor is a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies. If the binding activity to the Fcγ receptor is low, cross-linking between T cells and receptors expressed on NK cells and macrophages in a cancer antigen-independent manner does not occur. Therefore, cancer antigen-independent cytokine induction does not occur. Antibodies in which the Fc region has reduced binding activity to any of the Fcγ receptors FcγI, FcγIIA, FcγIIB, FcγIIIA, and / or FcγIIIB are desirable as antigen-binding molecules.

[0204] In this specification, a decrease in binding activity to the Fcγ receptor means, for example, that, based on the analytical method described above, the competitive activity of the test antigen-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the competitive activity of the control antigen-binding molecule.

[0205] As control antigen-binding molecules, antigen-binding molecules having the Fc region of IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate. Examples of the structure of the Fc region include the sequence with A added to the N-terminus of RefSeq registration number AAC82527.1, the sequence with A added to the N-terminus of RefSeq registration number AAB59393.1, the sequence with A added to the N-terminus of RefSeq registration number CAA27268.1, and the sequence with A added to the N-terminus of RefSeq registration number AAB59394.1. Furthermore, when using an antigen-binding molecule having a variant of the Fc region of a particular isotype of antibody as the test substance, the effect of the mutation in the variant on the binding activity to the Fcγ receptor can be verified by using an antigen-binding molecule having the Fc region of that particular isotype of antibody as a control. In this way, antigen-binding molecules having a variant of the Fc region that has been verified to have reduced binding activity to the Fcγ receptor can be appropriately constructed.

[0206] Examples of such mutants include deletions of amino acids 231A-238S, identified according to EU numbering (WO 2009 / 011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1(1), 47-54), and C226S, C229S, E233P, L234V, L235A (Blood (2007) 109, 1185-1192).

[0207] In other words, preferred antigen-binding molecules have an Fc region in which any of the following amino acids, identified according to EU numbering, among the amino acids constituting the Fc region of a particular isotype of antibody, are substituted at positions 220, 226, 229, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330, 331, and 332. The antibody isotype from which the Fc region originates is not particularly limited, and Fc regions originating from IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies can be used as appropriate, but Fc regions originating from IgG1 antibodies are preferred.

[0208] For example, among the amino acids constituting the Fc region of an IgG1 antibody, one of the following substitutions, identified according to EU numbering (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol after the number represents the amino acid residue before substitution); (a) L234F, L235E, P331S, (b) C226S, C229S, P238S, (c)C226S, C229S, (d)C226S, C229S, E233P, L234V, L235A (e) L234A, L235A or L235R, N297A (f) Antigen-binding molecules having an Fc region modified with L235A or L235R, S239K, or N297A, or an Fc region with a deletion in the amino acid sequence from position 231 to 238, may also be used as appropriate.

[0209] Furthermore, among the amino acids constituting the Fc region of the IgG2 antibody, any of the following substitutions, identified according to EU numbering, may occur (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol after the number represents the amino acid residue before substitution); (g)H268Q, V309L, A330S, P331S (h)V234A (i)G237A (j)V234A, G237A (k)A235E, G237A (l) Antigen-binding molecules having an Fc region with V234A, A235E, or G237A can also be used as appropriate.

[0210] Furthermore, among the amino acids constituting the Fc region of the IgG3 antibody, any of the following substitutions can be identified according to EU numbering (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol after the number represents the amino acid residue before substitution); (m)F241A (n)D265A Antigen-binding molecules having an Fc region with (o)V264A can also be used as appropriate.

[0211] Furthermore, among the amino acids constituting the Fc region of the IgG4 antibody, one of the following substitutions is identified according to EU numbering (the number represents the position of the amino acid residue identified according to EU numbering, the single amino acid symbol before the number represents the amino acid residue before substitution, and the single amino acid symbol after the number represents the amino acid residue before substitution); (p)L235A, G237A, E318A (q)L235E Antigen-binding molecules having an Fc region with (r)F234A and L235A can also be used as appropriate.

[0212] Other preferred examples include antigen-binding molecules having an Fc region in which any of the following amino acids, identified according to EU numbering, constitute the Fc region of an IgG1 antibody; the 233rd, 234th, 235th, 236th, 237th, 327th, 330th, and 331st positions are substituted with the corresponding amino acids in the corresponding IgG2 or IgG4.

[0213] Other preferred examples include antigen-binding molecules having an Fc region in which one or more of the following amino acids, identified according to EU numbering, constitute the Fc region of an IgG1 antibody; the 234th, 235th, and 297th positions are substituted with other amino acids. The type of amino acid present after substitution is not particularly limited, but antigen-binding molecules having an Fc region in which one or more of the 234th, 235th, and 297th positions are substituted with alanine are particularly preferred.

[0214] Other preferred examples include any of the following amino acids that make up the Fc region of an IgG1 antibody, as identified according to EU numbering; where the 265th position is substituted by another amino acid. Antigen-binding molecules having an Fc region are preferred. The type of amino acid present after substitution is not particularly limited, but antigen-binding molecules having an Fc region in which the amino acid at position 265 is substituted with alanine are particularly preferred.

[0215] Numerous studies have been conducted on the effector functions of IgG class antibodies, namely antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). Among human IgG class antibodies, IgG1 subclass antibodies exhibit the highest ADCC and CDC activity. Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is phagocytosis of target cells mediated by IgG class antibodies, has also been suggested as one of the effector functions of antibodies. Because IgG1 subclass antibodies can exert these effector functions against tumors, they are used as the basis for most antibody drugs targeting cancer antigens.

[0216] On the other hand, for IgG antibodies to mediate the effector functions of antibodies, such as ADCC, ADCP, or antibody-dependent cell-mediated phagocytosis (ADCP) activity, which is phagocytosis of target cells, binding of the Fc region of the IgG antibody to the Fcγ receptor (FcγR) present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages is necessary.

[0217] Enhancing cytotoxic effector functions such as ADCC and ADCP is a promising method for enhancing the antitumor effects of anticancer antibodies. Antibodies with Fc regions optimized for binding to the Fcγ receptor are suggested to mediate more potent effector functions, thereby exerting effective antitumor effects. Therefore, various antibody engineering techniques (e.g., WO2013047752) have been reported to enhance or improve the antitumor activity of antibody drugs against cancer antigens.

[0218] Regarding the binding of the Fc region to the Fcγ receptor, it has been shown that the hinge region of the antibody, several amino acid residues within the CH2 domain, and the glycan attached to EU numbering 297th Asn bound to the CH2 domain are important (Clark, M., Chemical Immunology (1997) 65, 88-110, Greenwood J, Clark M, Waldmann H., Eur. J. Immunol. (1993) 23, 1098-1104, Morgan A, Jones ND, Nesbitt AM, Chaplin L, Bodmer MW, Emtage JS., Immunology (1995) 86, 319-324). Focusing on this binding site, various mutants of the Fc region with different Fcγ receptor binding characteristics have been studied, and Fc region mutants with higher affinity for activated Fcγ receptors have been obtained (WO2000 / 042072, WO2006 / 019447).

[0219] Thus, in antibodies targeting membrane-bound antigens, binding activity to the Fcγ receptor plays a crucial role in cytotoxic activity. Therefore, when cytotoxic activity is required, isotypes of human IgG1 with high binding activity to FcγR are used, and further enhancing the binding activity to the Fcγ receptor is a widely used technique to enhance cytotoxic activity. Attempts have also been made to enhance the binding activity to the Fcγ receptor in antibodies targeting soluble antigens (WO2013047752).

[0220] The Fc region is the part of the Fc region represented by EU numbering; The amino acid at position 221 is either Lys or Tyr. The amino acid at position 222 is one of Phe, Trp, Glu, or Tyr. The amino acid at position 223 is one of Phe, Trp, Glu, or Lys. The amino acid at position 224 is one of Phe, Trp, Glu, or Tyr. The amino acid at position 225 is either Glu, Lys, or Trp. The amino acid at position 227 is one of Glu, Gly, Lys, or Tyr. The amino acid at position 228 is one of Glu, Gly, Lys, or Tyr. The amino acid at position 230 is one of Ala, Glu, Gly, or Tyr. The amino acid at position 231 is one of Glu, Gly, Lys, Pro, or Tyr. The amino acid at position 232 is one of Glu, Gly, Lys, or Tyr. The amino acid at position 233 is one of the following: Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 234 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 235 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 236 is one of the following: Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 237 is one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 238 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 239 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 240 is one of Ala, Ile, Met, or Thr. The amino acid at position 241 is one of Asp, Glu, Leu, Arg, Trp, or Tyr. The amino acid at position 243 is one of Leu, Glu, Leu, Gln, Arg, Trp, or Tyr. The amino acid at position 244 is His. The amino acid at position 245 is Ala. The amino acid at position 246 is one of Asp, Glu, His, or Tyr. The amino acid at position 247 is one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr. The amino acid at position 249 is one of Glu, His, Gln, or Tyr. The amino acid at position 250 is either Glu or Gln. The amino acid at position 251 is Phe, The amino acid at position 254 is either Phe, Met, or Tyr. The amino acid at position 255 is either Glu, Leu, or Tyr. The amino acid at position 256 is either Ala, Met, or Pro. The amino acid at position 258 is one of Asp, Glu, His, Ser, or Tyr. The amino acid at position 260 is one of Asp, Glu, His, or Tyr. The amino acid at position 262 is one of Ala, Glu, Phe, Ile, or Thr. The amino acid at position 263 is one of Ala, Ile, Met, or Thr. The amino acid at position 264 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. The amino acid at position 265 is one of the following: Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 266 is one of Ala, Ile, Met, or Thr. The amino acid at position 267 is one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 268 is one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp. The amino acid at position 269 is one of the following: Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 270 is one of the following: Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr. The amino acid at position 271 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acids at position 272 are Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, and V. Al, Trp, or Tyr The amino acid at position 273 is either Phe or Ile. The amino acid at position 274 is one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 275 is either Leu or Trp. The amino acid at position 276 is one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 278 is one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp. The amino acid ranked 279th is Ala. The amino acid at position 280 is one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr. The amino acid at position 281 is one of Asp, Lys, Pro, or Tyr. The amino acid at position 282 is one of Glu, Gly, Lys, Pro, or Tyr. The amino acid at position 283 is one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr. The amino acid at position 284 is one of Asp, Glu, Leu, Asn, Thr, or Tyr. The amino acid at position 285 is one of Asp, Glu, Lys, Gln, Trp, or Tyr. The amino acid at position 286 is one of Glu, Gly, Pro, or Tyr. The amino acid at position 288 is one of Asn, Asp, Glu, or Tyr. The amino acid at position 290 is one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr. The amino acid at position 291 is one of Asp, Glu, Gly, His, Ile, Gln, or Thr. The amino acid at position 292 is one of Ala, Asp, Glu, Pro, Thr, or Tyr. The amino acid at position 293 is one of the following: Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 294 is one of the following: Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 295 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 296 is one of the following: Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val. The amino acid at position 297 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 298 is one of the following: Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 299 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr. The amino acid at position 300 is one of the following: Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp. The amino acid at position 301 is one of Asp, Glu, His, or Tyr. The amino acid at position 302 is Ile. The amino acid at position 303 is either Asp, Gly, or Tyr. The amino acid at position 304 is one of Asp, His, Leu, Asn, or Thr. The amino acid at position 305 is one of Glu, Ile, Thr, or Tyr. The amino acid at position 311 is one of Ala, Asp, Asn, Thr, Val, or Tyr. The amino acid at position 313 is Phe, The amino acid at position 315 is Leu. The amino acid at position 317 is Glu or Gln. The amino acid at position 318 is one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr. The amino acid at position 320 is one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 322 is one of the following: Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 323 is Ile. The amino acid at position 324 is one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 325 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 326 is one of the following: Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 327 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 328 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 329 is one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 330 is one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 331 is one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr. The amino acid at position 332 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr. The amino acid at position 333 is one of the following: Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr. The amino acid at position 334 is one of Ala, Glu, Phe, Ile, Leu, Pro, or Thr. The amino acid at position 335 is one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr. The amino acid at position 336 is either Glu, Lys, or Tyr. The amino acid at position 337 is either Glu, His, or Asn. The amino acid at position 339 is one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr. The amino acid at position 376 is either Ala or Val. The amino acid at position 377 is either Gly or Lys. The amino acid at position 378 is Asp. The amino acid at position 379 is Asn. The amino acid at position 380 is either Ala, Asn, or Ser. The amino acid at position 382 is either Ala or Ile. The amino acid at position 385 is Glu, The amino acid at position 392 is Thr. The amino acid at position 396 is Leu. The amino acid ranked 421st is Lys. The amino acid at position 427 is Asn. The amino acid at position 428 is either Phe or Leu. The amino acid at position 429 is Met. The amino acid at position 434 is Trp. The amino acid at position 436 is Ile, and Preferably, the antibody is an IgG antibody having an Fc region in which the amino acid at position 440 contains at least one amino acid selected from the group Gly, His, Ile, Leu, or Tyr, and also an antibody having ADCC activity in which the IgG antibody-like molecule is a secondary molecule.

[0221] Examples of antigen-binding molecules of the present invention include multispecific antibodies. Heterochromic antibodies are antibodies that possess multiple different specificities. An example of a multispecific antibody is a bispecific antibody. IgG-type bispecific antibodies can be secreted by a hybrid hybridoma (quadroma) formed by fusing two hybridomas that produce IgG antibodies (Milstein C et al. Nature (1983) 305, 537-540). When using an Fc region with reduced binding activity to the Fcγ receptor as the Fc region of a bispecific antibody, an Fc region originating from a bispecific antibody may also be used as appropriate.

[0222] Methods for producing multispecific antibodies are not limited to these, but include, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); creating antibodies with two specificities using a leucine zipper (see Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); producing bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368). (See 1994); and may also be prepared by preparing a trispecific antibody as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0223] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0224] In one aspect of the present invention, the antigen-binding molecule includes a bispecific antigen-binding site having a target antigen recognition site and an immune receptor recognition site. Here, the antigen-binding molecule may have further antigen-binding sites in addition to the bispecific antigen-binding site. Examples of antigen-binding molecules in this embodiment include a bispecific antigen-binding molecule and a multispecific antigen-binding molecule having three or more antigen-binding sites. More specifically, examples include a bispecific antibody and a multispecific antibody having three or more antigen-binding sites.

[0225] IgG bispecific antibodies are secreted by introducing four genes—the genes for the light and heavy chains (L and H) of two target IgGs—into cells and co-expressing them. However, theoretically, there are as many as 10 possible combinations of H and L chains in the IgG produced by these methods. Purifying IgG with the desired H and L chain combination from these 10 types of IgG is difficult. Furthermore, the amount secreted with the desired combination is theoretically significantly reduced, requiring a large culture scale and further increasing manufacturing costs.

[0226] The bispecific antibodies of the present invention can be subjected to techniques to promote the association of desired combinations of H chains and L chain H chains. For example, to associate multispecific antibodies, a technique can be applied that introduces electrostatic repulsion at the interface of the second constant region (CH2) or the third constant region (CH3) of the antibody H chain to suppress the association of unintended H chains (WO2006 / 106905).

[0227] In a technique that suppresses unintended association of H chains by introducing electrostatic repulsion at the CH2 or CH3 interface, examples of amino acid residues that come into contact at the interface of other constant regions of the H chain include the regions corresponding to EU numbering residues 356, 439, 357, 370, 399, and 409 in the CH3 region. More specifically, for example, In antibodies containing two types of H chain CH3 regions, one to three sets of amino acid residues selected from the following sets of amino acid residues in the first H chain CH3 region (1) to (3) can be made to have the same charge: (1) amino acid residues included in the H chain CH3 region at EU numbering positions 356 and 439; (2) amino acid residues included in the H chain CH3 region at EU numbering positions 357 and 370; (3) amino acid residues included in the H chain CH3 region at EU numbering positions 399 and 409.

[0228] Furthermore, an antibody can be formed in which one to three sets of amino acid residues selected from the sets of amino acid residues shown in (1) to (3) above in a second H chain CH3 region different from the first H chain CH3 region, and which correspond to the sets of amino acid residues shown in (1) to (3) above that have the same charge in the first H chain CH3 region, have the opposite charge to the corresponding amino acid residues in the first H chain CH3 region.

[0229] The amino acid residues described in (1) to (3) above are in close proximity to each other when they associate. A person skilled in the art can find the site corresponding to the amino acid residues described in (1) to (3) above in a desired H chain CH3 region or H chain constant region by homology modeling using commercially available software, and can modify the amino acid residues in that site as appropriate.

[0230] In the above antibody, the "charged amino acid residue" is preferably selected from, for example, an amino acid residue belonging to either group (a) or (b) below; (a) Glutamic acid (E), aspartic acid (D), (b) Lysine (K), Arginine (R), Histidine (H). In the above antibodies, "having the same charge" means, for example, that any of the two or more amino acid residues are amino acid residues belonging to either group (a) or (b) above. "Having opposite charges" means, for example, that if at least one of the two or more amino acid residues is an amino acid residue belonging to either group (a) or (b) above, the remaining amino acid residues are amino acid residues belonging to different groups.

[0231] In a preferred embodiment, the antibody may have a first H chain CH3 region and a second H chain CH3 region crosslinked by a disulfide bond.

[0232] The amino acid residues to be modified in this invention are not limited to the amino acid residues in the variable region or constant region of the antibody as described above. Those skilled in the art can identify the amino acid residues that form an interface in polypeptide variants or heterologous polymers using homology modeling with commercially available software, and modify the amino acid residues at that site to control the association.

[0233] Furthermore, other known techniques can also be used for the association of the bispecific antibodies of the present invention. By substituting the amino acid side chain in the Fc region of one H chain of the antibody with a larger side chain (knob; projection) and substituting the amino acid side chain in the opposing Fc region of the other H chain with a smaller side chain (hole; void), the projection can be positioned within the void, thereby efficiently causing association between polypeptides having different amino acids and Fc regions (WO1996 / 027011, Ridgway JB et al., Protein Engineering (1996) 9, 617-621, Merchant AM et al. Nature Biotechnology (1998) 16, 677-681, US20130336973).

[0234] In addition, other known techniques can also be used to form the bispecific antibodies of the present invention. One technique involves replacing a portion of the CH3 of one H chain of the antibody with a sequence derived from IgA corresponding to that portion, and introducing a sequence derived from IgA corresponding to that portion into the complementary portion of the CH3 of the other H chain using a strand exchange. By using the ge-engineered domain CH3, the association of polypeptides with different sequences can be efficiently induced through complementary association of CH3 (Protein Engineering Design & Selection, 23; 195-202, 2010). This known technique can also be used to efficiently form the desired bispecific antibodies.

[0235] Other methods for forming bispecific antibodies include antibody production techniques utilizing the association of CH1 and CL, and VH and VL of antibodies, as described in WO2011 / 028952, WO2014 / 018572, and Nat Biotechnol. 2014 Feb;32(2):191-8.; techniques for producing bispecific antibodies using separately prepared monoclonal antibodies (Fab Arm Exchange) as described in WO2008 / 119353 and WO2011 / 131746; techniques for controlling the association between CH3 groups of antibody heavy chains as described in WO2012 / 058768 and WO2013 / 063702; techniques for producing bispecific antibodies composed of two types of light chains and one type of heavy chain as described in WO2012 / 023053; and Christoph et al. (Nature Biotechnology Vol. 31, p 753-758) Techniques such as those described in (2013) for producing bispecific antibodies using two bacterial cell lines that each express one of the antibody chains, consisting of one heavy chain and one light chain, can also be used.

[0236] Furthermore, even if it is not possible to efficiently form the desired bispecific antibody, the bispecific antibody of the present invention can also be obtained by separating and purifying the desired bispecific antibody from the produced antibody. For example, a method has been reported in which two homozygous antibodies and the desired heterozygous antibody can be purified by ion exchange chromatography by introducing amino acid substitutions into the variable regions of two types of H chains to create a difference in isoelectric point (pI) (WO2007114325). In addition, as a method for purifying heterozygous antibodies, a method has been reported in which a heterodimerized antibody consisting of a mouse IgG2a H chain that binds to protein A and a rat IgG2b H chain that does not bind to protein A is purified using protein A (WO98050431, WO95033844). Furthermore, by using H chains in which the IgG-Protein A binding sites, specifically the 435th and 436th amino acid residues in the EU numbering, are replaced with amino acids with different binding affinity to Protein A, such as Tyr and His, or by using H chains with different binding affinity to Protein A obtained according to the method described in Reference Example 5, the interaction between each H chain and Protein A can be altered, and by using a Protein A column, it is possible to efficiently purify only heterodimerized antibodies.

[0237] Alternatively, a common light chain capable of conferring binding ability to multiple different heavy chains may be obtained and used as the common light chain for a bispecific antibody. By introducing multiple heavy chain genes different from such a common light chain into cells, efficient expression of bispecific IgG becomes possible (Nature Biotechnology (1998) 16, 677-681). When selecting a common heavy chain, a method can also be used to select a common light chain that corresponds to any different heavy chain and exhibits high binding ability (WO2004 / 065611).

[0238] Furthermore, as the Fc region of the present invention, an Fc region in which the heterogeneity of the C-terminus of the Fc region is improved may be used as appropriate. More specifically, an Fc region is provided in which the glycine at position 446 and the lysine at position 447, which are identified according to EU numbering, are deleted from the amino acid sequences of two polypeptides constituting the Fc region originating from IgG1, IgG2, IgG3, or IgG4.

[0239] These techniques can be used in combination, for example, two or more. Furthermore, these techniques can be applied separately to the two H chains to be associated, as appropriate. In addition, these techniques can be used in combination with the Fc region where the binding activity to the Fcγ receptor is reduced. Note that the antigen-binding molecule of the present invention may also be a separately prepared antigen-binding molecule having the same amino acid sequence, based on the modified molecule described above.

[0240] In the present invention, the term "functionally equivalent" antibody variable region is not particularly limited as long as it satisfies the above-mentioned conditions for an antibody H chain variable region and / or antibody L chain variable region. For example, such an antibody variable region may have one or more amino acids in the amino acid sequence of the variable regions listed in Tables 1 to 3 above. (For example, 1, 2, 3, 4, 5, or 10 amino acids) may be substituted, deleted, added, and / or inserted. Methods well known to those skilled in the art for substituting, deleting, adding, and / or inserting one or more amino acids in an amino acid sequence include methods for introducing mutations into proteins. For example, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors.Methods Enzymol. 100, 468-500, Kramer,W, Drutsa,V, Jansen,HW, Kramer,B, Pflugfelder,M, and Fritz,HJ(1984) The gapped duplex DNA approach to oligonucleotide-directed mutation construction. Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ(1987) Oligonucleotide-directed construction of mutations via gapped duplex DNA Methods. By appropriately introducing mutations into the amino acid sequence using methods such as Enzymol. 154, 350-367 and Kunkel, TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci US A. 82, 488-492), a variable region functionally equivalent to the antibody variable region having the above-mentioned functions can be prepared.

[0241] When modifying amino acid residues, it is desirable to mutate them with other amino acids whose amino acid side chain properties are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the letters in parentheses represent the single-letter abbreviations of the amino acids). Amino acid substitutions within each of these groups are called conservative substitutions. It is already known that polypeptides having amino acid sequences modified by the deletion, addition, and / or substitution of one or more amino acid residues in a given amino acid sequence maintain their biological activity (Mark, DF et al., Proc.Natl.Acad.Sci.USA (1984)81:5662-6; Zoller, MJ and Smith, M., Nucleic Acids Res.(1982)10:6487-500; Wang, A. et al., Science(1984)224:1431-3; Dalbadie-McFarland, G. et al. al., Proc.Natl.Acad.Sci.USA (1982)79:6409-13). The variable region of the present invention, including such amino acid modifications, has at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity with the CDR sequence, FR sequence, or the entire amino acid sequence of the variable region before modification. In this specification, sequence identity is defined as the percentage of residues identical to the residues of the original H-chain variable region or L-chain variable region after the sequence has been aligned as necessary to maximize sequence identity and gaps have been introduced as appropriate. Amino acid sequence identity can be determined by the methods described below.

[0242] Furthermore, "functionally equivalent antibody variable regions" can also be obtained, for example, from nucleic acids that hybridize under stringent conditions to nucleic acids consisting of a base sequence encoding the amino acid sequence of the variable region described in Tables 1-3 above. Examples of stringent hybridization conditions for isolating nucleic acids that hybridize under stringent conditions to nucleic acids consisting of a base sequence encoding the amino acid sequence of the variable region include 6 M urea, 0.4% SDS, 0.5 x SSC, 37°C, or hybridization conditions with equivalent stringency. Using conditions with higher stringency, for example, 6 M urea, 0.4% SDS, 0.1 x SSC, 42°C, can be expected to isolate nucleic acids with higher homology. Washing conditions after hybridization include, for example, 0.5xSSC (1xSSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), and 0.1% SDS, washing at 60°C, more preferably 0.2xSSC, and 0.1% SDS, washing at 60°C, more preferably 0.2xSSC, and 0.1% SD Washing is performed with S, at 62°C, more preferably with 0.2xSSC, and 0.1% SDS, at 65°C, more preferably with 0.1xSSC, and 0.1% SDS, and at 65°C. The sequencing of the isolated nucleic acids can be performed by known methods described later. The homology of the isolated nucleic acids is such that there is sequence identity of at least 50%, more preferably 70%, and even more preferably 90% (e.g., 95%, 96%, 97%, 98%, 99% or more) of the entire base sequence.

[0243] Alternatively, instead of using the hybridization techniques described above, it is also possible to isolate nucleic acids that hybridize with nucleic acids consisting of the base sequence encoding the amino acid sequence of the variable region under stringent conditions by using gene amplification methods, such as polymerase chain reaction (PCR), which utilize primers synthesized based on the base sequence information encoding the amino acid sequence of the variable region.

[0244] The identity of base sequences and amino acid sequences can be determined by the BLAST algorithm developed by Karlin and Altschul (Proc.Natl.Acad.Sci.USA(1993)90:5873-7). Programs called BLASTN and BLASTX have been developed based on this algorithm (Altschul). et al., J.Mol.Biol.(1990)215:403-10). When analyzing the base sequence using BLASTN based on BLAST, the parameters should be, for example, score = 100 and wordlength = 12. When analyzing the amino acid sequence using BLASTX based on BLAST, the parameters should be, for example, score = 50 and wordlength = 3. When using the BLAST and Gapped BLAST programs, use the default parameters of each program. The specific methods for these analyses are publicly known (see the NCBI (National Center for Biotechnology Information) BLAST (Basic Local Alignment Search Tool) website; http: / / www.ncbi.nlm.nih.gov).

[0245] The Fc region included in the bispecific antibody of the present invention is not particularly limited as long as it is an Fc region with reduced binding activity to the Fcγ receptor. However, preferred Fc regions of the present invention include, for example, the combination of the Fc region portion of E22Hh and the Fc region portion of E22Hk described in WO2016 / 047722A1, the combination of the Fc region portion of E2702GsKsc and the Fc region portion of E2704sEpsc, and the combination of the Fc region portion of E2702sKsc and the Fc region portion of E2704sEpsc.

[0246] Furthermore, it is conceivable to incorporate the gene encoding the antigen-binding molecule of the present invention into a gene therapy vector and perform gene therapy. Regarding administration methods, in addition to direct administration via naked plasmid, the molecule can be packaged in liposomes or the like, formed as various viral vectors such as retroviral vectors, adenovirus vectors, vaccinia virus vectors, poxvirus vectors, adenovirus-related vectors, and HVJ vectors (see Adolph, "Viral Genome Method," CRC Press, Florid (1996)), or coated onto a bead carrier such as colloidal gold particles (WO93 / 17706, etc.) for administration. The nucleic acid encoding the antigen-binding molecule of the present invention may also be administered directly to a living organism, or by electroporation. For example, the antigen-binding molecule of the present invention can be administered by chemically modifying the mRNA encoding the antigen-binding molecule to enhance its stability in vivo, directly administering the mRNA to a human, and expressing the antigen-binding molecule in vivo (see EP2101823B, WO2013 / 120629). However, the antigen-binding molecule may be administered by any method as long as it is expressed in vivo and exerts its effects. Preferably, a sufficient amount is administered via an appropriate parenteral route (injection, infusion, gas-induced particle shock therapy (using an electron gun, etc.), nasal spray, etc., via a mucosal route, such as intravenous, intraperitoneal, subcutaneous, intradermal, intraadipose tissue, intramammary tissue, inhalation, or intramuscular routes). The gene encoding the antigen-binding molecule of the present invention may also be administered ex vivo by liposome transfection, particle shock therapy (U.S. Patent No. 4,945,050), or by utilizing viral infection to administer it to blood cells and bone marrow-derived cells, etc., and then reintroducing the cells into animals.

[0247] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the pharmaceutical compositions of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0248] In this invention, a pharmaceutical composition generally refers to an agent used for the treatment or prevention of a disease, or for examination and diagnosis. In this invention, when a pharmaceutical composition is used in combination with the administration of other components, the pharmaceutical composition may be administered simultaneously, separately, or consecutively with the other components. This pharmaceutical composition may contain other components as components.

[0249] Pharmaceutical compositions containing the antigen-binding molecule of the present invention can be formulated using methods known to those skilled in the art. For example, they can be administered parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or as an injectable suspension. For example, they can be formulated by mixing them in appropriate combination with a pharmacokinetically acceptable carrier or medium, specifically sterile water or saline solution, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., in a unit dose form generally required for pharmaceutical practice. The amount of active ingredient in these formulations is set so as to yield an appropriate volume within the indicated range.

[0250] Sterile compositions for injection can be formulated in accordance with standard formulation procedures using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizers, such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may be used in combination.

[0251] Examples of oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may also be used as solubilizers. Furthermore, buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may be added. The prepared injection solution is usually filled into appropriate ampoules.

[0252] The pharmaceutical composition containing the antigen-binding molecule of the present invention is preferably administered by parenteral administration. For example, compositions in the form of injection, nasal administration, pulmonary administration, or transdermal administration may be administered. For example, it may be administered systemically or locally by intravenous injection, intra-arterial injection, intramuscular injection, intraperitoneal injection, intramedullary injection, intra-articular injection, intra-bursal injection, intracranial injection, intrathecal injection, subarachnoid injection, intradermal injection, subcutaneous injection, intracardiac injection, injection into lesions such as tumors, or by methods utilizing catheters.

[0253] The method of administration may be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition of the present invention may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, a dosage of 0.001 to 100,000 mg per patient may be set, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and method of administration considering these conditions.

[0254] Depending on the requirements of the present invention, the pharmaceutical composition of the present invention may be contained in microcapsules (hydroxymethylcellulose). The drug may be encapsulated in microcapsules (such as those made from gelatin or poly[methylmethacrylate]) and incorporated into a colloidal drug delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) (see Remington's Pharmaceutical Science 16th edition, Oslo Ed. (1980), etc.). Furthermore, methods for making the drug a sustained-release drug are also known, and these methods can be applied to the bispecific antigen-binding molecule of the present invention (J. Biomed. Mater. Res. (1981) 15, 267-277, Chemtech. (1982) 12, 98-105, U.S. Patent No. 3,773,719, European Patent Publications EP58481 and EP133988, Biopolymers (1983) 22, 547-556).

[0255] The term "chimeric receptor" refers to a recombinant polypeptide that, when expressed on immune effector cells, exhibits specificity towards target cells, such as cancer cells, and produces intracellular signals, and includes at least an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0256] The extracellular domain of a chimeric receptor means any protein molecule or part thereof that can specifically bind to a given molecule. In the present invention, the extracellular domain of a chimeric receptor is a polypeptide comprising a sequence of the extracellular domain of an immune receptor or a variant thereof. In one aspect of the present invention, the extracellular domain of a chimeric receptor may be a polypeptide consisting solely of a sequence of the extracellular domain of an immune receptor or a variant thereof, or it may include additional polypeptides or amino acid residues in the sequence of the extracellular domain of an immune receptor or a variant thereof. In another aspect of the present invention, the extracellular domain of a chimeric receptor may comprise a sequence of fragments of the extracellular domain of an immune receptor or a variant thereof.

[0257] In this invention, the extracellular domain of an immune receptor refers to the extracellular portion of an immune receptor that can bind to an endogenous immune ligand. For example, in the case of CD137, it refers to SEQ ID NOs. 129, 130, 131, and 132.

[0258] In the present invention, an extracellular domain variant of an immune receptor refers to a polypeptide in which a portion of the extracellular domain of an immune receptor is added, deleted, or substituted. Preferably, an extracellular domain variant of an immune receptor has reduced binding to an endogenous immune ligand, and refers to a variant in which the binding site of the extracellular domain of the immune receptor to the endogenous ligand is modified based on, for example, the binding site of the immune receptor to the endogenous ligand identified by X-ray crystal structure analysis described in the literature. For example, an example of an extracellular domain variant of an immune receptor with reduced binding to an endogenous immune ligand is an extracellular domain variant of the CD137 immune receptor in which the cysteine-rich domain 3, cysteine-rich domain 4, and stalk (Genbank NM001561.6, Cys88~Gln186) connected to the transmembrane region are deleted.

[0259] In the present invention, the extracellular domain fragment of an immune receptor can be any fragment of a polypeptide constituting the extracellular domain of an immune receptor, and the number of constituent amino acids is not particularly limited as long as the antigen-binding molecule can recognize it. Examples of such fragments include polypeptides or fragments thereof that constitute an epitope recognized by an antibody having agonist activity in an immune receptor. In the present invention, the extracellular domain of a chimeric receptor may contain a modified version of the epitope, and the polypeptide fragment constituting the extracellular domain of the immune receptor contained in the epitope also falls under the category of "fragment of the extracellular domain of an immune receptor."

[0260] The term "transmembrane domain" includes polypeptides that are located between the extracellular domain and the intracellular signaling domain and have the function of transmembrane.

[0261] The term "intracellular signaling domain" refers to any oligopeptide domain or polypeptide known to transmit signals that trigger the activation or inhibition of biological processes within a cell, such as the activation of immune cells like T cells or NK cells. This refers to the main domain, which includes a "stimulus molecule signaling domain" derived from at least one T cell stimuli molecule, as described later, and may also include a "co-stimulus molecule signaling domain" derived from at least one T cell co-stimulus molecule, as described later.

[0262] As used in this disclosure, “domain” means, for example, a region of a polypeptide that folds into a specific structure independently of other regions and / or has a specific function. A domain may be, for example, the cytoplasmic portion or part thereof of a molecule. As used in this disclosure, “cytoplasmic domain” of a molecule means the full-length cytoplasmic domain or a portion thereof that transmits intracellular signals when activated.

[0263] In one embodiment, the chimeric receptor is a molecule containing a domain defined below. In one embodiment, the chimeric receptor comprises a chimeric fusion protein including an extracellular domain, an extracellular hinge domain, a transmembrane domain, and an intracellular signaling domain including a stimulus molecule-derived signaling domain.

[0264] In one embodiment, the chimeric receptor comprises a chimeric fusion protein including an extracellular domain, an extracellular hinge domain, a transmembrane domain, and an intracellular signaling domain including a co-stimulatory molecule-derived co-stimulatory molecule signaling domain and a stimulatory molecule-derived functional signaling domain.

[0265] In one embodiment, the chimeric receptor comprises a chimeric fusion protein including an extracellular domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

[0266] In one embodiment, the chimeric receptor comprises a chimeric fusion protein comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain comprising at least two co-stimulatory signaling domains derived from one or more co-stimulatory molecules, a stimulatory signaling domain derived from a stimulatory molecule, and other functional domains and / or motifs.

[0267] In one embodiment, a chimeric receptor is disclosed comprising: i) an extracellular domain capable of binding to an immunoreceptor recognition site of an antigen-binding molecule; ii) a transmembrane domain; and iii) an intracellular segment comprising a CD3 zeta intracellular signaling domain comprising one or more intracellular signaling domains selected from a cytoplasmic costimulatory domain and / or the cytoplasmic domain of an interleukin receptor chain, and an exogenous STAT3-related motif (wherein the intracellular segment comprises an endogenous or exogenous JAK-binding motif and a STAT5-related motif). In one embodiment, these domains are fused directly or indirectly, optionally starting from the N-terminus and in the above order. In one embodiment, these domains within the intracellular segment are fused in the reverse order.

[0268] In one embodiment, an extracellular hinge domain and a transmembrane domain may be included between the extracellular domain and the intracellular signaling domain. The term "extracellular hinge domain" refers to a domain that connects the extracellular domain and the transmembrane domain. The extracellular hinge domain is not particularly limited as long as it can connect the extracellular domain and the transmembrane domain. It may be derived from a natural protein or be artificially designed. The extracellular hinge domain can be composed of, for example, about 10 to 300 amino acids, preferably about 20 to 100 amino acids. It is preferable that the extracellular hinge domain does not interfere with the binding ability of the extracellular domain to the antigen-binding molecule of this disclosure and does not interfere with signal transduction via the intracellular signaling domain. The term "transmembrane domain" refers to a domain located between the extracellular domain and the intracellular signaling domain that has the function of transmembrane. The polypeptide having the transmembrane domain is not particularly limited. The transmembrane domain may be derived from a natural protein or may be artificially designed. Transmembrane domains derived from natural proteins can be obtained from any membrane-binding protein or transmembrane protein.

[0269] In one embodiment, the chimeric receptor includes an additional leader sequence at the amino terminus (N-terminus) of the chimeric receptor fusion protein. In one embodiment, the chimeric receptor further includes a leader sequence at the N-terminus of the extracellular domain, where the leader sequence may be cleaved from the extracellular domain during cell processing and localization of the chimeric receptor to the cell membrane.

[0270] The term "immune receptor" refers to a receptor expressed on immune cells that is involved in the activation or suppression of immune cells. Examples of immune cells include T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells, or hematopoietic cells (neutrophils, basophils). In the present invention, the chimeric receptor has an extracellular domain of an immune receptor or a modified version thereof. Here, the immune receptor is preferably a receptor involved in the activation of immune cells, such as a costimulatory molecule belonging to the tumor necrosis factor receptor superfamily (TNFRSF), specifically CD137, CD40, OX40, RANK, GITR, etc. The immune receptor may also be a stimulating molecule or a costimulatory molecule, specific examples of which are described below.

[0271] The immunoreceptor recognition site of the antigen-binding molecule in the present invention can recognize the extracellular domain or a fragment of the immunoreceptor contained in the extracellular binding domain of the chimeric receptor. More preferably, the immunoreceptor recognition site of the antigen-binding molecule recognizes a different portion from the binding site of the endogenous ligand. The endogenous ligand binding site on the immunoreceptor is identified, for example, by structural biological analysis described in the literature. For example, in the case of CD137, the X-ray crystal structure of the complex with CD137L has been revealed (Chin SM et al (2018) Nat Commun. 9, 4679), and it has been reported that F36 on CRD1, P49, S52, Q59, T61, C62, D63, I64, Q67, K69, V71, F72 on CRD2, and S100, M101, C102 on CRD3 are involved in the interaction with CD137L. Of these, mutants in which I64 or V71 is mutated to Arg have been created, and both have been shown to reduce binding to CD137L. Mutants in which amino acid residues involved in interaction with CD137L are mutated to other amino acid residues can be used as extracellular domain modifiers that reduce binding to CD137L.

[0272] In one embodiment, the extracellular domain of the chimeric receptor includes the extracellular domain or a fragment of a costimulatory molecule belonging to TNFRSF, and the immunoreceptor recognition site of the antigen-binding molecule can use an agonist antibody against a costimulatory molecule belonging to TNFRSF (hereinafter referred to as a TNFRSF agonist antibody) or an antigen-binding fragment thereof. In various specific examples, the antigen-binding molecule used as the pharmaceutical composition of the present invention can activate the activity of the chimeric receptor-expressing cell by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 750%, 1000%, or more.

[0273] In one embodiment of the present invention, the extracellular domain of the chimeric receptor includes a target molecule or fragment thereof of a TNFRSF agonist antibody. The target molecule of the TNFRSF agonist antibody is not particularly limited as long as it is a factor that activates cells expressing the TNF receptor superfamily (e.g., T cells and NK cells). Preferred factors include, for example, CD137 and CD40. A more preferred factor is, for example, CD137. For example, examples of CD137 agonist antibodies include Urelumab (CAS registry number: 934823-49-1) and various other known CD137 agonist antibodies.

[0274] In one embodiment of the present invention, the extracellular domain of the chimeric receptor contains an epitope of an antibody having agonist activity against the immune receptor as a fragment of the immune receptor. Examples of immune receptors are as described above, and CD137 is a preferred example.

[0275] In one embodiment of the present invention, the extracellular domain of the chimeric receptor comprises the target molecule or a fragment thereof of a CD137 agonist antibody. Examples of CD137 agonist antibodies include the following antibodies, such as those indicated in the sequence number described in WO2015 / 156268: [1] An antibody having the amino acid sequence described in SEQ ID NO: 66 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 85 as the light chain variable region; [2] An antibody having the amino acid sequence described in SEQ ID NO: 67 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 86 as the light chain variable region; [3] An antibody having the amino acid sequence described in SEQ ID NO: 70 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 89 as the light chain variable region; [4] An antibody having the amino acid sequence described in SEQ ID NO: 76 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 95 as the light chain variable region; [5] An antibody having the amino acid sequence described in SEQ ID NO: 77 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 96 as the light chain variable region; [6] An antibody having the amino acid sequence described in SEQ ID NO: 78 as the heavy chain variable region and the amino acid sequence described in SEQ ID NO: 97 as the light chain variable region; [7] An antibody described in any of [1] to [6], having the amino acid sequence described in SEQ ID NO: 99 as the heavy chain constant region, and the amino acid sequence described in SEQ ID NO: 59 or SEQ ID NO: 60 as the light chain constant region; [8] An antibody having activity equivalent to that of any of the antibodies described in [1] to [7]; [9] An antibody that binds to the same epitope as the antibody described in any of [1]~[7].

[0276] In the antibodies described in [8] above, "equivalent activity" means that the agonist activity to CD137 is 70% or more, preferably 80% or more, and more preferably 90% or more, of the binding activity of the antibody described in any of [1] to [7] above.

[0277] A preferred example of an antibody that binds to the same epitope as the antibody described in any of [1] to [7] above is an antibody that recognizes the region in the CD137 protein having the sequence SPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGC. Furthermore, an antibody that recognizes the region in the CD137 protein having the sequence DCTPGFHCLGAGCSMCEQDCKQGQELTKKGC can also be mentioned.

[0278] In one embodiment, examples of extracellular hinge domains include those of CD8 alpha, CD8 beta, CD28, CD4, NKp30, NKp44, and NKp46. Alternatively, the hinge region of an immunoglobulin (e.g., IgG4) may be used.

[0279] In one embodiment, examples of proteins from which the transmembrane domain is derived include the alpha and beta chains of the T cell receptor, CD3 zeta, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 alpha, CD8 beta, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, GITR, NKp30, NKp44, NKp46, etc. In one embodiment, the protein from which the transmembrane domain is derived is CD8 alpha or It's CD28.

[0280] The term "signaling domain" refers to the functional portion of a protein that acts by transmitting information within an cell to regulate cellular activity via a defined signaling pathway, either by generating a second messenger or by functioning as an effector in response to such a messenger.

[0281] When used in this disclosure, the term “intracellular signaling domain” refers to the intracellular portion of a molecule. Intracellular signaling domains can generate signals that promote the immune effector functions of cells containing chimeric receptors, such as chimeric receptor-expressing T cells. Examples of immune effector functions in chimeric receptor-expressing T cells include cytolytic activity and helper activity, such as cytokine secretion. In embodiments, the intracellular signaling domain is a portion of a protein that transmits effector function signals, causing cells to perform specialized functions. While the entire intracellular signaling domain may be employed, it is often not necessary to use the entire chain. As long as a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, provided that it transmits effector function signals. Therefore, the term intracellular signaling domain means encompassing any truncated portion of an intracellular signaling domain sufficient to transmit effector function signals.

[0282] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. Typical primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent simulation. In another embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. Typical co-stimulatory intracellular signaling domains include those derived from molecules involved in co-stimulatory signals or antigen-independent stimulation. For example, in the case of chimeric receptor-expressing T cells, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0283] As used in this disclosure, the term “CD3 zeta” refers to the group 3 (CD3) T cell coreceptor of all mammalian species, preferably human. In mammals, CD3 comprises a CD3 zeta chain, a CD3 delta chain, and two CD3 epsilon chains. The CD3 zeta chain (e.g., NCBI RefSeq: NP_932170.1) comprises an intracellular signaling domain that can be used to manipulate the chimeric receptor. In the specific chimeric receptors of this disclosure, the primary signaling sequence of CD3-zeta is either the full-length or partial cytoplasmic region sequence (nucleotide sequences 299-634) of Genbank NM000734.3, or equivalent residues from non-human species, such as mice, rodents, monkeys, apes, and their congeners.

[0284] In one embodiment, the intracellular signaling domain may include the cytoplasmic domain of the interleukin receptor chain. In one embodiment, the intracellular signaling domain may be CD28, 4-1BB, ICOS, or CD3 zeta-CD28-4-1BB or CD3 zeta-CD28-OX40, which are composed of multiple signaling domains linked together. In one embodiment, the protein from which the transmembrane domain originates is CD8 alpha or CD28, and the intracellular signaling domain may be CD28, 4-1BB, ICOS, or CD3 zeta-CD28-4-1BB or CD3 zeta-CD28-OX40, which are composed of multiple signaling domains linked together.

[0285] In one embodiment, a chimeric receptor is disclosed comprising: i) an extracellular domain capable of binding to an immunoreceptor recognition site of an antigen-binding molecule; ii) a transmembrane domain; and iii) an intracellular segment comprising one or more intracellular signaling domains selected from a cytoplasmic costimulatory domain and / or the cytoplasmic domain of an interleukin receptor chain, and a CD3 zeta intracellular signaling domain comprising an exogenous STAT3-related motif (wherein the intracellular segment comprises an endogenous or exogenous JAK-binding motif and a STAT5-related motif). In some embodiments, these domains are fused directly or indirectly, optionally starting from the N-terminus and in the above order. In some embodiments, these domains within the intracellular segment are fused in the reverse order.

[0286] The term "stimulus" refers to the primary response induced by the binding of a stimulating molecule (e.g., a TCR / CD3 complex or a chimeric receptor) to its ligand (or, in the case of a chimeric receptor, the immunoreceptor recognition site of the antigen-binding molecule), thereby mediating a signaling event, including, but not limited to, signaling via the TCR / CD3 complex or signaling via the appropriate NK receptor or signaling domain of a chimeric receptor. Stimuli may also mediate the altered expression of a particular molecule.

[0287] The term “stimulating molecule” refers to a molecule expressed by immune cells, such as T cells, NK cells, or B cells, that gives rise to an intracellular signaling sequence that modulates the activation of immune cells in the form of a stimulus, with respect to at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated, for example, by the binding of a TCR / CD3 complex to an MHC molecule presenting a peptide, thereby mediating T cell responses such as proliferation, activation, differentiation, and homogeneous reactions, but not limited to these. The primary intracellular signaling sequence acting in the form of a stimulus (also referred to as the “primary signaling domain”) may contain a signaling motif, which is known as an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs containing intracellular signaling sequences with specific applications in this disclosure include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc epsilon RI, CD66d, CD32, DAP10, DAP12, CLEC2, CLEC7A (Dectin1), CLEC9A, EZRIN, RADIXIN, and MOESIN. In the specific chimeric receptors of this disclosure, the intracellular signaling domain in any one or more chimeric receptors of this disclosure comprises an intracellular signaling sequence, such as the primary signaling sequence of CD3-zeta. The extracellular domain of the chimeric receptor of the present invention may be the extracellular domain of the above-mentioned stimulating molecule.

[0288] The term "costimulatory molecule" refers to a co-stimulatory partner on a T cell that, by specifically binding to a costimulatory ligand, mediates a costimulatory response by T cells, such as proliferation (secondary signaling), although this does not apply to costimulatory ligands. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. The term "costimulatory intracellular signaling domain" refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain may include the entire intracellular portion, the entire intrinsic intracellular signaling domain of the molecule from which it is obtained, or functional fragments or derivatives thereof. Co-stimulatory molecules are not limited to these, but include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, OX40 (CD134), CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD5, CD8 Alpha, CD8 Beta, IL2R Beta, IL2R Gamma, IL7R Alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2 Ligands that specifically bind to D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD154, and CD83 are examples. The extracellular domain of the chimeric receptor of the present invention may be the extracellular domain of the above-mentioned co-stimulatory molecule.

[0289] In one embodiment, the co-stimulatory molecule is selected from, for example, 4-1BB (i.e., CD137), CD27, CD28, and / or OX40 to enhance T cell receptor stimulation.

[0290] The terms "4-1BB" or "CD137" refer to a member of the TNFR superfamily having the sequence amino acid sequence provided as GenBank accession number AAA62478.2, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes and their congeners, and the "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes and their congeners. In one embodiment, the "4-1BB costimulatory domain" is the full length or a portion of nucleotide sequence 886-1026 of Genbank NM001561.5, or equivalent residues from non-human species, e.g., mice, rodents, monkeys, apes and their congeners.

[0291] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in either single-stranded or double-stranded forms. The term “nucleic acid” encompasses genes, cDNA, or mRNA. In one embodiment, a nucleic acid molecule is a synthetic (e.g., chemically synthesized) nucleic acid molecule or a recombinant nucleic acid molecule. Unless otherwise specified, the term encompasses nucleic acids containing analogs or derivatives of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also substantially encompasses its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, in addition to sequences explicitly presented. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a hybrid base and / or deoxyinosine residue [Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. [Probes 8:91-98 (1994)].

[0292] As used in this disclosure, the term “nucleic acid sequence” means a sequence of nucleosides or nucleotide monomers consisting of natural bases, sugars, and intersugar (backbone) bonds. The term also includes modified or substituted sequences comprising non-natural monomers or parts thereof. The nucleic acid sequences of this application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain natural bases including adenine, guanine, cytosine, thymine, and uracil. These sequences may also contain modified bases. Examples of such modified bases include aza and deaza. It contains adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

[0293] As used in this disclosure, the term “isolated nucleic acid” means a nucleic acid that is substantially free from cell material or culture medium if produced by recombinant DNA technology, or from chemical precursors or other chemicals if chemically synthesized. An isolated nucleic acid also substantially does not contain the sequences naturally adjacent to the nucleic acid from which it originates (i.e., the sequences located at the 5' and 3' ends of the nucleic acid). The term “nucleic acid” includes DNA and RNA, which may be double-stranded or single-stranded, and may correspond to sense or antisense strands. Furthermore, the term “nucleic acid” includes complementary nucleic acid sequences, such as cDNA.

[0294] The term “coding” refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which has either a specific sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a specific sequence of amino acids, and the biological properties that result from it, serving as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, in cells or other biological systems, when proteins are produced by the transcription and translation of mRNA corresponding to a gene, that gene, cDNA, or RNA codes for a protein. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually shown in sequence listings, and the non-coding strand used as a template for the transcription of a gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0295] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" encompasses all nucleotide sequences that are degenerate of each other or that encode the same amino acid sequence. Furthermore, the phrase "nucleotide sequence encoding a protein or RNA" may, in some cases, contain introns to the extent that the nucleotide sequence encoding that protein may contain introns. Additionally, nucleic acid molecules may be operably bound to at least one regulatory element for the expression of a chimeric receptor.

[0296] The terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide encompasses any peptide or protein containing two or more amino acids linked together by peptide bonds. As used in this disclosure, this term refers to both short chains, commonly referred to in the industry as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the industry as proteins, of which many types exist. Examples of “polypeptides” include, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Examples of polypeptides include native peptides, recombinant peptides, or combinations thereof.

[0297] In this disclosure, polypeptides typically refer to peptides and proteins having a length of about 10 amino acids or more. When a series of amino acids linked by peptide bonds from the N-terminus to the C-terminus is considered a peptide chain, the polypeptides in this disclosure may also be complex proteins formed by interactions such as disulfide bonds, hydrophobic interactions, and ionic bonds between multiple series of peptide chains.

[0298] The term "isolated polypeptide," also known as "isolated protein," is produced using recombinant DNA technology, or is derived from cell material or culture medium, or is chemically synthesized. In this context, it refers to polypeptides that are substantially free of chemical precursors or other chemical substances.

[0299] The term "amino acid" includes all natural and modified amino acids.

[0300] A "conservative amino acid variation," as used in this disclosure, is a variation in which one amino acid residue is substituted with another amino acid residue without impairing the desired properties of the protein.

[0301] The amino acid sequences specifically provided in this application may include conservative sequence modifications. “Conservative sequence modification” refers to amino acid mutations that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of this disclosure by standard techniques known in the industry, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the industry. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the chimeric receptors of this disclosure may be replaced with other amino acid residues of the same side chain family, and the modified chimeric receptors can be tested using the functional assays described herein.

[0302] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0303] In this disclosure, “host cells,” “host cell lines,” and “host cell cultures” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those used when the original transformed cells were screened or selected are also included in this disclosure.

[0304] In this disclosure, “vector” refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to in this disclosure as “expression vectors.”

[0305] Transfection refers to the uptake of an expression vector by a host cell, where it is uncertain whether the desired encoding sequence will actually be expressed. Many transfection methods are known to technicians with the usual skills, such as CaPO4 precipitation and electroporation. Generally, successful transfection is recognized when signs of the vector's activity appear in the host cell.

[0306] The term "promoter" refers to a DNA sequence recognized by a cellular synthetic mechanism, or the introduced synthetic mechanism, that is necessary to initiate the specific transcription of a polynucleotide sequence.

[0307] The term "lentivirus" refers to a genus of retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and because they can deliver a significant amount of genetic information to the host cell's DNA, they are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0308] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, particularly self-inactivating lentiviral vectors as described in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used in clinical settings include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX® vector system, and similar products. Non-clinical lentiviral vectors are also available, and the selection and preparation of vectors can be appropriately carried out by those skilled in the art.

[0309] In this disclosure, the term “pharmaceutically acceptable” means that the molecular entities and other components of such compositions are physiologically tolerable and do not typically produce undesirable reactions when administered to mammals (e.g., humans). Preferably, “pharmaceutically acceptable” in this disclosure means that it is approved by a federal or state regulatory authority or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, more specifically in humans.

[0310] In one embodiment, the chimeric receptor of this disclosure comprises two signaling domains described herein, namely CD3 zeta and 4-1BB / CD137, or CD3 zeta plus one or more signaling domains. In one particular embodiment, several signaling domains are fused with one another for additive or synergistic effects. Non-limiting examples of useful additional signaling domains include one or more of the following: TCR zeta chain, CD27, CD28, OX40 / CD134, 4-1BB / CD137, Fc epsilon RIy, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, DAP-10, and CD40.

[0311] This disclosure provides a chimeric receptor comprising: i) an extracellular domain capable of binding to a given antigen via an antigen-binding molecule; ii) a transmembrane domain; and iii) an intracellular segment comprising a CD3 zeta intracellular signaling domain comprising one or more intracellular signaling domains selected from a cytoplasmic costimulatory domain and / or the cytoplasmic domain of an interleukin receptor chain, and an exogenous STAT3-related motif (wherein the intracellular segment comprises an endogenous or exogenous JAK-binding motif and a STAT5-related motif). In some embodiments, these domains are fused directly or indirectly, optionally starting from the N-terminus and in the above order. In some embodiments, these domains within the intracellular segment are fused in the reverse order.

[0312] The disclosure also includes a chimeric receptor comprising i) an extracellular domain that can be bound via an antigen-binding molecule, ii) a transmembrane domain, and iii) an intracellular segment comprising one or more intracellular signaling domains including the cytoplasmic domain of an interleukin (IL) receptor chain and optionally at least one supplemental cytoplasmic domain. In some embodiments, these domains are fused directly or indirectly, optionally starting from the N-terminus and in the above order. In one embodiment, these domains within the intracellular segment are They are fused in the reverse order.

[0313] In some embodiments, the IL receptor chain is located proximal to the transmembrane domain and / or near the N-terminus of the intracellular segment of the chimeric receptor, or forms the N-terminus. In other embodiments, the IL receptor chain is located near the C-terminus of the intracellular segment of the chimeric receptor, or forms the C-terminus. In some embodiments, the IL receptor chain is located upstream or N-terminal to the CD3 zeta intracellular signaling domain, which comprises the exogenous STAT3-associated motif YXXQ.

[0314] In embodiments in which the intracellular segment comprises only the signaling domain of the IL receptor chain, the chimeric receptor-expressing cell can be activated, for example, by B cells, by a predetermined antigen present in the MHC complex via the endogenous TCR and / or by the CD80 / 86 molecule via endogenous CD28.

[0315] Furthermore, cells expressing the chimeric receptor of this disclosure are also provided. Such cells may exhibit higher cytotoxic activity against cells having a predetermined / pre-selected antigen on their surface to which the chimeric receptor binds via an antigen-binding molecule, compared, for example, with parental cells that do not express the chimeric receptor. For example, as shown in the examples, cells expressing the chimeric receptor of this disclosure provide an antigen-binding molecule-dependent and cancer / tumor antigen-dependent antitumor effect upon administration of an antigen-binding molecule. Therefore, an antitumor effect in humans is also expected.

[0316] In cells expressing the chimeric receptor of this disclosure, it is sufficient that the chimeric receptor of this disclosure is expressed; other transduction may also occur.

[0317] The intracellular segment of the chimeric receptor described herein may comprise one or more intracellular signaling domains, and is a proteinaceous molecule capable of transmitting signals to cells when an extracellular domain located within the same molecule binds to (interacts with) its cognitive antigen / ligand.

[0318] In one aspect, the intracellular segment of the chimeric receptor comprises a CD3 zeta intracellular signaling domain comprising an exogenous STAT3-related motif. In addition, the intracellular segment of the chimeric receptor comprises one or more intracellular signaling domains selected from the cytoplasmic domain and / or cytoplasmic costimulatory domain of the IL receptor chain, and the intracellular segment comprises an endogenous or exogenous JAK-binding motif and a STAT5-related motif.

[0319] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex. For example, the intracellular signaling domain of CD3 zeta provides primary cytoplasmic signaling. Primary cytoplasmic signaling sequences include immune receptor tyrosine activation motifs (ITAMs) [Nature, vol. 338, The domain may contain a signaling motif known as [pp. 383-384 (1989)]. On the other hand, the inhibitory primary cytoplasmic signaling sequence may contain a signaling motif known as the immune receptor tyrosine inhibitory motif (ITIM) [J Immunol., vol. 162, No. 2, pp. 897-902 (1999)]. In this disclosure, intracellular signaling domains having ITAM and / or ITIM can be used.

[0320] In this disclosure, the intracellular domain of CD3 zeta comprises an immune receptor tyrosine activation motif (ITAM). Examples of intracellular signaling domains having usable ITAMs include, for example, intracellular signaling domains having ITAMs derived from FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, instead of or replacing CD3 zeta. Specifically, intracellular domains comprising one or more ITAMs Examples include amino acid numbers 52-164 of CD3 zeta (NCBI RefSeq: NP_932170.1), amino acid numbers 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP_004097.1), amino acid numbers 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP_000130.1), amino acid numbers 139-182 of CD3 gamma (NCBI RefSeq: NP_000064.1), amino acid numbers 128-171 of CD3 delta (NCBI RefSeq: NP_000723.1), amino acid numbers 153-207 of CD3 epsilon (NCBI RefSeq: NP_000724.1), and CD5 (NCBI This includes peptides having the sequences of amino acid numbers 402-495 of CD22 (NCBI RefSeq:NP_055022.2), amino acid numbers 707-847 of CD22 (NCBI RefSeq:NP_001762.2), amino acid numbers 166-226 of CD79a (NCBI RefSeq:NP_001774.1), amino acid numbers 182-229 of CD79b (NCBI RefSeq:NP_000617.1), and amino acid numbers 177-252 of CD66d (NCBI RefSeq:NP_001806.2), as well as their variants having the same function as these peptides. The amino acid numbers based on the NCBI RefSeq ID or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor of each protein (including signal peptide sequences, etc.).

[0321] In one embodiment, the amino acid residue represented by "X" in the STAT3-related motif YXXQ can be any native amino acid, including any modified native amino acid that holds a STAT3 bond. In one embodiment, amino acid X is independently selected from leucine, arginine, histidine, phenylalanine, lysine, proline, methionine, valine, glutamine, threonine, and aspartic acid. For example, amino acid X is arginine. For example, amino acid X is histidine.

[0322] In one embodiment, the two amino acid residues adjacent to the tyrosine residue of the STAT3-related motif YXXQ are arginine-histidine. In yet another embodiment, the exogenous STAT3-related motif is YRHQ.

[0323] The exogenous STAT3-associated motif YXXQ can be introduced into any part of the intracellular domain of CD3 zeta, although in some embodiments, the YXXQ-associated motif is inserted near the C-terminal region. While we do not wish to be bound by any particular theory, many endogenous YXXQ motifs are thought to be located around 100aa from the C-terminus or within that range. Furthermore, YXXQ motifs located near the C-terminal region have been shown to be more functional when more proximal in GP130 and LIFR studies (Schmitz J et al. J Immunol. 2000;164:848-54; Tomida M et al. Blood. 1999;93:1934-41).

[0324] In one embodiment, the exogenous STAT3-related motif YXXQ is introduced into any portion of the intracellular domain of CD3 zeta within 200 amino acid residues from the C-terminus of the chimeric receptor. For example, the STAT3-related motif may be introduced within 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acid residues from the C-terminus of the chimeric receptor. In one embodiment, the exogenous STAT3-related motif is introduced at a location other than the ITAM.

[0325] In one embodiment, the CD3 zeta intracellular domain comprising an exogenous STAT3-related motif comprises at least one ITAM motif. In another embodiment, the CD3 zeta intracellular domain comprising an exogenous STAT3-related motif comprises two ITAM motifs. In yet another embodiment, comprising an exogenous STAT3-related motif The intracellular domain of CD3 zeta protein consists of three ITAM motifs.

[0326] Those skilled in the art will recognize that several methods are available for introducing STAT3-related motifs into the intracellular signaling domain of CD3 zeta. For example, exogenous STAT3-related motifs can be introduced by substituting the amino acid residue Leu-His-Met at positions 104–106 of the intracellular signaling domain of CD3 zeta with the tyrosine at position 104 and any two other amino acid residues adjacent to that tyrosine residue at positions 105 and 106. Amino acid residues 104–105–106 of the intracellular signaling domain of CD3 zeta correspond to amino acid residues 156–157–158 of the full-length CD3 zeta (e.g., NCBI RefSeq: NP_932170.1).

[0327] As described, in one embodiment, the chimeric receptor comprises an intracellular segment comprising one or more intracellular signaling domains selected from the IL receptor chain and the cytoplasmic domain of the cytoplasmic costimulatory domain.

[0328] In this disclosure, the cytoplasmic domain of the IL receptor chain can be selected from any chain of the IL receptor. For example, a cytoplasmic domain comprising amino acids 266-551 of the IL-2 receptor beta chain (NCBI REFSEQ:NP_000869.1) (amino acid numbers 256-538 of the IL-21 receptor alpha chain (NCBI REFSEQ:NP_068570.1)), amino acid numbers 284-369 of the common IL-2 receptor gamma chain (NCBI REFSEQ:NP_000197.1), amino acid numbers 265-459 of the IL-7R alpha (NCBI REFSEQ:NP_002176.2), amino acid numbers 292-521 of the IL-9R alpha (NCBI REFSEQ:NP_002177.2), or amino acid numbers 257-825 of the IL-4R alpha (NCBI REFSEQ:NP_000409.1) can be used. The entire cytoplasmic domain of the IL receptor chain is available for use.

[0329] Alternatively, terminal cleavage fragments of the cytoplasmic domain of the IL receptor (ILR) chain can also be used. For example, the terminal cleavage fragment may consist of up to 250 amino acids of the ILR cytoplasmic domain, or 50-200 amino acids or 80-150 amino acids.

[0330] In one embodiment, the cytoplasmic domain of the IL receptor chain, or optionally a terminal cleavage fragment of the cytoplasmic domain of the IL receptor chain, comprises at least a STAT-related motif, optionally a STAT5-related motif, and a JAK-binding motif (also known as a box-1 motif). In another embodiment, the cytoplasmic domain of the IL receptor chain or its terminal cleavage fragment comprises a STAT5-related motif and a JAK-binding motif.

[0331] In one embodiment, the cytoplasmic domain and / or terminal cleavage fragments of the IL receptor chain include variants having the same function, such as variants that induce STAT signaling, and optionally STAT5 signaling and / or JAK signaling.

[0332] In one aspect of this disclosure, the cytoplasmic domain of the IL-2 receptor (IL-2R) beta chain is available. An example of the cytoplasmic domain of the IL-2R beta chain available in this disclosure is amino acid numbers 266-551 of the IL-2R beta chain (NCBI RefSeq: NP_000869.1). In one embodiment, a peptide having either the sequence of amino acid numbers 266-337 or 530-551 is included. In one aspect of this disclosure, terminal cleavage fragments of the cytoplasmic domain of the IL-2R beta chain are available. These terminal cleavage fragments i) enable association with the tyrosine kinase JAK1, a JAK-binding motif also known as the BOX-1 motif (e.g., NCBI RefSeq : Amino acid numbers 278-286 of NP_000869.1, and ii) STAT-related motifs, which may optionally include STAT5 or STAT3-related motifs. Other parts of the IL receptor chain can be modified, for example, by conservative amino acid variations.

[0333] In some embodiments, the intracellular segment may include an exogenous JAK-binding motif or a signaling molecule comprising a JAK-binding motif. For example, the JAK-binding motif may be derived from IL2R gamma (IL2RG), erythropoietin receptor (EpoR), thrombopoietin receptor (TpoR), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), and growth hormone receptor (GHR).

[0334] The IL-2R beta chain comprises three functional STAT5-binding motifs, YFFF, YCTF, and YLSL, used for STAT5 association. Mutations in these tyrosine residues can invalidate the IL-2 reactivity of the IL-2R beta chain (Friedmann et al., 1996). The erythropoietin receptor (EpoR) comprises two tyrosine residues, Y343 and Y401, which mediate STAT5 activation, and both have been described as possessing a YXXL motif (Klingmuller et al., 1996). Therefore, YXXL may be a preferred motif for STAT5 recruitment. Other amino acid residues are also functional, for example, as shown in the IL-2R beta chain STAT5-binding motif. In one embodiment, the STAT5-related motif is an IL-2R beta-chain STAT5-related motif and comprises tyrosine residue -510 (tyrosine residue 510 is amino acid number 536 in NCBI RefSeq: NP_000869.1).

[0335] In one embodiment, the STAT5-related motif may be derived from IL2R gamma, EpoR, TpoR, GM-CSFR, and GHR.

[0336] In one embodiment, the STAT5-associated motif of the IL-2R beta chain comprises amino acid residues YXXL. In one embodiment, the amino acid residue represented by "X" in the STAT5-associated motif may be any native amino acid, including any modified native amino acid that holds a STAT5 bond.

[0337] Similarly, the intracellular segment comprises one or more JAK-binding motifs that can be positioned or introduced into any of the intracellular signaling domains.

[0338] In one aspect of this disclosure, the cytoplasmic domain of the IL-21 receptor (IL-21R) alpha chain is available. An example of the IL-21R alpha chain cytoplasmic domain used in this disclosure is an intracellular signaling domain comprising amino acids 256-538 of the IL-21R alpha chain (NCBI RefSeq: NP_068570.1). In one aspect of this disclosure, terminal cleavage fragments of the IL-21R alpha chain cytoplasmic domain are available. The terminal cleavage fragments include a box-1 motif (amino acids 266-274 of NCBI RefSeq: NP_068570.1) necessary for association with tyrosine kinase JAK1, as well as a STAT-related motif. In one embodiment, the STAT-related motif comprises tyrosine residue -500 (amino acid number 519 in NCBI RefSeq: NP_000869.1) and tyrosine residue 500 required for STAT1 / 3 association, i.e., three residues adjacent to the C-terminus of YLRQ.

[0339] Other examples of intracellular signaling domains include cytoplasmic regions derived from TCR complexes and / or costimulatory molecules, and any variants having the same function as those sequences. Further examples are found in Table 2 of Sadelain et al 2009, which are incorporated herein by reference. The listed cytoplasmic signaling domains are included.

[0340] Activation of native T cells is transmitted by two distinct types of intracellular signaling domains: a domain for inducing antigen-dependent primary activation (e.g., primary cytoplasmic signaling, provided by CD3 zeta, for example) via the TCR complex, and an antigen-independent domain for providing secondary or co-stimulatory signals (secondary cytoplasmic signaling).

[0341] Examples of intracellular domains available in this disclosure that comprise secondary or co-stimulatory cytoplasmic signaling domains include sequences derived from CD2, CD4, CD5, CD8 alpha, CD8 beta, CD28, CD134, CD137 (4-1BB), ICOS, and CD154, for example, their terminal cleavage fragments comprising a signaling motif. Specific examples include amino acid numbers 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acid numbers 421-458 of CD4 (NCBI RefSeq: NP_000607.1), amino acid numbers 402-495 of CD5 (NCBI RefSeq: NP_055022.2), amino acid numbers 207-235 of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acid numbers 196-210 of CD8 beta (GenBank: AAA35664.1), amino acid numbers 180-220 of CD28 (NCBI RefSeq: NP_006130.1), amino acid numbers 214-255 of CD137 (4-1BB, NCBI RefSeq: NP_001552.2), and CD134 (OX40, NCBI This includes peptides having either amino acid sequences 241-277 of ICOS (RefSeq:NP_003318.1) or amino acid sequences 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as their variants having the same function as these peptides.

[0342] A preferred disclosure is, in one aspect, a chimeric receptor comprising an intracellular signaling domain of CD3 zeta comprising an exogenous STAT3-related motif, in addition to an intracellular segment having one or more, for example, two or three intracellular signaling domains.

[0343] The disclosure also includes chimeric receptors comprising intracellular segments having two or more identical intracellular signaling domains in series. In one aspect, the disclosure provides a chimeric receptor comprising a chimeric receptor in which the cytoplasmic domain of the IL receptor is located at the N-terminal end of the intracellular signaling domain of CD3 zeta, i.e., a chimeric receptor comprising a chimeric receptor in which the cytoplasmic domain of the IL receptor and the intracellular signaling domain of CD3 zeta are linked in this order from the N-terminal end. The disclosure also includes a chimeric receptor obtained by further adding the intracellular domain of CD28 (e.g., the cytoplasmic costimulatory domain of CD28) to the above-described chimeric receptor, i.e., a chimeric receptor comprising a chimeric receptor in which the intracellular signaling domain of CD28, the cytoplasmic domain of the IL receptor, and the intracellular signaling domain of CD3 zeta comprising an exogenous STAT3 motif are linked in this order from the N-terminal end.

[0344] In one embodiment, the chimeric receptor comprises an intracellular segment comprising a CD3 zeta intracellular signaling domain, which includes an exogenous STAT3-related motif and an intracellular signaling domain selected from the cytoplasmic domain and cytoplasmic costimulatory domain of an interleukin receptor chain, wherein at least one of the intracellular signaling domains comprises an endogenous or exogenous JAK-binding motif and a STAT5-related motif.

[0345] In one embodiment, the chimeric receptor comprises a CD3 zeta intracellular signaling domain having an exogenous STAT3-related motif, and a cytoplasmic domain of an IL receptor chain fragment comprising an endogenous or exogenous JAK-binding motif and a STAT5-related motif, and CD It comprises 28 cytoplasmic costimulatory domains.

[0346] In the chimeric receptors of this disclosure, oligopeptide linkers or polypeptide linkers can be inserted between domains of intracellular segments, so as to link the domains thereto and / or link them to other domains. For example, linkers having a length of 2 to 10 amino acids can be used. In particular, linkers having a glycine-serine sequence can be used. For example, the linker IDGGGGSGGGGSGGGGS can be inserted between the CD28 cytoplasmic domain and the partial cytoplasmic IL-2 receptor beta domain. For example, the linker KLGGSGP can be inserted between the partial cytoplasmic IL-2 receptor beta domain and the intracellular domain of the CD3 zeta chain.

[0347] In another aspect, a chimeric receptor is provided, comprising i) an extracellular domain capable of binding to an immunoreceptor recognition site of an antigen-binding molecule, ii) a transmembrane domain, and iii) an intracellular segment comprising one or more intracellular signaling domains including a cytoplasmic domain and optionally a supplemental cytoplasmic domain of an interleukin receptor chain.

[0348] The cytoplasmic domain of the IL receptor chain may be selected from any of the IL receptor chains described herein. The entire region of the cytoplasmic domain of the IL receptor chain is available. Alternatively, terminal cleavage fragments of the cytoplasmic domain of the IL receptor chain are also available. Examples of the full-length and terminal cleavage fragments are shown herein.

[0349] In some embodiments, the terminal cleavage fragment may include at least one tyrosine kinase-related motif (also known as a box-1 motif) and a STAT (signaling activator)-related motif as described herein. For example, the terminal cleavage fragment may consist of up to 250 amino acids of the ILR cytoplasmic domain, or 50 to 200 amino acids or 80 to 150 amino acids.

[0350] The STAT-related motif of the IL-2R beta chain comprises tyrosine residue -510 (tyrosine residue 510 is amino acid number 536 in NCBI RefSeq: NP_000869.1). In one embodiment, the STAT-related motif comprises tyrosine residue 510 and four residues adjacent to the C-terminus of tyrosine residue 510, i.e., YLSLQ.

[0351] Other STAT-related motifs are also known, including, for example, YXXQ and possibly YXPQ of IL-6, YXXQ of IL-10, YLPSNID of IL-12, YLSLQ, YCTFP, YFFFH of IL-2, YVTMS of IL-7, YLPQE of IL-9, and YKAFS and YKPFQ of IL-4. Any of these STAT signaling domains are available and / or can be introduced into the ILR chain.

[0352] In some embodiments, in addition to the cytoplasmic domain of the IL receptor, the chimeric receptor intracellular segment comprises at least one supplemental signaling domain other than those present within the IL receptor. Examples of intracellular signaling domains include cytoplasmic regions and / or costimulatory molecules derived from the TCR complex, and any variants having the same function as such sequences. Other examples include the cytoplasmic signaling domains listed in Table 2 of Sadelain et al 2009, which are incorporated herein by reference.

[0353] This disclosure includes a chimeric receptor comprising an intracellular segment comprising one or more, for example, two or three intracellular signaling domains in addition to the cytoplasmic domain of the IL receptor. For example, the chimeric receptor comprises the cytoplasmic domain of the IL receptor and the intracellular signaling domain of CD3 zeta. For example, the chimeric receptor comprises the cytoplasmic domain of the IL receptor It comprises the intracellular signaling domain of CD3 zeta and the cytoplasmic costimulatory domain of CD28.

[0354] In one embodiment, the chimeric receptor comprises an intracellular segment comprising a CD3 zeta intracellular signaling domain, and one or more cytoplasmic costimulatory domains, wherein the intracellular segment comprises a JAK-binding motif, STAT5 and / or STAT3-related motifs.

[0355] The chimeric receptors of this disclosure comprise a transmembrane domain. The transmembrane domain may be derived from a natural polypeptide or may be artificially designed. Transmembrane domains derived from natural polypeptides can be obtained from membrane-bound or transmembrane proteins. For example, transmembrane domains of T cell receptor alpha or beta chains, CD3 zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR are available. Artificially designed transmembrane domains are primarily polypeptides comprising hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan, and valine may be found at each end of a synthetic transmembrane domain. Optionally, short oligopeptide linkers or polypeptide linkers, for example, linkers 2 to 10 amino acids long, may be positioned between the transmembrane domain and the intracellular segment described herein. In particular, linker sequences having a glycine-serine sequence can be used.

[0356] For example, a transmembrane domain having any of the sequences between amino acid numbers 153 and 179 of CD28 (NCBI RefSeq: NP_006130.1) can be used as a transmembrane domain.

[0357] In the chimeric receptors of this disclosure, the spacer domain can be positioned between the extracellular domain and the transmembrane domain, or between the intracellular segment and the transmembrane domain. The spacer domain means any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the transmembrane domain to the intracellular segment. The spacer domain comprises up to 300 amino acids, for example, about 10 to 100 amino acids, or about 25 to 50 amino acids.

[0358] The spacer domain preferably has a sequence that promotes the binding of the chimeric receptor to the antigen via an antigen-binding molecule and enhances signaling to the cell. Examples of amino acids expected to promote binding include cysteine, charged amino acids, and serine and threonine within the potential glycosylation site, and these amino acids can be used as amino acids constituting the spacer domain.

[0359] In one embodiment, the spacer domain is a polypeptide comprising or consisting of amino acids 118-178 of CD8 alpha (NCBI RefSeq: NP_001759.3), i.e., the hinge region of CD8 alpha, amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), amino acids 114-152 of CD28 (NCBI RefSeq: NP_006130.1), or a portion thereof. Furthermore, the spacer domain may be an artificially synthesized sequence.

[0360] The chimeric receptors of this disclosure can be designed to form polymers, in particular dimers. For example, the chimeric receptors can be polymerized (dimerized) via disulfide bonds. Therefore, cysteine ​​is inserted into the spacer domain and / or transmembrane domain.

[0361] Furthermore, the chimeric receptors of this disclosure can have a signal peptide sequence ligated to their N-terminus. Signal peptide sequences are present at the N-terminus of many secretory and membrane proteins and have a length of 15 to 30 amino acids. Many of the protein molecules having an intracellular domain described herein are membrane proteins and possess a signal peptide sequence. Signal peptides derived from such secretory and membrane proteins can be used as signal peptides for the chimeric receptors of this disclosure. Any signal peptide can be used. For example, the signal peptide may be an oncostatin M signal peptide. The signal peptide may be of human origin or non-human origin, such as from insect cells or viruses. In one embodiment, the signal peptide is a human signal peptide.

[0362] This disclosure provides nucleic acids encoding the chimeric receptor described herein. The nucleic acids encoding the chimeric receptor can be readily prepared by conventional methods from the amino acid sequence of the indicated chimeric receptor. The nucleotide sequence encoding the amino acid sequence can be obtained from the aforementioned NCBI RefSeq ID or GenBank accession number for the amino acid sequence of each domain, and the nucleic acids of this disclosure can be prepared using standard molecular biological and / or chemical procedures. For example, nucleic acids can be synthesized based on nucleotide sequences, and the nucleic acids of this disclosure can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0363] The nucleic acids of this disclosure can be ligated to another nucleic acid so as to be expressed under the control of a suitable promoter. Examples of promoters include promoters that constitutively promote the expression of a gene or operably ligated construct, and promoters that induce the expression of a gene or operably ligated construct by the action of a drug (e.g., tetracycline or doxorubicin). The nucleic acids of this disclosure can also be ligated to a nucleic acid comprising other regulatory elements that cooperate with the promoter or transcription start site, such as an enhancer sequence or a terminator sequence, in order to obtain efficient transcription of the nucleic acid. In addition to the nucleic acids of this disclosure, genes that can serve as markers for confirming nucleic acid expression (e.g., drug resistance genes, genes encoding reporter enzymes, or genes encoding fluorescent proteins) may be incorporated.

[0364] In one embodiment, the nucleic acid is a nucleic acid whose codons have been optimized for expression in a specific host.

[0365] A method for producing cells expressing the chimeric receptor of this disclosure comprises the step of introducing the nucleic acid encoding the chimeric receptor described herein into cells. This step is performed ex vivo. For example, cells are ex vivo to produce cells expressing the chimeric receptor of this disclosure. The nucleic acids of this disclosure can be transformed in vivo with a viral or non-viral vector carrying them.

[0366] The methods of this disclosure may use cells derived from mammals, such as human cells, or cells derived from non-human mammals such as monkeys, mice, rats, pigs, horses, or dogs.

[0367] In one embodiment, the mammal is a human.

[0368] The present disclosure provides a chimeric receptor, a nucleic acid encoding the chimeric receptor, a cell expressing the chimeric receptor, and a composition comprising the cell. In one embodiment, One or more of the above may be used in the field of adoptive immunotherapy targeting antigens such as tumor antigens, and / or in screening or other in vitro assays. The chimeric receptors of this disclosure can be introduced into cells to, for example, enhance or increase the expression level of the chimeric receptor in those cells. Such cells may exert cytotoxic activity against cells expressing the target antigen.

[0369] a) Isolating immune cells from mammals (in some cases, these immune cells are T cells); b) Transfecting isolated immune cells (or T cells, if applicable) with nucleic acids encoding chimeric receptors as described herein; and c) A method for producing cells expressing the chimeric receptor of the present disclosure is provided, which optionally comprises isolating and / or expanding the chimeric receptor-expressing cells (optionally, chimeric receptor-expressing T cells) after transfection or transduction.

[0370] In one embodiment, autologous T lymphocytes or autologous NK cells, or autologous macrophages are activated and / or proliferated ex vivo before reintroduction into the subject. In one embodiment, the T lymphocytes or NK cells are allogeneic T lymphocytes or allogeneic NK cells. In one embodiment, allogeneic T lymphocytes are T lymphocytes in which the expression of endogenous T cell receptors is blocked or eliminated. In one embodiment, allogeneic T lymphocytes or allogeneic NK cells are activated and / or proliferated ex vivo before introduction into the subject. In one embodiment, the chimeric receptor is introduced into T lymphocytes or NK cells or macrophages by a method selected from the group consisting of retroviral transduction, lentiviral transduction, DNA electroporation and RNA electroporation, DNA or RNA transfection, or gene editing.

[0371] The NK cells used in the methods of this disclosure can be preferentially proliferated by exposure to cells that lack or express little to no major histocompatibility antigen I and / or II molecules and are genetically modified to express membrane-bound IL-15 and 4-1BB ligand (CDI37L). Such cell lines include K562 [ATCC, CCL 243; Lozzio et al., Blood 45(3): 321-334 (1975); Klein et al., Int. J. Cancer 18: 421-431 (1976)] and Wilms tumor cell line HFWT [Fehniger TA, Caligiuri M A. Int Rev Immunol 20(3-4):503-534 (2001); Harada H, et al., Exp Hematol 32(7):614-621 (2004)], endometrial tumor cell line HHUA, melanoma cell line HMV-II, hepatoblastoma cell line HuH-6, lung small cell carcinoma cell lines Lu-130 and Lu-134-A, neuroblastoma cell lines NB 19 and N1369, and testicular NEC The cell lines used include, but are not limited to, embryonic carcinoma cell lines derived from 14, the cervical carcinoma cell line TCO-2, and the myelomere metastatic neuroblastoma cell line TNB 1 [Harada H., et al., Jpn. J. Cancer Res 93: 313-319 (2002)]. Preferably, the cell lines used are those that lack or express little to both MHC I and II molecules, such as K562 and HFWT cell lines. A solid support may be used instead of a cell line. Such a support preferably binds to NK cells and is capable of binding molecules that induce an initial activation event and / or a proliferative response, or have such an effect, thereby having at least one molecule bound to its surface that acts as a scaffold. The support may have a CD137 ligand protein, CD137 antibody, IL-15 protein, or IL-15 receptor antibody bound to its surface. Preferably, the support has an IL-15 receptor antibody and a CD137 antibody bound to its surface.

[0372] In one embodiment, in any of the methods of the present disclosure, including T lymphocyte activation, T lymphocytes can be activated in the presence of one or more agents selected from the group consisting of anti-CD3 / CD28, IL-2, and phytohemagglutinin. In any of the methods of the present disclosure, including NK cell activation, NK cells can be activated in the presence of CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, I It can be activated in the presence of one or more drugs selected from the group consisting of L-21 protein and K562 cell lines.

[0373] The cells used in the methods of this disclosure are not particularly limited, and any cells may be used. For example, cells collected, isolated, or purified from bodily fluids, tissues, or organs, such as blood (peripheral blood, umbilical cord blood, etc.) or bone marrow, or cells obtained by differentiating the aforementioned cells or reprogramming them to produce pluripotent stem cells (iPSCs) may be used (see, for example, Themeli et al 2013). Peripheral blood mononuclear cells (PBMCs), immune cells [including, for example, T cells, dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes, NK cells, or hematopoietic cells (neutrophils, basophils)], umbilical cord blood mononuclear cells, fibroblasts, adipocyte precursors, hepatocytes, cutaneous keratinocytes, mesenchymal stem cells, adipocytes, various cancer cell lines, or neural stem cells may be used. For example, NK cells or T cells, T cell precursors (hematopoietic stem cells, lymphocyte precursors, etc.) or cell populations containing them may be used. Examples of T cells include CD8-positive T cells, CD4-positive T cells, regulatory T cells, cytotoxic T cells, and tumor-infiltrating lymphocytes. Cell populations containing T cells and T cell precursors include PBMCs. The above-mentioned cells may be collected from living organisms, obtained by expanding culture of cells collected from living organisms, or established as cell lines. If transplantation of produced chimeric receptor-expressing cells or cells differentiated from produced chimeric receptor-expressing cells into a living organism is desired, nucleic acids can be introduced into the organism itself or cells collected...

Claims

1. A pharmaceutical composition comprising cells expressing a chimeric receptor, for use in combination with the administration of an antigen-binding molecule, A chimeric receptor includes an extracellular domain, which comprises the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. A pharmaceutical composition in which the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor.

2. A pharmaceutical composition containing an antigen-binding molecule for use in combination with the administration of cells expressing a chimeric receptor, The chimeric receptor includes an extracellular domain, which includes the extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or a fragment thereof. A pharmaceutical composition in which the antigen-binding molecule is a multispecific antigen-binding molecule having a target antigen recognition site and an immune receptor recognition site that recognizes the immune receptor.

3. The pharmaceutical composition according to claim 1 or 2, wherein the immune receptor recognition site recognizes the extracellular domain of a chimeric receptor and an endogenous immune receptor.

4. The pharmaceutical composition according to any one of claims 1 to 3, comprising an extracellular domain variant of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to the ligand of the endogenous immune receptor.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the immune receptor is a co-stimulatory molecule.

6. A pharmaceutical composition according to any one of claims 1 to 5, wherein the immune receptor is human CD137.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the extracellular domain of the chimeric receptor comprises a human CD137 extracellular domain variant or a fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the intracellular signaling domain of the chimeric receptor comprises the intracellular signaling domain of CD3 zeta.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the target antigen is a receptor, a tumor antigen, an MHC antigen, or a differentiation antigen.

10. The pharmaceutical composition according to any one of claims 1 to 8, wherein the target antigen is a tumor antigen.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the antigen-binding molecule is a bispecific antibody.

12. A pharmaceutical composition according to any one of claims 1 to 11, for use in the treatment or prevention of cancer.

13. A chimeric receptor comprising an extracellular domain of an immune receptor, a modified extracellular domain of an immune receptor, or an extracellular domain containing a fragment thereof.

14. The chimeric receptor according to claim 13, wherein the extracellular domain of the chimeric receptor comprises a modified extracellular domain of an immune receptor or a fragment thereof, wherein the extracellular domain of the chimeric receptor has reduced binding to the ligand of an endogenous immune receptor.

15. The chimeric receptor according to claim 13 or 14, wherein the immune receptor is a co-stimulatory molecule.

16. A chimeric receptor according to any one of claims 13 to 15, wherein the immune receptor is human CD137.

17. The chimeric receptor according to any one of claims 13 to 16, wherein the extracellular domain of the chimeric receptor is a modified human CD137 extracellular domain or a fragment thereof, wherein at least part or all of the cysteine-rich domain 3 and cysteine-rich domain 4 of the human CD137 extracellular domain are deleted.

18. The chimeric receptor according to any one of claims 13 to 17, wherein the intracellular signaling domain of the chimeric receptor includes the intracellular signaling domain of CD3 zeta.

19. A cell expressing the chimeric receptor according to any one of claims 13 to 18.

20. A composition comprising the cells of claim 19.

21. A nucleic acid encoding a chimeric receptor according to any one of claims 13 to 18.

22. A vector into which the nucleic acid described in claim 21 is inserted.

23. A method for producing the cells according to claim 19, comprising transfecting or transducing cells using the nucleic acid according to claim 21 or the vector according to claim 22.

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