Binding substances that bind to PD-L1 and CD137 and their use

Abstract

To provide bispecific antibodies which block PD1-(PD-L1) inhibitory signaling and, at the same time, co-stimulate T cells via trans binding to the CD137 molecules expressed on activated T cells, with activation to occur through the trans binding.SOLUTION: The invention discloses bispecific antibodies binding to human PD-L1 and also binding to human CD137. The invention further discloses uses of the antibodies of the invention and methods, nucleic acid constructs and host cells for producing antibodies of the invention.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of Invention This invention relates to novel conjugates and their use in medicine. In particular, this invention relates to conjugates that bind to human PD-L1 and human CD137, such as bispecific antibodies. This invention further relates to the use of the conjugates of the present invention, as well as methods for producing the antibodies of the present invention, nucleic acid constructs, and host cells. [Background technology]

[0002] Background of the Invention CD137 (4-1BB, TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor (TNFR) family. CD137 is a member of the CD8 family. + T cells and CD4 + CD137 is a co-stimulatory molecule found on the surface of T cells, regulatory T cells (Tregs), natural killer (NK) cells and NKT cells, B cells, and neutrophils. While CD137 is not constitutively expressed on T cells, it is induced upon activation of the T cell receptor (TCR). Stimulation via its native ligand, 4-1BBL, or agonist antibodies, signaling occurs using TNFR-related factor (TRAF)-2 and TRAF-1 as adapters. Initial signaling by CD137 involves a K-63 polyubiquitin binding reaction that ultimately activates the nuclear factor (NF)-κB and mitogenic factor-activated protein (MAP)-kinase pathways. Signaling increases T cell co-stimulation, proliferation, cytokine production, and maturation, and CD8 +T cell survival was prolonged. Agonist antibodies against CD137 have been shown to promote T cell-mediated antitumor management in various preclinical models (Murillo et al. 2008 Clin. Cancer Res. 14(21): 6895-6906 (Non-patent Literature 1)). Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing the antitumor immune response. Antibodies that stimulate CD137 have been disclosed in the prior art and include the human IgG4 antibody urelumab (WO2005035584 (Patent Literature 1)) and the human IgG2 antibody utomilumab (Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733 (Non-patent Literature 2)).

[0003] Programmed cell death ligand 1 (PD-L1, PDL1, CD274, B7H1) is a 33kDa 1-pass transmembrane type I membrane protein. Three PD-L1 isoforms have been described based on alternative splicing. PD-L1 belongs to the immunoglobulin (Ig) superfamily and contains one Ig-like C2 domain and one Ig-like V domain. Freshly isolated T and B cells express only very small amounts of PD-L1, and CD14 + A portion of monocytes (approximately 16%) constitutively express PD-L1. However, interferon-γ (IFNγ) is known to upregulate PD-L1 on tumor cells.

[0004] PD-L1 tolerates tumor-reactive T cells by (1) binding to programmed cell death protein 1 (PD-1) (CD279), a PD-L1 receptor on activated T cells; and (2) mediated by PD-1 signaling via tumor cell-expressed PD-L1, leading to CD8 +PD-L1 interferes with antitumor immunity by (3) making tumor cells resistant to T cell and Fas ligand-mediated lysis; by (4) tolerating T cells through reverse signaling via T cell-expressed CD80 (B7.1); and by (5) promoting the development and maintenance of induced T regulatory cells. PD-L1 is expressed in many human cancers, including melanoma, ovarian cancer, lung cancer, and colon cancer (Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6 (Non-Patent Literature 3)).

[0005] PD-L1 blocking antibodies have shown clinical activity in several cancers known to overexpress PD-L1 (including melanoma and NSCLC). For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. Currently, atezolizumab is being clinically tested as an immunotherapy for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., 2017 Lancet 389:255-265 (Non-Patent Literature 4)), and is approved for non-small cell lung cancer and bladder cancer. Avelumab, a PD-L1 antibody (Kaufman et al Lancet Oncol. 2016;17(10):1374-1385 (Non-Patent Literature 5)), is FDA approved for the treatment of adult patients and children aged 12 years or older with metastatic Merkel cell carcinoma and is being clinically developed for several cancer indications, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, NSCLC, ovarian cancer, and kidney cancer. Durvalumab, another PD-L1 antibody, is approved for locally advanced or metastatic urothelial carcinoma and is being clinically developed for several solid tumors and hematological malignancies (see, for example, Massard et al., 2016 J Clin Oncol. 34(26):3119-25 (Non-Patent Literature 6)). Further anti-PD-L1 antibodies are described in WO2004004771 (Patent Document 2), WO2007005874 (Patent Document 3), WO2010036959 (Patent Document 4), WO2010077634 (Patent Document 5), WO2013079174 (Patent Document 6), WO2013164694 (Patent Document 7), WO2013173223 (Patent Document 8), and WO2014022758 (Patent Document 9).

[0006] Horton et al (J Immunother Cancer. 2015; 3(Suppl 2): ​​O10(Non-Patent Literature 7)) disclose a combination of an agonist 4-1BB antibody and a PD-L1 neutralizing antibody.

[0007] Currently, combination therapy with utomirumab and avelumab is being tested in clinical settings (Chen et al., J Clin Oncol 35, 2017 suppl; abstr TPS7575 (Non-Patent Literature 8), and clinical trial NCT02554812).

[0008] However, despite advances in this field, there is a need for multispecific antibodies that can bind to both PD-L1 and CD137. These can simultaneously bind to antigen-presenting cells (APCs) or tumor cells expressing PD-L1 and T cells expressing CD137, resulting in conditional activation of (cytotoxic) T cells. When PD-L1 binds to PD1 expressed on activated T cells, T cells are inhibited. Therefore, the object of the present invention is to provide a PD-L1 × CD137 bispecific binding agent, for example, a bispecific antibody that blocks PD1-(PD-L1) inhibitory signaling and simultaneously co-stimulates T cells by trans-binding to the CD137 molecule expressed on activated T cells, with activation occurring via trans-binding. This could lead to efficient induction of anti-tumor immunity. A further object of the present invention is to provide a PD-L1×CD137 bispecific conjugate having an inactive Fc region or lacking an Fc-binding region, thereby providing a bispecific conjugate that, when bound to CD137, does not induce complement-dependent cytotoxicity (CDC) or other Fc-mediated effector functions on T cells. A further object of the present invention is to provide a PD-L1×CD137 bispecific conjugate suitable for T cell activation. A further object of the present invention is to provide a PD-L1×CD137 bispecific conjugate suitable for the activation of tumor-specific T cells, such as tumor-infiltrating T cells. A further object of the present invention is to provide a bispecific conjugate having a toxicological profile that is improved compared to current treatment options in the art. A further object of the present invention is to provide a conjugate having an improved potency profile compared to current treatment options in the art. [Prior art documents] [Patent Documents]

[0009]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Patent document 8

Patent document 9

Non-licensed literature

[0010]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0011] In the first aspect, the present invention provides a binding substance comprising a first antigen-binding domain that binds to human CD137 and a second antigen-binding domain that binds to human PD-L1, wherein the second antigen-binding domain inhibits the binding of human PD-L1 to human PD-1. Such a binding substance comprising a first antigen-binding domain and a second antigen-binding domain has a dual effect.

[0012] Firstly, the binding agent binds to PD-L1-expressing tumor cells or antigen-presenting cells (APCs) via its PD-L1 binding domain, while binding to T cells via its CD137 binding domain, thereby activating the T cells and, consequently, conditionally activating them. Therefore, although not limited to theory, the binding agent according to the present invention may mediate CD137 clustering when it binds simultaneously to both PD-L1 and CD137. CD137 clustering by the binding agent is necessary for this receptor to be sufficiently activated and for T cells to be co-stimulated via CD137. Secondly, the binding agent thus brings T cells closer to tumor cells, thereby facilitating tumor cell death by T cells. Furthermore, although not limited to a specific theory, PD-L1-expressing tumor cells and effector T cells, such as CD8 + The proximity of T cells to each other may initiate interferon-□ release, which in turn may upregulate PD-L1 on tumor cells, thus facilitating the recruitment of more binding agents to the tumor and potentially further enhancing tumor death.

[0013] Finally, the binding substance of the present invention inhibits the binding of human PD-L1 to human PD-1, and therefore prevents PD-L1 from interfering with PD-1-mediated antitumor immunity. Thus, the binding substance prevents T cells from receiving inhibitory signals via PD-1 / PD-L1 interaction, while T cells receive activation signals by binding to the CD137 molecule, which leads to signaling that enhances T cell proliferation, activation, effector function, and memory function.

[0014] The PD-L1 × CD137 conjugate of the present invention is particularly useful in therapeutic settings where T cells can be simultaneously stimulated with an activating receptor, such as CD137, and inhibited with an inhibitory signal, such as PD-1 / PD-L1 blocking. This can result in a larger-scale T cell proliferation, activation, and survival than if CD137 stimulation and PD-L1-mediated PD-1 / PD-L1 blocking were performed separately.

[0015] In one embodiment of the present invention, the PD-L1 × CD137 conjugate is a bispecific antibody.

[0016] In another aspect of the present invention, the binding substance is a bispecific antibody having a first antigen-binding domain that binds to human CD137 and a second antigen-binding domain that binds to human PD-L1, wherein the second antigen-binding domain inhibits the binding of human PD-L1 to human PD-1.

[0017] In a further embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, wherein (a) the first antigen-binding region comprises a heavy chain variable region (VH) including the HCDR1 sequence shown in SEQ ID NO:9, the HCDR2 sequence shown in SEQ ID NO:10, and the HCDR3 sequence shown in SEQ ID NO:11, and a light chain variable region (VL) including the LCDR1 sequence shown in SEQ ID NO:13, the LCDR2 sequence shown as GAS, and the LCDR3 sequence shown in 14, and (b) the second antigen-binding region comprises a heavy chain variable region (VH) including the HCDR1 sequence shown in SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:19, and the HCDR3 sequence shown in SEQ ID NO:20, and a light chain variable region (VL) including the LCDR1 sequence shown in SEQ ID NO:22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in 23.

[0018] Alternatively, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, wherein (a) the first antigen-binding region comprises a heavy chain variable region (VH) including the HCDR1 sequence shown in SEQ ID NO: 50, the HCDR2 sequence shown in SEQ ID NO: 51, and the HCDR3 sequence shown in SEQ ID NO: 52, and a light chain variable region (VL) including the LCDR1 sequence shown in SEQ ID NO: 54, the LCDR2 sequence shown as SAS, and the LCDR3 sequence shown in 55, and (b) the second antigen-binding region comprises a heavy chain variable region (VH) including the HCDR1 sequence shown in SEQ ID NO: 18, the HCDR2 sequence shown in SEQ ID NO: 19, and the HCDR3 sequence shown in SEQ ID NO: 20, and a light chain variable region (VL) including the LCDR1 sequence shown in SEQ ID NO: 22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in 23.

[0019] In another embodiment of the present invention, the binding substance comprises a first antigen-binding region derived from a humanized antibody that binds to human CD137, and / or a second antigen-binding region derived from a human antibody that binds to human PD-L1.

[0020] In a further aspect, the present invention relates to the use of the conjugates of the present invention in medicine, and in particular to the use of the conjugates of the present invention for treating cancer.

[0021] Such conjugates, for example, nucleic acids encoding the amino acid sequence of an antibody; expression vectors containing such nucleic acids; cells containing such nucleic acids or expression vectors; compositions containing such conjugates, nucleic acids, expression vectors, or cells; such conjugates, nucleic acids, expression vectors, cells, or compositions for use in the treatment of cancer or other diseases; such conjugates, for example, methods for producing bispecific antibodies; and such conjugates, for example, multispecific antibodies, in particular diagnostic methods and kits based on bispecific antibodies, these and other aspects and embodiments of the present invention are described in further detail below. [Invention 1001] A binding substance comprising a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, wherein the second antigen-binding region inhibits the binding of human PD-L1 to human PD-1. [Invention 1002] The conjugate of the present invention 1001, wherein the second antigen-binding region is bound to cynomolgus monkey (Macaca fascicularis) PD-L1 or its mature polypeptide, as shown in SEQ ID NO:29. [Invention 1003] The second antigen-binding region that binds to human PD-L1, a. A heavy chain variable region including heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:20 or a sequence in which up to one amino acid has been modified in SEQ ID NO:20, and b. Light chain variable region including light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:23 or a sequence in which up to two amino acids are modified in SEQ ID NO:23. A conjugate according to the present invention 1001 or 1002, comprising the heavy chain variable region and light chain variable region of an antibody that competes with an antibody for human PD-L1 binding. [Invention 1004] The second antigen-binding region that binds to human PD-L1, a. A heavy chain variable region containing HCDR3 having the sequence shown in SEQ ID NO:20 or a sequence in which up to one amino acid has been modified in SEQ ID NO:20, and b. Light chain variable region containing LCDR3 having the sequence shown in SEQ ID NO:23 or a sequence in which up to two amino acids are modified in SEQ ID NO:23. A conjugating substance according to any one of the present invention 1001 to 1003, comprising the heavy chain variable region and the light chain variable region of an antibody having specificity for PD-L1 of the antibody containing the above. [Invention 1005] A conjugating substance according to any of the present invention 1001 to 1004, wherein the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing HCDR3 having the sequence shown in SEQ ID NO:20 or a sequence in which up to one amino acid is modified in SEQ ID NO:20. [Invention 1006] A conjugate according to any of the invention 1001 to 1005, wherein the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing HCDR2 having the sequence shown in SEQ ID NO:19 or a sequence in which up to one amino acid is modified in SEQ ID NO:19. [Invention 1007] A conjugating substance according to any of the present invention 1001 to 1006, wherein the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing HCDR1 having the sequence shown in SEQ ID NO:18 or a sequence in which up to one amino acid is modified in SEQ ID NO:18. [Invention 1008] A binding substance according to any of the present invention 1001 to 1007, wherein the second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) including an HCDR1 sequence, an HCDR2 sequence, and an HCDR3 sequence, wherein the HCDR1 sequence includes the sequence shown in SEQ ID NO:18, the HCDR2 sequence includes the sequence shown in SEQ ID NO:19, and the HCDR3 sequence includes the sequence shown in SEQ ID NO:20. [Invention 1009] A binding substance according to any of the invention 1001 to 1008, wherein the second antigen-binding region that binds to human PD-L1 comprises a light chain variable region (VL) including an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO:22, the LCDR2 sequence includes the sequence shown as DDN, and the LCDR3 sequence includes the sequence shown in 23. [Invention 1010] A binding substance according to any of the invention 1001 to 1009, wherein the second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) including HCDR1, HCDR2, and HCDR3 sequences, and a light chain variable region (VL) including LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1 sequence is the sequence shown in SEQ ID NO:18, the HCDR2 sequence is the sequence shown in SEQ ID NO:19, the HCDR3 sequence is the sequence shown in SEQ ID NO:20, the LCDR1 sequence is the sequence shown in SEQ ID NO:22, the LCDR2 sequence is the sequence shown as DDN, and the LCDR3 sequence is the sequence shown in 23. [Invention 1011] A conjugating substance according to any one of the present invention 1001 to 1010, wherein the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:17. [Invention 1012] A binding substance according to any of the present invention 1001 to 1011, wherein the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH), and the VH includes the sequence shown in SEQ ID NO:17. [Invention 1013] A conjugating substance according to any one of the invention 1001 to 1012, wherein the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:21. [Invention 1014] A binding substance according to any of the present invention 1001 to 1013, wherein the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL), and the VL includes the sequence shown in SEQ ID NO:21. [Invention 1015] Any of the binding materials of the present invention, wherein the second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) and a variable region (VL), the VH comprising the sequence shown in SEQ ID NO:17, and the VL comprising the sequence shown in SEQ ID NO:21. [Invention 1016] The first antigen-binding region of the present invention binds to cynomolgus monkey (Macaca fascicularis) CD137 or its mature polypeptide as shown in SEQ ID NO:31, any of the binding substances of the present invention. [Invention 1017] Any of the binding substances of the present invention, wherein the first antigen-binding region binds to the human CD137 shown in SEQ ID NO:30 or its mature polypeptide to a greater extent than it binds to the mutant human CD137 shown in SEQ ID NO:33 or its mature polypeptide. [Invention 1018] Any of the binding materials of the present invention, wherein the first antigen-binding region binds to the mutant human CD137 or its mature polypeptide shown in SEQ ID NO:34 to the same extent as it binds to the human CD137 or its mature polypeptide shown in SEQ ID NO:30. [Invention 1019] The first antigen-binding region that binds to human CD137, a. A heavy chain variable region including heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:11 or a sequence in which up to three amino acids are modified in SEQ ID NO:11, and A light chain variable region including light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:14 or a sequence in which up to four amino acids are modified in SEQ ID NO:14; or b. Heavy chain variable region including heavy chain complementarity determination region 3 (HCDR3), which has the sequence shown in SEQ ID NO:52 or a sequence in which up to three amino acids are modified in SEQ ID NO:52, and Light chain variable region including light chain complementarity determination region 3 (LCDR3), which has the sequence shown in SEQ ID NO:55 or a sequence in which up to four amino acids are modified in SEQ ID NO:55. A conjugate substance according to the present invention, comprising the heavy chain variable region and light chain variable region of an antibody that competes with an antibody for human CD137 binding. [Invention 1020] Any of the binding materials of the present invention, wherein the first antigen-binding region that binds to human CD137 binds to at least one amino acid in the amino acid sequence shown in SEQ ID NO:40. [Invention 1021] The first antigen-binding region that binds to human CD137, a. The antibody containing the VH sequence shown in SEQ ID NO:15 and the VL sequence shown in SEQ ID NO:16 binds to the same human CD137 epitope, or b. The antibody containing the VH sequence shown in SEQ ID NO:49 and the VL sequence shown in SEQ ID NO:53 binds to the same human CD137 epitope. Any of the aforementioned binding substances of the present invention. [Invention 1022] The first antigen-binding region that binds to human CD137, a. A heavy chain variable region and a light chain variable region of an antibody having specificity for CD137, comprising a heavy chain variable region containing HCDR3 having the sequence shown in SEQ ID NO:11 or a sequence in which up to three amino acids are modified in SEQ ID NO:11, and a light chain variable region containing LCDR3 having the sequence shown in SEQ ID NO:14 or a sequence in which up to four amino acids are modified in SEQ ID NO:14; or b. Heavy chain variable region and light chain variable region of an antibody having specificity for CD137, comprising a heavy chain variable region containing HCDR3 having the sequence shown in SEQ ID NO:52 or a sequence in which up to 3 amino acids are modified in SEQ ID NO:52, and a light chain variable region containing LCDR3 having the sequence shown in SEQ ID NO:55 or a sequence in which up to 4 amino acids are modified in SEQ ID NO:55. A binding substance containing any of the above-mentioned present inventions. [Invention 1023] The first antigen-binding region that binds to human CD137, a. A heavy chain variable region (VH) containing HCDR3 having the sequence shown in SEQ ID NO:11 or a sequence in which up to three amino acids have been modified in SEQ ID NO:11; or b. Heavy chain variable region (VH) containing HCDR3 having the sequence shown in SEQ ID NO:52 or a sequence in which up to three amino acids have been modified in SEQ ID NO:52. A binding substance containing any of the above-mentioned present inventions. [Invention 1024] The first antigen-binding region that binds to human CD137, a. HCDR2 having the sequence shown in SEQ ID NO:10 or a sequence in which up to two amino acids are modified in SEQ ID NO:10; b. HCDR2 having the sequence shown in SEQ ID NO:52 or a sequence in which up to three amino acids have been modified in SEQ ID NO:52. A binding substance of the present invention that includes a heavy chain variable region (VH) containing the above. [Invention 1025] The first antigen-binding region that binds to human CD137, a. Heavy chain variable region (VH) containing HCDR1 having the sequence shown in SEQ ID NO:9 or a sequence in which up to three amino acids have been modified in SEQ ID NO:9; or b. Heavy chain variable region (VH) containing HCDR1 having the sequence shown in SEQ ID NO:50 or a sequence in which up to three amino acids have been modified in SEQ ID NO:50. A binding substance containing any of the above-mentioned present inventions. [Invention 1026] The first antigen-binding region that binds to human CD137, a. The HCDR1 sequence includes the sequence shown in SEQ ID NO:9, the HCDR2 sequence includes the sequence shown in SEQ ID NO:10, and the HCDR3 sequence includes the sequence shown in SEQ ID NO:11. Heavy chain variable region (VH) including the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence; or b. The HCDR1 sequence includes the sequence shown in SEQ ID NO:50, the HCDR2 sequence includes the sequence shown in SEQ ID NO:51, and the HCDR3 sequence includes the sequence shown in SEQ ID NO:52. The heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence. A binding substance containing any of the above-mentioned present inventions. [Invention 1027] The first antigen-binding region that binds to human CD137, a. The LCDR1 sequence includes the sequence shown in SEQ ID NO:13, the LCDR2 sequence includes the sequence shown as GAS, and the LCDR3 sequence includes the sequence shown in 14. Light chain variable region (VL) including the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence; or b. The LCDR1 sequence includes the sequence shown in SEQ ID NO: 54, the LCDR2 sequence includes the sequence shown as SAS, and the LCDR3 sequence includes the sequence shown in 55. The light chain variable region (VL) includes the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence. A binding substance containing any of the above-mentioned present inventions. [Invention 1028] The first antigen-binding region that binds to human CD137, a. The HCDR1 sequence is the sequence shown in SEQ ID NO:9, the HCDR2 sequence is the sequence shown in SEQ ID NO:10, the HCDR3 sequence is the sequence shown in SEQ ID NO:11, the LCDR1 sequence is the sequence shown in SEQ ID NO:13, the LCDR2 sequence is the sequence shown as GAS, and the LCDR3 sequence is the sequence shown in 14. A heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a light chain variable region (VL) comprising the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence; or b. The HCDR1 sequence is the sequence shown in SEQ ID NO:50, the HCDR2 sequence is the sequence shown in SEQ ID NO:51, the HCDR3 sequence is the sequence shown in SEQ ID NO:52, the LCDR1 sequence is the sequence shown in SEQ ID NO:54, the LCDR2 sequence is the sequence shown as SAS, and the LCDR3 sequence is the sequence shown in 55. A heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a light chain variable region (VL) comprising the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence. A binding substance containing any of the above-mentioned present inventions. [Invention 1029] The first antigen-binding region that binds to human CD137, a. A heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:15; or b. Heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:49. A binding substance containing any of the above-mentioned present inventions. [Invention 1030] The first antigen-binding region that binds to human CD137, a. Heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15; or b. Heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49 A binding substance containing any of the above-mentioned present inventions. [Invention 1031] The first antigen-binding region that binds to human CD137, a. A light chain variable region (VL) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:16; or b. Light chain variable region (VL) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:53. A binding substance containing any of the above-mentioned present inventions. [Invention 1032] The first antigen-binding region that binds to human CD137, a. Light chain variable region (VL) containing the sequence shown in SEQ ID NO:16; or b. Light chain variable region (VL) containing the sequence shown in SEQ ID NO:53 A binding substance containing any of the above-mentioned present inventions. [Invention 1033] The first antigen-binding region that binds to human CD137, a. The heavy chain variable region (VH) contains the sequence shown in SEQ ID NO:15 and the variable region (VL) contains the sequence shown in SEQ ID NO:16, VH and VL; or b. The heavy chain variable region (VH) contains the sequence shown in SEQ ID NO:49 and the light chain variable region (VL) contains the sequence shown in SEQ ID NO:53, and the VH and VL A binding substance containing any of the above-mentioned present inventions. [Invention 1034] It comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, a. The first antigen-binding region includes a heavy chain variable region (VH) containing the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10, and the CDR3 sequence shown in SEQ ID NO:11, or a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:50, the HCDR2 sequence shown in SEQ ID NO:51, and the HCDR3 sequence shown in SEQ ID NO:52; and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the CDR1 sequence shown in SEQ ID NO:18, the CDR2 sequence shown in SEQ ID NO:19, and the CDR3 sequence shown in SEQ ID NO:20. Any of the aforementioned binding substances of the present invention. [Invention 1035] It comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, a. The first antigen-binding region is - Heavy chain variable region (VH) including the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10, and the CDR3 sequence shown in SEQ ID NO:11, and light chain variable region (VL) including the CDR1 sequence shown in SEQ ID NO:13, the CDR2 sequence shown as GAS, and the CDR3 sequence shown in 14, or - Heavy chain variable region (VH) including the HCDR1 sequence shown in SEQ ID NO:50, the HCDR2 sequence shown in SEQ ID NO:51, and the HCDR3 sequence shown in SEQ ID NO:52, and light chain variable region (VL) including the LCDR1 sequence shown in SEQ ID NO:54, the LCDR2 sequence shown as SAS, and the LCDR3 sequence shown in 55. Includes; and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the CDR1 sequence shown in SEQ ID NO:18, the CDR2 sequence shown in SEQ ID NO:19, and the CDR3 sequence shown in SEQ ID NO:20, and a light chain variable region (VL) containing the CDR1 sequence shown in SEQ ID NO:22, the CDR2 sequence shown as DDN, and the CDR3 sequence shown in 23. Any of the aforementioned binding substances of the present invention. [Invention 1036] It comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, a. The first antigen-binding region is - Heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15, or - Heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49 Includes; and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17. Any of the aforementioned binding substances of the present invention. [Invention 1037] It comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, a. The first antigen-binding region is - Heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15, and light chain variable region (VL) containing the sequence shown in SEQ ID NO:16, or - Heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49, and light chain variable region (VL) containing the sequence shown in SEQ ID NO:53. Includes; and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21. Any of the aforementioned binding substances of the present invention. [Invention 1038] A conjugating substance according to any of the present invention, which is a multispecific antibody. [Invention 1039] A binding substance according to the present invention, which takes the form of a full-length antibody or an antibody fragment. [Invention 1040] Any of the binding materials of the present invention, wherein the first antigen-binding region comprises a first heavy chain variable region (VH) and a first light chain variable region (VL), and the second antigen-binding region comprises a second heavy chain variable region (VH) and a second light chain variable region (VL). [Invention 1041] Any of the binding materials of the present invention, wherein each variable region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4). [Invention 1042] The binding substance of the present invention 1041, wherein the complementarity determining region and the framework region are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. [Invention 1043] (i) a polypeptide comprising the first heavy chain variable region (VH) and further comprising a first heavy chain steady region (CH), and (ii) a polypeptide comprising the second heavy chain variable region (VH) and further comprising a second heavy chain steady region (CH), any of the binding materials of the present invention. [Invention 1044] (i) a polypeptide comprising the first light chain variable region (VL) and further comprising the first light chain constant region (CL), and (ii) a polypeptide comprising the second light chain variable region (VL) and further comprising the second light chain constant region (CL), any of the binding materials of the present invention. [Invention 1045] The antibody comprises a first binding arm and a second binding arm. a. The first binding arm comprises (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain steady region (CH), and (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain steady region (CL); and b. The second binding arm comprises (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain steady region (CH), and (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain steady region (CL), A binding substance according to invention 1043 or 1044. [Invention 1046] A binding material according to any of the present invention 1043 to 1045, wherein each of the first heavy chain steady region and the second heavy chain steady region (CH) comprises one or more of the steady region domain 1 region (CH1 region), hinge region, CH2 region, and CH3 region, preferably at least the hinge region, CH2 region, and CH3 region. [Invention 1047] A binding substance according to any of the present invention, which is an isotype binding substance selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [Invention 1048] A binding substance according to any of the present inventions, which is a full-length IgG1 antibody. [Invention 1049] a. The first antigen-binding region that binds to CD137 is derived from a chimeric antibody, and / or b. The second antigen-binding region that binds to human PD-L1 is derived from a chimeric antibody. Any of the aforementioned binding substances of the present invention. [Invention 1050] a. The first antigen-binding region that binds to CD137 is derived from a humanized antibody, and / or b. The second antigen-binding region that binds to human PD-L1 is derived from a humanized antibody. Any of the aforementioned binding substances of the present invention. [Invention 1051] a. The first antigen-binding region that binds to human CD137 is derived from a human antibody, and / or b. The second antigen-binding region that binds to human PD-L1 is derived from a human antibody. Any of the aforementioned binding substances of the present invention. [Invention 1052] a. The first antigen-binding region that binds to human CD137 is derived from a humanized antibody, and / or b. The second antigen-binding region that binds to human PD-L1 is derived from a human antibody. Any of the aforementioned binding substances of the present invention. [Invention 1053] Any of the binding materials of the present invention, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) contains a CH3 region, and the two CH3 regions contain asymmetric mutations. [Invention 1054] Any of the binding materials of the present invention, wherein in the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and the first and second heavy chains are not substituted at the same position. [Invention 1055] (i) The amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) The amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain, wherein the conjugate of the present invention 1054. [Invention 1056] Any of the conjugates of the present invention, wherein the antibody induces effector function via Fc to a lower degree compared to another antibody comprising the same first antigen-binding region and second antigen-binding region and two heavy chain constant regions (CH) including the human IgG1 hinge, CH2 region, and CH3 region. [Invention 1057] The conjugate of the present invention 1056, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified to a lower extent than that of an antibody identical to the antibody except that it contains an unmodified first heavy chain constant region (CH) and a second heavy chain constant region (CH), so that the antibody induces effector function via Fc. [Invention 1058] The binding substance of the present invention 1056 or 1057, wherein the effector function via Fc is measured by binding to the Fcγ receptor, by binding to C1q, or by inducing Fc-mediated crosslinking of the Fcγ receptor. [Invention 1059] The binding substance of the present invention 1058, wherein the effector function via Fc is measured by binding with C1q. [Invention 1060] The conjugate of the present invention 1053, wherein the first and second heavy chain constant regions are modified so that the binding of C1q to the antibody is reduced, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, compared to the wild-type antibody, and the C1q binding is preferably measured by ELISA. [Invention 1061] Any of the binding materials of the present invention, wherein in at least one of the first heavy chain constant region (CH) and the second heavy chain constant region (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. [Invention 1062] The binding substance of the present invention 1061, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E in the first heavy chain and the second heavy chain, respectively. [Invention 1063] The binding substance of the present invention 1061, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A in the first heavy chain steady region (HC) and the second heavy chain steady region (HC), respectively. [Invention 1064] A binding substance of the present invention 1063, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain steady region and the second heavy chain steady region are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain steady region is L and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain steady region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is L. [Invention 1065] The binding substance of the present invention 1062, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain steady region and the second heavy chain steady region are F and E, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain steady region is L and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain steady region is R and the position corresponding to F405 in the human IgG1 according to the EU numbering of the second heavy chain is L. [Invention 1066] A conjugating substance according to any of the present invention, which is a multispecific antibody, for example, a bispecific antibody. [Invention 1067] Any of the above-described conjugating substances of the present invention that induce and / or enhance the proliferation of T cells. [Invention 1068] The aforementioned T cells are CD4 + T cells and / or CD8 + The binding substance of the present invention 1067 is a T cell. [Invention 1069] Any of the binding substances of the present invention, wherein the binding substance activates CD137 signaling only when the second antigen-binding domain is bound to PD-L1. [Invention 1070] The conjugate according to invention 1067 or 1068, wherein T cells expressing a specific T cell receptor (TCR) are co-cultured with dendritic cells (DCs) that present the corresponding antigen recognized by the TCR on the major histocompatibility complex, thereby measuring the proliferation of T cells. [Invention 1071] A nucleic acid encoding any of the binding substances or polypeptide chains of the present invention 1001 to 1070. [Invention 1072] An expression vector comprising the nucleic acid of the present invention 1071. [Invention 1073] A cell comprising the nucleic acid of Invention 1071 or the expression vector of Invention 1072. [Invention 1074] The cells of the present invention 1073, which are mammalian cells, for example, Chinese hamster ovary cells. [Invention 1075] A composition comprising any binding substance of Invention 1001 to 1070, the nucleic acid of Invention 1071, the expression vector of Invention 1072, or the cells of Invention 1073 or 1074. [Invention 1076] A pharmaceutical composition, the composition of the present invention 1075. [Invention 1077] A composition of the present invention 1076, further comprising a pharmaceutically acceptable carrier and / or excipient. [Invention 1078] A conjugating substance according to any of Invention 1001 to 1070, nucleic acid according to Invention 1071, expression vector according to Invention 1072, cell according to Invention 1073 or 1074, or a composition according to any of Invention 1075 to 1077, for use as a pharmaceutical. [Invention 1079] A conjugate, nucleic acid, expression vector, cell, or composition of the present invention 1078 for use in the treatment of cancer. [Invention 1080] A method for treating a disease, comprising the step of administering to a subject requiring such treatment any of the following: a binding substance according to any of Invention 1001 to 1070, a nucleic acid according to Invention 1071, an expression vector according to Invention 1072, cells according to Invention 1073 or 1074, or a composition according to any of Invention 1075 to 1077. [Invention 1081] The method of the present invention 1080, wherein the disease is cancer. [Invention 1082] A conjugate, nucleic acid, expression vector, cell, or composition for use in Invention 1079 or the method of Invention 1081, wherein the cancer is characterized by the presence of a solid tumor or is selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer. [Invention 1083] A conjugate, nucleic acid, expression vector, cell, or composition or method of the present invention 1081 for use in the said cancer being non-small cell lung cancer (NSCLC). [Invention 1084] Use of any of the conjugates 1001 to 1070, nucleic acids of the 1071, expression vectors of the 1072, cells of the 1073 or 1074, or any of the compositions 1075 to 1077 for the manufacture of pharmaceuticals such as pharmaceuticals for treating cancer, for example, cancer characterized by the presence of solid tumors, or cancer selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer. [Invention 1085] Use of the present invention 1084, wherein the lung cancer is non-small cell lung cancer (NSCLC). [Invention 1086] Use of the method of the present invention 1080 or 1081 or the use of the present invention 1084, including one or more additional therapeutic substances, such as in combination with chemotherapeutic agents. [Invention 1087] a. A step of culturing host cells that produce a first antibody containing an antigen-binding region that binds to human CD137 as defined in any of invention 1001 and 1016-1036, and optionally purifying the first antibody from the culture; b. A step of culturing host cells that produce a second antibody containing an antigen-binding region that binds to human PD-L1 as defined in any of invention 1001-1015 and 1049-1052, and optionally purifying the second antibody from the culture; c. The step of incubating the first antibody together with the second antibody under conditions of sufficient reducing to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization; and d. Steps to obtain CD137×PD-L1 bispecific antibodies A method for producing the bispecific antibody of the present invention 1066, including the above. [Invention 1088] An anti-idiotype antibody that binds to a first antigen-binding region and / or a second antigen-binding region as defined in any one of claims 1001 to 1070 of the present invention. [Brief explanation of the drawing]

[0022] [Figure 1] Sequence alignments of human CD137, African elephant CD137, and wild boar CD137. Amino acids in African elephant CD137 and wild boar CD137 that differ from those in the human sequence are highlighted in black. [Figure 2] CD137 shuffle constructs containing the African elephant (shuffle 5) CD137 domain or the wild boar (shuffle 1-4, 6) CD137 domain. [Figure 3] Expression of the CD137 shuffle construct on HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137 shuffle construct. Cell surface expression of the construct was measured by flow cytometry using polyclonal anti-CD137 antibodies that recognize human CD137, wild boar CD137, and African elephant CD137. [Figure 4]Binding of antibody CD137-009 to CD137 shuffle constructs expressed on HEK293-T17 cells. HEK293-T17 cells were transfected with CD137 shuffle constructs and human CD137 (hCD137wt), African elephant CD137, or wild boar CD137. Binding of antibody CD137-009 to these constructs expressed on HEK293-T17 cells was measured by flow cytometry. Staining with polyclonal anti-CD137 antibody is shown as a control. [Figure 5] Effect of the monovalent antibody b12-FEAL×PD-L1-547-FEAR on PD-1 / PD-L1 interaction. The effect of b12-FEAL×PD-L1-547-FEAR was measured using a PD-1 / PD-L1 inhibitory bioassay. The data shown are the induction ratios compared to the control (without antibody treatment) in a single representative experiment. [Figure 6] Schematic diagram of the expected mechanism of action of the CD137×PD-L1 bispecific antibody. (A) PD-L1 is expressed on the surface of antigen-presenting cells (APCs) and tumor cells. Binding of PD-L1 to T cells expressing the negative regulatory molecule PD-1 effectively inactivates T cell activation signals, ultimately inhibiting T cells. (B) When the CD137×PD-L1 bispecific antibody is added, the inhibitory PD-1:PD-L1 interaction is blocked by the PD-L1-specific arm. At the same time, this bispecific antibody induces agonist signaling to CD137 expressed on T cells via intercellular interactions, resulting in potent T cell co-stimulation. [Figure 7A]Release of PD-1 / PD-L1-mediated T cell inhibition and further co-stimulation of CD8+ T cell proliferation by CD137-009-FEAL×PD-L1-547-FEAR in antigen-specific T cell assays using the active PD-1 / PD-L1 axis. CFSE-labeled T cells electroporated with claudin-6-specific TCRs and PD-1 in vitro translation (IVT)-RNA were incubated for 5 days with immature dendritic cells electroporated with claudin-6-IVT-RNA in the presence of 0.1 μg / mL and 0.02 μg / mL of CD137-009-FEAL×PD-L1-547-FEAR, b12-FEAL×PD-L1-547-FEAR, or b12 control antibody. CD8+ T cell proliferation was measured by flow cytometry. The data shown are (A and C) representative CFSE histograms from two different donors, and (B and D) the corresponding dividing cell percentage and proliferation index calculated using FlowJo software. (B) shows the analysis of data from donor 1 representative shown in (A). (D) shows the analysis of data from donor 2 representative shown in (C). Error bars (SD) indicate intra-experimental variability (three repetitions using cells from one donor). [Figure 7B] See the explanation in Figure 7A. [Figure 7C] See the explanation in Figure 7A. [Figure 7D] See the explanation in Figure 7A. [Figure 8]Analysis of EC50 values ​​of the bispecific antibody CD137-009-FEAL×PD-L1-547-FEAR in an antigen-specific T cell assay using an active PD-1 / PD-L1 axis. CFSE-labeled T cells electroporated with claudin-6-specific TCRs and PD-1-IVT-RNA were incubated for 5 days with immature dendritic cells electroporated with claudin-6-IVT-RNA in the presence of CD137-009-FEAL×PD-L1-547-FEAR (3-fold serial dilutions from 1 to 0.00015 μg / mL). CD8+ T cell proliferation was measured by flow cytometry. The data shown are the percentage of dividing cells (outlined diamonds) and the proliferation index (solid triangles) as a function of antibody concentration. Error bars (SD) indicate intra-experimental variability (6 replicates using cells from one donor). The curve was fitted using nonlinear regression, and the EC50 value was obtained using GraphPad Prism software. [Figure 9A]Comparison of CD137-009-FEAL×PD-L1-547-FEAR and combinations of two monovalent CD137 antibodies (CD137-009-FEAL×b12-FEAR+b12-FEAL×PD-L1-547-FEAR) or two parental antibodies (CD137-009+PD-L1-547) in antigen-specific T cell assays using an active PD-1 / PD-L1 axis. CFSE-labeled T cells electroporated with claudin-6-specific TCRs and PD1-IVT-RNA were incubated for 5 days with immature dendritic cells electroporated with claudin-6-IVT-RNA in the presence of 0.25 μg / mL of (i) CD137-009-FEAL×PD-L1-547-FEAR, (ii) CD137-009-FEAL×b12+b12-FEAL×PD-L1-547-FEAR, (iii) CD137-009-FEAL×b12, (iv) b12-FEAL×PD-L1-547-FEAR, (v) CD137-009+PD-L1-547, (vi) CD137-009, (vii) PD-L1-547, or (viii) b12 control antibodies. CD8+ T cell proliferation was measured by flow cytometry. The data shown are (A) a representative CFSE histogram and (B and C) the corresponding mean values ​​of dividing cell percentage and proliferation index calculated using FlowJo software. Error bars (SD) indicate intra-experimental variability (three replicates using cells from one donor). [Figure 9B] See the explanation in Figure 9A. [Figure 9C] See the explanation in Figure 9A. [Figure 10]Ex vivo amplification of tumor-infiltrating lymphocytes (TILs) from human non-small cell lung cancer tissue resection using CD137-009-FEAL×PD-L1-547-FEAR. Tumor fragments from resected tissue were cultured with 10 U / mL IL-2 and the indicated concentrations of CD137-009-FEAL×PD-L1-547-FEAR. After 10 days of culture, cells were collected and analyzed by flow cytometry. (A) TIL count as amplification factor compared to untreated control, (B) CD3+CD8+ T cell count as amplification factor compared to untreated control, (C) CD3+CD4+ T cell count as amplification factor compared to untreated control, (D) CD3-CD56+ NK cell count as amplification factor compared to untreated control. Bars represent the mean ± SD of individual wells for n=5, with 2 tumor fragments per well used as starting material. [Figure 11] Effect of mCD137-3H3×mPD-L1-MPDL3280A mouse surrogate antibody on antigen-specific T cell proliferation in OT-I adoptive cell transfer setups. Ovalbumin (OVA)-specific OT1+Thy1.1+ double-positive cytotoxic T cells isolated from donor mice were injected (ro) into the posterior orbit of naive C57BL / 6 recipient mice. The day after adoptive cell transfer, recipient mice were ro-injected with 100 μg of OVA as antigen stimulation, followed by ro-injection of 100 μg or 20 μg per mouse of mCD137-3H3×mPD-L1-MPDL3280A, mCD137-3H3×b12, or mPD-L1-MPDL3280A×b12 antibody. PBS injection (shown as OVA alone in the figure) was used as a baseline reference, and untreated animals were used as negative controls. Six days later, 100 μL of blood was collected via the ro pathway and analyzed for Thy1.1+CD8+ T cells. The data shown are (A) a schematic diagram of the OT-I adoptive cell transfer experiment and (B) the Thy1.1+CD8+ T cell frequency for each treatment group on day 6. Squares represent individual animals, and error bars (SD) indicate intra-experimental variability (n=5 mice / group). Statistical analysis was performed using one-way Anova and Tukey multiple comparison tests. ns = no significant difference between groups. *** = P<0.001. [Figure 12]Antitumor efficacy of mCD137-3H3×mPD-L1-MPDL3280A mouse surrogate antibody in a subcutaneous syngeneic CT26 mouse tumor model. Female BALB / c mice with subcutaneous CT26 tumors were treated with 20 μg per mouse of either (i) mCD137-3H3×mPD-L1-MPDL3280A, (ii) mCD137-3H3×b12, or (iii) mPD-L1-MPDL3280A×b12 antibody, or (iv) PBS intraperitoneally. The administration schedule was every 2-3 days for the first 8 injections, and then every 7 days until the end of the experiment. On day 29, 100 μL of blood was collected via the ro pathway and analyzed for gp70-specific CD8+ T cells. The data shown are (A) tumor growth curves, where each line represents one mouse; (B) the resulting Kaplan-Meier survival analysis; and (C) the gp70-specific CD8+ T cell frequency for each treatment group at 29 days post-transplant. PFS = progression-free survival. [Figure 13] Binding of monospecific bivalent PD-L1 antibody and monovalent b12×PD-L1 antibody to tumor cells. Binding of PD-L1-547 and b12-FEAL×PD-L1-547-FEAR to MDA-MB-231(A), PC-3(B), and SK-MES-1(C) cells. The data shown are the average fluorescence intensity (MFI) determined by flow cytometry. Monospecific bivalent b12 antibody was included as a negative control. [Figure 14]The binding of the CD137 antibody to CD137 variants with alanine mutations at positions 1-163. Binding was expressed as a z-score (magnitude of change), a measure of the change in binding compared to the control antibody. The z-score (magnitude of change) was defined as (standardized gMFIaa position - μ) / σ. In the formula, μ and σ are the mean and standard deviation of the standardized gMFI across all variants. Residues with a binding z-score less than -1.5 (shown by a dotted line) were considered "bind loss variants." Residues with a positive binding z-score are bind loss residues compared to the non-cross-blocking CD137-specific control antibody. The numbers on the x-axis indicate amino acid positions. (A) Z-score of binding between b12-FEAL×CD137-009-FEAR-A488 and CD137 variants with alanine or glycine mutations at positions 1-163, using CD137-005-FEAR-A488 as a non-cross-blocking CD137-specific control antibody for standardization. (B) Z-score of binding between CD137-005-FEAR-A488 and CD137 variants with alanine or glycine mutations at positions 1-163, using CD137-MOR7480-FEAR-A488 as a non-cross-blocking CD137-specific control antibody for standardization. (C) Z-score of binding of CD137-MOR7480-FEAR-A488 to CD137 variants with alanine or glycine mutations at positions 1-163, using CD137-005-FEAR-A488 as a non-cross-blocking CD137-specific control antibody for standardization. [Figure 15A]Comparison of PD-L1-547-FEAL×CD137-009-HC7LC2-FEAR with a combination of two monovalent controls (b12-FEAL×CD137-009-HC7LC2-FEAR+b12-FEAL×PD-L1-547-FEAR) or a combination of two parental antibodies (CD137-009-HC7LC2-FEAR+PD-L1-547-FEAR) in a non-antigen-specific T cell proliferation assay. CFSE-labeled PBMCs were incubated with suboptimal concentrations of anti-CD3 antibody (0.03 μg / mL and 0.1 μg / mL), or incubated without anti-CD3 antibody (w / o) (as a negative control for T cell activation), and (i) PD-L1-547-FEAL×CD137-009-HC7LC2-FEAR and (ii) b12-FEAL×CD137-009-HC7LC2-FEAR+b12-FEAL×PD at 0.2 μg / mL. (iii)b12-FEAL×CD137-009-HC7LC2-FEAR, (iv)b12-FEAL×PD-L1-547-FEAR, (v)CD137-009-HC7LC2-FEAR+PD-L1-547-FEAR, (vi)CD137-009-HC7LC2-FEAR, (vii)PD-L1-547-FEAR, or (viii)b12-IgG-FEAL control antibody were cultured for 4 days. CD4+(A) and CD8+(B) T cell proliferation was measured by flow cytometry. Data from three donors are shown as the average of the growth index over three replicates, calculated using FlowJo v10.4 software. Error bars (SD) indicate intra-experimental variability (three replicates using cells from one donor). [Figure 15B] See the explanation in Figure 15A. [Figure 16]Determination of EC50 values ​​for T cell proliferation induction by PD-L1-547-FEAL×CD137-009-HC7LC2-FEARx in a non-antigen-specific T cell proliferation assay. CFSE-labeled PBMCs were incubated for 4 days with suboptimal concentrations of anti-CD3 antibody and serial dilutions of PD-L1-547-FEAL×CD137-009-HC7LC2-FEAR (1~0.00015 μg / mL) or the control antibody 1 μg / mL b12 IgG. Data from two representative donors are shown. PBMCs from donor 1 were stimulated with 0.03 μg / mL anti-CD3 (A, B), and PBMCs from donor 2 were stimulated with 0.09 μg / mL anti-CD3 (C, D). CD4+ (A and C) and CD8+ (B and D) T cell proliferation was measured by flow cytometry. The data shown represents the average of the growth index values ​​over three iterations, calculated using FlowJo v10.4 software and fitted using a 4-parameter logarithmic fit. Error bars (SD) indicate intra-experimental variability (three iterations using cells from a single donor). [Figure 17] The effect of PD-L1-547-FEAL×CD137-009-HC7LC2-FEAR on the secretion of 10 pro-inflammatory cytokines in antigen-specific T cell assays with or without PD-1 electroporation of T cells. T cells electroporated with CLDN6-specific TCR and 2 μg PD1-IVT-RNA, or T cells electroporated with CLDN6-specific TCR, were incubated with iDCs electroporated with CLDN6-IVT-RNA in the presence of different concentrations of CD137-009-HC7LC2-FEAL×PD-L1-547-FEAR (3-fold serial dilutions; 1 μg / mL to 0.00015 μg / mL) or the b12 control antibody b12-IgG-FEAL. 48 hours after antibody addition, cytokine levels in the supernatant were measured using a multiplex sandwich immunoassay with the MSD V-Plex Human Proinflammatory Panel 1 (10-Plex) kit. Each data point represents the mean ± SD of three individual wells. [Figure 18]Effect of PD-L1-547-FEAL×CD137-009-HC7LC2-FEAR on the secretion of 10 pro-inflammatory cytokines in an antigen-nonspecific T cell assay. Human PBMCs were sub-optimally stimulated with anti-CD3 antibody in the presence of different concentrations of PD-L1-547-FEAL×CD137-009-HC7LC2-FEAR (3-fold serial dilution; 1 μg / mL to 0.00015 μg / mL) or the b12 control antibody b12-IgG-FEAL. Forty-eight hours after antibody addition, cytokine levels in the supernatant were measured by multiplex sandwich immunoassay using the MSD V-Plex Human Proinflammatory panel 1 (10-Plex) kit. Each data point represents the mean ± SD of 3 individual wells.

Mode for Carrying Out the Invention

[0023] Detailed Description of the Invention definition The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low molecular weight light chains (L) and one pair of heavy chains (H), all four being interconnected by disulfide bonds. The structure of immunoglobulins has been clearly characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as V H or VH) and a heavy chain constant region (abbreviated herein as C H or CH). The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly mobile. The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in the IgG molecule. Each light chain typically consists of a light chain variable region (abbreviated herein as V L or VL) and a light chain constant region (abbreviated herein as CL The light chain constant region is typically composed of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions whose sequence can change significantly and / or which can take the form of structure-defined loops), also called complementarity-determining regions (CDRs), which contain more conserved regions than the hypervariable regions, called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol.). 196 See also 901-917 (1987). Unless otherwise specified or inconsistent with the context, CDR sequences herein are identified using DomainGapAlign in accordance with the IMGT rules (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); Internet http address www.imgt.org / (See also). Unless otherwise specified or inconsistent with the context, the description of amino acid positions in the constant region in this invention follows EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). For example, in this specification, SEQ ID NO:93 indicates amino acid positions 118-447 in the constant region of the IgG1m(f) heavy chain, according to EU numbering.

[0024] As used herein, the term “amino acid corresponding to position…” refers to the amino acid position number of the human IgG1 heavy chain. The corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that “corresponds” to an amino acid or segment in another sequence is an amino acid or segment that aligns with the other amino acid or segment using a standard sequence alignment program, e.g., ALIGN, ClustalW, or an analogue, typically with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Methods for aligning sequences or segments within a sequence and thereby determining the positions within a sequence that correspond to the amino acid positions according to the present invention are considered well known in the art.

[0025] In the context of the present invention, the term “binding substance” refers to any active substance capable of binding to a desired antigen. In certain embodiments of the present invention, the binding substance is an antibody, an antibody fragment, or a construct thereof. The binding substance may also include a synthetic portion, a modified portion, or a non-natural portion, in particular a non-peptide portion. Such portions may, for example, link a desired antigen-binding function or antigen-binding region, such as an antibody or antibody fragment. In one embodiment, the binding substance is a synthetic construct comprising an antigen-binding CDR or variable region.

[0026] In the context of the present invention, the term "antibody" (Ab) means an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which, under typical physiological conditions, has the ability to specifically bind to an antigen for a fairly long half-life, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or more, or any other relevant, function-defined period (e.g., enough time to induce, promote, enhance, and / or modulate a physiological response related to antibody-antigen binding, and / or enough time for the antibody to enhance effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule include a binding domain that interacts with the antigen. As used herein, the term "antigen-antibody binding region" means the region that interacts with the antigen and includes both the VH region and the VL region. When the term antibody is used herein, it includes not only monospecific antibodies but also multispecific antibodies that contain multiple, for example, two or more, or three or more, different antigen-binding regions. The constant region of an antibody (Ab) can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and complement system components, such as C1q, the first component of the classical pathway of complement activation, in host tissue or host factors. As stated above, unless otherwise specified or clearly contrary to the context, the term antibody herein includes antigen-binding fragments, i.e., antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments included in the term "antibody" include (i) a monovalent fragment consisting of Fab' or Fab fragment, VL, VH, CL, and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) an F(ab')2 fragment, a bivalent fragment in which two Fab fragments are linked by disulfide crosslinking at the hinge region; (iii) an Fd fragment essentially consisting of VH and CH1 domains; (iv) an Fv fragment essentially consisting of the VL and VH domains of a single arm of an antibody; and (v) a domain antibody essentially consisting of the VH domain (Holt et al; Trends Biotechnol. 2003 Nov;). 21 (11):484-90) also known as the dAb fragment (Ward et al., Nature) 341 , 544-546(1989)); (vi) Camelid or nanobody molecule (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24), and (vii) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the VL and VH regions pair to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)). For example, Bird et al., Science 242 , 423-426 (1988) and Huston et al., PNAS USA 85The fragments may be linked using recombination by a synthetic linker, which allows them to be made as a single protein chain forming (see 5879-5883 (1988)). Such single-chain antibodies are included in the term antibody unless otherwise specified or clearly indicated by the context. Such fragments, while generally included in the meaning of antibody, are a unique feature of the present invention, exhibiting different biological properties and utility, both collectively and independently. These antibody fragments and other useful antibody fragments, as well as the bispecificity types of such fragments, in the context of the present invention will be discussed further herein. The term antibody should also be understood, unless otherwise specified, to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, e.g., chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, provided by any known technique, e.g., enzymatic cleavage, peptide synthesis, and recombination techniques. The antibodies produced may have any isotype. As used herein, the term “isotype” refers to an immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by a heavy chain constant region gene. When a specific isotype, e.g., IgG1, is referred to herein, the term is used to indicate that the antibody sequence is more similar to that isotype, e.g., IgG1, than to other isotypes, although it is not limited to a specific isotype sequence, e.g., a specific IgG1 sequence. Accordingly, for example, the IgG1 antibody of the present invention may be a sequence variant of a natural IgG1 antibody, including a change in the constant region.

[0027] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody molecule having only one molecular composition. A monoclonal antibody composition exhibits only one binding specificity and affinity for a particular epitope. Therefore, the term "human monoclonal antibody" refers to an antibody that exhibits only one binding specificity, having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced by hybridomas obtained by fusing B cells from transgenic non-human animals or transchromosomal non-human animals, such as transgenic mice having a genome containing human heavy chain transgenes and light chain transgenes, with immortalized cells.

[0028] In the context of the present invention, the terms “bispecific antibody” or “bs” refer to an antibody having two distinct antigen-binding regions defined by different antibody sequences. In some embodiments, the distinct antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the distinct antigen-binding regions bind to different target antigens. A bispecific antibody may be any type of bispecific antibody, including any bispecific antibody type described below herein.

[0029] As used herein, unless otherwise inconsistent with the context, the terms “Fab arm” or “arm” refer to a single heavy-light chain pair and are used herein as synonymous with “half molecule.”

[0030] Where it is stated that a bispecific antibody comprises a halpomole antibody "derived from" a first antibody and a halpomole antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody was produced by recombining the halpos derived from the first and second antibodies, respectively, by any known method to produce the resulting bispecific antibody. In this context, "recombining" is not intended to be limited to a specific recombination method, and therefore includes all methods for producing bispecific antibodies as described below herein, including, for example, recombination by halpom exchange, recombination at the nucleic acid level, and / or recombination by co-expression of two types of halpos in the same cell.

[0031] In the context of this invention, the term "monovalent antibody" means that an antibody molecule can bind to only one type of antigen molecule and therefore cannot crosslink an antigen or cell.

[0032] When used in the context of antibodies, the term "full length" indicates that the antibody is not a fragment but contains all of the domains of a particular isotype that are naturally and normally found for that isotype. For example, in the case of an IgG1 antibody, this would include the VH, CH1, CH2, CH3, hinge, VL, and CL domains.

[0033] As used herein, unless otherwise inconsistent with the context, the term “Fc region” means an antibody region consisting of two Fc sequences in the heavy chain of an immunoglobulin, wherein the Fc sequences include at least a hinge region, a CH2 domain, and a CH3 domain.

[0034] As used herein, the term “heterodimer interaction between the first CH3 region and the second CH3 region” refers to the interaction between the first CH3 region and the second CH3 region in a first CH3 / second CH3 heterodimer protein.

[0035] As used herein, the term “homodimerative interaction between the first CH3 region and the second CH3 region” refers to the interaction between the first CH3 region in a first CH3 / first CH3 homodimer protein and another first CH3 region, and the interaction between the second CH3 region in a second CH3 / second CH3 homodimer protein and another second CH3 region.

[0036] With regard to the binding of an antibody to a given antigen or epitope, the terms “binding” or “can bind” as used herein typically refer to measurements using biolayer interferometry (BLI), or, for example, measurements using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument, where the antigen is used as the ligand and the antibody as the analyte, approximately 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, approximately 10 -10 M or less, or about 10 -11 M, or even less than K D The binding is at an affinity corresponding to the antibody. The antibody has a K for binding to a nonspecific antigen other than a predetermined antigen or a closely related antigen (e.g., BSA, casein). D K is at least 1 / 10 of, for example, at least 1 / 100, for example, at least 1 / 1,000, for example, at least 1 / 10,000, for example, at least 1 / 100,000. D It binds to a predetermined antigen with an affinity corresponding to the antibody's K. D It depends on the antibody K D If the affinity is very low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than the affinity for a nonspecific antigen may be at least 1 / 10,000.

[0037] The term "k" used in this specification d (sec -1 The term ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is koff Also called a value.

[0038] The term "K" used in this specification D The term (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0039] In a preferred embodiment, the antibody of the present invention is isolated. As used herein, “isolated antibody” is intended to mean an antibody that substantially does not contain other antibodies with different antigen specificities. In a preferred embodiment, an isolated bispecific antibody that specifically binds to PD-L1 and CD137 substantially does not contain a monospecific antibody that specifically binds to PD-L1 or CD137. In another preferred embodiment, the antibody, or a pharmaceutical composition containing the antibody, substantially does not contain naturally occurring antibodies that cannot bind to PD-L1. In a further preferred embodiment, the antibody of the present invention has a modified amino acid sequence structure compared to the structure of a naturally occurring anti-PD-L1 antibody, and this structural modification results in the antibody exhibiting modified functionality compared to the functionality exhibited by the naturally occurring anti-PD-L1 antibody, the functionality being selected from the group consisting of (i) PD-L1 binding affinity, (ii) ability to inhibit the binding of PD-L1 to PD-1, (iii) ability to induce effector function via Fc, and (iv) ability not to induce effector function via Fc.

[0040] As used herein, the term "PD-L1" refers to the programmed death ligand 1 protein. PD-L1 is found in humans and other species, and therefore, unless otherwise specified, the term "PD-L1" is not limited to human PD-L1. Human PD-L1 sequences, macaque (cynomolgus monkey) PD-L1 sequences, African elephant PD-L1 sequences, wild boar PD-L1 sequences, and mouse PD-L1 sequences are found by Genbank accession numbers NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively. The sequence of human PD-L1 is also shown at SEQ ID NO:28. Amino acids 1-18 are predicted to be the signal peptide. The sequence of macaque (cynomolgus monkey) PD-L1 is also shown at SEQ ID NO:29. Amino acids 1-18 are predicted to be signal peptides.

[0041] As used herein, the term "PD-L2" refers to the human programmed death 1-ligand 2 protein (Genbank accession number NP_079515).

[0042] As used herein, the term "PD-1" refers to the human programmed death-1 protein, also known as CD279.

[0043] As used herein, the term "CD137" refers to the human surface antigen classification 137 protein. CD137(4-1BB), also known as TNFRSF9, is the receptor for the ligand TNFSF9 / 4-1BBL. CD137 is thought to be involved in T cell activation. In one embodiment, CD137 is human CD137 having UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO:30. Amino acids 1-23 are predicted to be the signal peptide. In another embodiment, CD137 is cynomolgus monkey (Macaca fascicularis) CD137 having UniProt accession number A9YYE7-1. The sequence of cynomolgus monkey CD137 is shown in SEQ ID NO:31. Amino acids 1-23 are predicted to be the aa signal peptide. The wild boar (Sus scrofa) CD137 is shown as SEQ ID NO:38. Amino acids 1-23 are predicted to be the aa signal peptide. The African elephant (Loxodonta africana) CD137 is shown as SEQ ID NO:39. Amino acids 1-23 are predicted to be the aa signal peptide.

[0044] A "PD-L1 antibody" or "anti-PD-L1 antibody" is an antibody that specifically binds to the antigen PD-L1, particularly human PD-L1, as described above.

[0045] A "CD137 antibody" or "anti-CD137 antibody" is an antibody that specifically binds to the antigen CD137, as described above.

[0046] "CD137×PD-L1 antibody," "anti-CD137×PD-L1 antibody," "PD-L1×CD137 antibody," or "anti-PD-L1×CD137 antibody" are bispecific antibodies containing two different antigen-binding regions, one of which specifically binds to the antigen PD-L1, and the other specifically binds to CD137.

[0047] The present invention also provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the antibodies of the examples. Even if the functional variant of VL, VH, or CDR used with respect to an antibody, such antibody still retains at least a significant proportion (at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or greater proportions) of the affinity and / or specificity / selectivity of the “reference” or “parent” antibody, and in some cases such antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent antibody.

[0048] Such functional variants typically retain significant sequence identity with the parent antibody. The percentage of identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences (i.e., homology % = number of identical positions / total number of positions × 100). The percentage of identity between two nucleotide sequences or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0049] Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of the parent antibody sequence primarily in terms of conservative substitutions. For example, 10 of the substitutions in a variant, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1, are conservative amino acid residue exchanges.

[0050] In the context of the present invention, a conservative substitution may be defined as a substitution within the amino acid classes reflected in the following table.

[0051] Amino acid residue classes for conservative substitutions TIFF0007836786000001.tif53139

[0052] In the context of this invention, unless otherwise specified, the following notation is used to describe mutations: (i) A substitution of an amino acid at a particular position is written as K409R, for example, meaning that lysine at position 409 of the protein is substituted with arginine. (ii) For specific variants, a specific three-letter or one-letter notation is used, including the symbols Xaa and X to indicate any amino acid residue. Thus, a substitution of lysine to arginine at position 409 is indicated by K409R, and a substitution of lysine to any amino acid residue at position 409 is indicated by K409X. In the case of a deletion of lysine at position 409, this is K409 * This is shown.

[0053] In the context of the present invention, "inhibition of PD-L1-PD-1 binding" means that the binding of PD-L1 to PD-1 is detected to be significantly reduced in the presence of an antibody capable of binding to PD-L1. Typically, inhibition means a reduction of at least about 10% of the binding between PD-L1 and PD-1 caused by the presence of an anti-PD-L1 antibody, e.g., at least about 15%, e.g., at least about 20%, e.g., at least 40%. Inhibition of PD-L1-PD-1 binding can be measured by any suitable technique. In one embodiment, inhibition is measured as described in Example 6 herein.

[0054] As used herein, the term “specificity” is intended to have the following meaning, unless otherwise inconsistent with the context: Two antibodies have “same specificity” if they bind to the same antigen and the same epitope.

[0055] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes typically consist of surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge properties. Conformational epitopes and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not. Epitopes may contain amino acid residues directly involved in binding, and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or covered by peptides that specifically bind to the antigen (in other words, these amino acid residues are within the footprint of the peptides that specifically bind to the antigen).

[0056] As used herein, the term “chimeric antibody” refers to an antibody in which the variable region originates from a non-human species (e.g., a rodent) and the constant region originates from a different species, such as a human. To reduce antibody immunogenicity, chimeric monoclonal antibodies have been developed for therapeutic purposes. The term “variable region” or “variable domain” as used in relation to chimeric antibodies refers to the region containing the CDR and framework regions of both the heavy and light chains of immunoglobulin. Chimeric antibodies can be prepared using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch.15. Chimeric antibodies may be genetically engineered recombinant antibodies or enzymatically engineered recombinant antibodies. The preparation of chimeric antibodies is within the knowledge of those skilled in the art, and therefore, the preparation of chimeric antibodies according to the present invention may be carried out by methods other than those described herein.

[0057] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology to the human variable domain. This can be achieved by bridging six non-human antibody complementarity-determining regions (CDRs) that together form an antigen-binding site to a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). To completely reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace (reverse-mutate) framework residues derived from the parent antibody (i.e., the non-human antibody) with human framework residues. Structural homology modeling may be helpful in identifying amino acid residues within the framework region that are important for the binding characteristics of the antibody. Accordingly, a humanized antibody may comprise a human framework region, which optionally contains one or more amino acid reverse mutations into non-human CDR sequences, primarily non-human amino acid sequences, and a fully human constant domain. Optional, and not necessarily reverse mutations, further amino acid modifications may be applied to obtain humanized antibodies with desirable characteristics, such as affinity and biochemical properties.

[0058] As used herein, the term “human antibody” refers to an antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies may include amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or mutations introduced by in vivo somatic mutation). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse or rat, has been transplanted into a human framework sequence. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic hybridization technique described in Kohler and Milstein, Nature 256: 495 (1975). While somatic hybridization is preferred in principle, other techniques for producing monoclonal antibodies, such as viral transformation or carcinogenesis of B lymphocytes or phage display techniques using human antibody gene libraries, can be used. The mouse system is a suitable animal system for preparing hybridomas that secrete human monoclonal antibodies. Hybridoma production in mice is a well-established technique. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion techniques are also known. Thus, human monoclonal antibodies can be produced, for example, using a mouse system or a transgenic or transchromosome mouse or rat that has a part of the human immune system rather than a rat system. In one embodiment, a human antibody is obtained from a transgenic animal, e.g., a mouse or rat, that has a human germline immunoglobulin sequence instead of an animal immunoglobulin sequence. In such an embodiment, the antibody originates from a human germline immunoglobulin sequence introduced into the animal, but the final antibody sequence is the result of further modification of the human germline immunoglobulin sequence by somatic hypermutation and affinity maturation through an endogenous animal antibody mechanism.See, for example, Mendez et al. 1997 Nat Genet. 15(2):146-56. The term “reducing conditions” or “reducing environment” refers to conditions or environments in which the cysteine ​​residues in the hinge region of a substrate, in this case an antibody, are more likely to be reduced than oxidized.

[0059] As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to mean a cell into which an expression vector, for example, an expression vector encoding the antibody of the present invention, has been introduced. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NS0 cells, and lymphocytes.

[0060] The term "treatment" means administering an effective amount of the therapeutically active antibody of the present invention for the purpose of alleviating, improving, suppressing, or eradicating (curing) symptoms or disease conditions.

[0061] The term "effective dose" or "therapeutic effective dose" refers to the amount of an antibody that is effective in achieving the desired therapeutic outcome, given the dosage and time required to obtain that outcome. The therapeutic effective dose of an antibody may vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the antibody's ability to induce the desired response in that individual. The therapeutic effective dose is also the amount in which the therapeutically beneficial effects of the antibody or antibody portion outweigh the toxic or adverse effects.

[0062] The term "anti-idiotype antibody" refers to an antibody that recognizes a unique determinant that is generally associated with the antigen-binding site of the antibody.

[0063] The terms "competition" and "rivalry" refer to competition between a first antibody and a second antibody for the same antigen. Alternatively, "competition" and "rivalry" may also refer to competition between an antibody and an endogenous ligand for binding to its corresponding receptor. If an antibody interferes with the binding of an endogenous ligand to its receptor, such an antibody is said to block the endogenous interaction between the ligand and its receptor, and therefore competes with the endogenous ligand. Methods for testing antibody competition for binding to a target antigen are well known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be performed, for example, as an ELISA or by flow cytometry. Alternatively, competition can be measured using biolayer interferometry.

[0064] Further aspects and embodiments of the present invention As described above, in the first aspect, the present invention relates to a binding substance comprising a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, wherein the second antigen-binding region inhibits the binding of human PD-L1 to human PD-1.

[0065] Therefore, such a binding substance contains two different antigen-binding regions, one of which can bind to PD-L1 and the other antigen-binding region can bind to CD137.

[0066] As shown by the inventors of the present invention, the binding substance according to the present invention can activate and / or induce proliferation by binding to CD137 on one cell and simultaneously binding to PD-L1 on the surface of another cell. In humans, CD137 is used to activate T cells, for example, CD8 + T cells and CD4 +While PD-L1 is expressed on the surface of T cells, PD-L1 is primarily expressed on the surface of antigen-presenting cells (APCs), such as dendritic cells or tumor cells. Therefore, a binding agent according to the present invention, such as a bispecific antibody, which can bind to both CD137 and PD-L1, can simultaneously bind to T cells and APCs or to T cells and tumor cells. Thus, a binding agent according to the present invention, such as a bispecific antibody, may mediate intercellular interactions between APCs and T cells by simultaneously binding to PD-L1 and CD137 on the surface of these cells. This may lead to the proliferation of antigen-specific T cells. Furthermore, a binding agent according to the present invention, such as a bispecific antibody, may mediate intercellular interactions between tumor cells and T cells by simultaneously binding to PD-L1 on the surface of tumor cells and CD137 on the surface of T cells. This may lead to further activation of T cells in the presence of tumor cells by binding to CD137 on the surface of T cells, while simultaneously bringing T cells and tumor cells into close proximity by binding to PD-L1 on the surface of tumor cells. Therefore, when T cells are activated in the presence of tumor cells, the killing of tumor cells by T cells may increase. Furthermore, the PD-L1 antigen-binding domain of the binding substance according to the present invention has the ability to inhibit the binding of PD-L1 on the surface of tumor cells to PD-1 on the surface of T cells, thereby inducing T cell inhibition in tumor cells, and thus preventing them from escaping the antitumor effect of activated T cells.

[0067] Therefore, the conjugate substance of the present invention, for example, a bispecific antibody, may be used in the treatment of diseases in which the reactivation of T cells can be beneficial, such as cancer.

[0068] In one embodiment of the present invention, the second antigen-binding domain binds to human PD-L1 or its mature polypeptide, as shown in SEQ ID NO:28.

[0069] In one embodiment of the present invention, the second antigen-binding domain binds to cynomolgus monkey (Macaca fascicularis) PD-L1 or its mature polypeptide, as shown in SEQ ID NO:29. Therefore, a conjugate having an antigen-binding domain that is cross-specific to human PD-L1 and cynomolgus monkey PD-L1 is suitable for preclinical trials in cynomolgus monkeys.

[0070] Different binding agents, such as antibodies, that can bind to the same antigen, for example, PD-L1, may bind to different regions of the antigen. In some cases, even if one PD-L1 antibody binds to PD-L1, another PD-L1 antibody may still bind to PD-L1. However, in other cases, the binding of one PD-L1 antibody to PD-L1 may compete with (block) the binding of a different PD-L1 antibody to PD-L1. Therefore, competition experiments can provide information about the sites on the target antigen where antibodies bind, which may affect the functional action of antibody binding.

[0071] In one embodiment of the present invention, the second antigen-binding domain that binds to human PD-L1 does not bind to human PD-L2.

[0072] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 is, a. A heavy chain variable region including the sequence shown in SEQ ID NO:20 or a heavy chain complementarity determination region 3 (HCDR3) having up to one amino acid, for example, one amino acid modified in SEQ ID NO:20, and b. Light chain variable region including light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:23 or a sequence in which up to two amino acids, e.g., two amino acids or e.g., one amino acid, are modified in SEQ ID NO:23. It includes the heavy chain variable region and light chain variable region of an antibody that competes with an antibody for human PD-L1 binding.

[0073] In one embodiment of the present invention, the modified amino acid may be an amino acid substitution, for example, a conservative amino acid substitution. In one embodiment of the present invention, up to one amino acid in SEQ ID NO:20, for example, up to one conservative amino acid substitution in SEQ ID NO:20, is modified. In one embodiment of the present invention, up to two amino acids in SEQ ID NO:23, for example, one, for example, two conservative amino acid substitutions in SEQ ID NO:23, are modified.

[0074] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 is, a. A heavy chain variable region including heavy chain complementarity determination region 1 (HCDR1) having the sequence shown in SEQ ID NO:18, heavy chain complementarity determination region 2 (HCDR2) having the sequence shown in SEQ ID NO:19, and heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:20, and b. Light chain variable region including light chain complementarity determination region 1 (LCDR1) having the sequence shown in SEQ ID NO:22, light chain complementarity determination region 2 (LCDR2) having the sequence shown as DDN, and light chain complementarity determination region 3 (LCDR3) having the sequence shown in 23. It includes the heavy chain variable region and light chain variable region of an antibody that competes with an antibody for human PD-L1 binding.

[0075] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody that compete for binding to human PD-L1, wherein VH includes the sequence shown in SEQ ID NO:17 and VL includes the sequence shown in SEQ ID NO:21. Antibodies competing for binding to a target antigen may bind to different epitopes on the antigen, and since the epitopes are very close to each other, the first antibody that binds to one epitope will prevent the second antibody from binding to the other epitope. However, in other situations, two different antibodies may bind to the same epitope on the antigen and are considered to compete for binding in competitive binding assays.

[0076] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region containing HCDR3 having the sequence shown in SEQ ID NO:20 or a sequence in which up to one amino acid is modified in SEQ ID NO:20, and a light chain variable region containing LCDR3 having the sequence shown in SEQ ID NO:23 or a sequence in which up to two amino acids are modified in SEQ ID NO:23, and an antibody having specificity for PD-L1.

[0077] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes the heavy chain variable region and light chain variable region of an antibody that binds to an epitope on PD-L1, the same as an antibody that includes a heavy chain variable region containing an HCDR1 having the sequence shown in SEQ ID NO:18, an HCDR2 having the sequence shown in SEQ ID NO:19, and an HCDR3 having the sequence shown in SEQ ID NO:20, or a sequence in which up to one amino acid is modified overall across the entire HCDR sequence shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, and an LCDR1 having the sequence shown in SEQ ID NO:22, an LCDR2 having the sequence shown as DDN, and an LCDR3 having the sequence shown in 23, or a light chain variable region containing an LCDR sequence shown in SEQ ID NO:22, an LCDR sequence shown as DDN, and an LCDR sequence shown in 23, in which up to two amino acids are modified overall across the entire LCDR sequence shown in SEQ ID NO:22, an LCDR sequence shown as DDN, and an LCDR sequence shown in 23. Here, an embodiment is described that enables modification of up to one amino acid across the entire three HCDR sequences of VH and modification of up to two amino acids across the entire three LCDR sequences of VL.

[0078] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 binds to the same human PD-L1 epitope as the antibody containing VH, which includes the sequence shown in SEQ ID NO:17, and VL, which includes the sequence shown in SEQ ID NO:21. Thus, in one embodiment, the antigen-binding region that binds to human PD-L1 and binds to the same epitope as the antibody containing specific VH and VL sequences is understood to be the binding substance of the present invention, and this antibody binds to the same amino acid on the PD-L1 molecule. The binding of the binding substance and the antibody to the same epitope on the target antigen can be confirmed by standard alanine scanning or antibody-antigen crystallization experiments known to those skilled in the art.

[0079] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing HCDR3 having the sequence shown in SEQ ID NO:20 or a sequence in which up to one amino acid is modified in SEQ ID NO:20.

[0080] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing HCDR2 having the sequence shown in SEQ ID NO:19 or a sequence in which up to one amino acid is modified in SEQ ID NO:19.

[0081] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing HCDR1 having the sequence shown in SEQ ID NO:18 or a sequence in which up to one amino acid is modified in SEQ ID NO:18.

[0082] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, wherein the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence each comprise the sequence shown in SEQ ID NO:18, the sequence shown in SEQ ID NO:19, and the sequence shown in SEQ ID NO:20, respectively, and up to three amino acids are modified in total across the three HCDR sequences, for example, two amino acids, for example, one amino acid. In one embodiment of the present invention, up to one amino acid is modified in total across the three HCDR sequences. In one embodiment of the present invention, up to two amino acids are modified in total across the three HCDR sequences. In one embodiment, up to two amino acids are modified in the same HCDR sequence. In one embodiment, up to two amino acids are modified in different HCDR sequences. In one embodiment, up to three amino acids are modified in total across the three HCDR sequences, for example, three amino acids, for example, two amino acids, for example, one amino acid. In one embodiment, up to three amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid, are modified in the same HCDR sequence. In one embodiment, up to three amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid, are modified in different HCDR sequences.

[0083] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, wherein the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence each include the sequences shown in SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0084] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL) comprising LCDR3 having the sequence shown in SEQ ID NO:23 or a sequence in which up to two amino acids, for example two amino acids or for example one amino acid, are modified in SEQ ID NO:23.

[0085] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL) comprising an LCDR2 having a DDN sequence or a sequence in which up to one amino acid, for example one amino acid, is modified in the DDN.

[0086] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VL) comprising LCDR1 having the sequence shown in SEQ ID NO:22 or a sequence in which up to two amino acids, for example two amino acids or for example one amino acid, are modified in SEQ ID NO:22.

[0087] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL) comprising an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO:22, the LCDR2 sequence includes the sequence shown as DDN, and the LCDR3 sequence includes the sequence shown in 23, and up to two amino acids in total, e.g., two amino acids, e.g., one amino acid, are modified across the three LCDR sequences.

[0088] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL) comprising an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO:22, the LCDR2 sequence includes the sequence shown as DDN, and the LCDR3 sequence includes the sequence shown in 23.

[0089] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a light chain variable region (VL) containing the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence, wherein the HCDR1 sequence is the sequence shown in SEQ ID NO:18, the HCDR2 sequence is the sequence shown in SEQ ID NO:19, and the HCDR3 sequence is the sequence shown in SEQ ID NO:20, and the LCDR1 sequence includes the sequence shown in SEQ ID NO:22, the LCDR2 sequence includes the sequence shown as DDN, and the LCDR3 sequence is the sequence shown in 23.

[0090] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:17.

[0091] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL) having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:21.

[0092] Therefore, for example, the antigen-binding region that can bind to human PD-L1 is, A VH sequence having at least 70% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 70% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 75% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 75% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 80% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 80% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 85% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 85% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 90% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 90% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 95% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 95% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 97% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 97% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 99% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 99% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21, or A VH sequence having at least 100% amino acid sequence identity with the VH sequence shown in SEQ ID NO:17, and a VL sequence having at least 100% amino acid sequence identity with the VL sequence shown in SEQ ID NO:21. Includes.

[0093] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a heavy chain variable region (VH), the VH includes the sequence shown in SEQ ID NO:17.

[0094] In one embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 includes a light chain variable region (VL), the VL containing the sequence shown in SEQ ID NO:21.

[0095] In a preferred embodiment of the present invention, the second antigen-binding region that binds to human PD-L1 comprises a heavy chain variable region (VH) and a variable region (VL), wherein VH comprises the sequence shown in SEQ ID NO:17 and VL comprises the sequence shown in SEQ ID NO:21.

[0096] In a further embodiment, the VH sequence and the VL sequence each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, wherein each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequence is unmutated; and each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequence is unmutated. In the context of this embodiment, identity % refers to the percentage identity obtained when the framework sequences are combined as a single continuous sequence without intermediate CDR sequences.

[0097] CD137 As described above, the binding substance of the present invention includes a first antigen binding region that binds to human CD137. Therefore, the binding substance according to the present invention may also be a bispecific antibody having a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, wherein the second antigen-binding region inhibits the binding of human PD-L1 to human PD-1.

[0098] In one embodiment of the present invention, the first antigen-binding domain binds to human CD137 or its mature polypeptide, as shown in SEQ ID NO:30.

[0099] In one embodiment of the present invention, the first antigen-binding domain binds to cynomolgus monkey (Macaca fascicularis) CD137 or its mature polypeptide, as shown in SEQ ID NO:31. Therefore, a conjugate having an antigen-binding domain that is cross-specific to both human CD137 and cynomolgus monkey CD137 is suitable for preclinical trials in cynomolgus monkeys.

[0100] In one embodiment of the present invention, the first antigen-binding region binds to the mutant human CD137 or its mature polypeptide shown in SEQ ID NO:30 to a higher degree than it binds to the mutant human CD137 or its mature polypeptide shown in SEQ ID NO:33, as measured by cell binding with cells transfected with the following construct. The mutant human CD137 (Shuffle 5 / Elephant) shown in SEQ ID NO:33 corresponds to the amino acid sequence of human CD137 in which amino acids 48-88 are replaced with corresponding amino acids derived from elephant CD137. In one embodiment of the present invention, the first antigen-binding region has reduced binding to the mutant human CD137 (Shuffle 5 / Elephant) or its mature polypeptide shown in SEQ ID NO:33 compared to the human CD137 or its mature polypeptide shown in SEQ ID NO:30. In one embodiment of the present invention, the first antigen-binding region does not bind to the mutant human CD137 or its mature polypeptide (Shuffle 5 / Elephant) shown in SEQ ID NO:33. Accordingly, in one embodiment of the present invention, the first antigen-binding domain that binds to human CD137 binds to a human CD137 epitope, which is an epitope located in an amino acid sequence defined by positions 48-88 of SEQ ID NO:30, corresponding to positions 25-65 of SEQ ID NO:41, for example, an epitope that contains or requires one or more amino acids C, P, P, N, S, F, S, S, A, G, G, Q, R, T, C, D, I, C, R, Q, C, K, G, V, F, R, T, R, K, E, C, S, S, T, S, N, A, E, C, D, C.The binding of the first antigen-binding region to human CD137 may depend on any amino acid residues in the amino acid sequence defined by positions 48-88 of SEQ ID NO:30, corresponding to positions 25-65 of SEQ ID NO:41, for example, one or more amino acids C, P, P, N, S, F, S, S, A, G, G, Q, R, T, C, D, I, C, R, Q, C, K, G, V, F, R, T, R, K, E, C, S, S, T, S, N, A, E, C, D, C located at positions 48-88 of SEQ ID NO:30, corresponding to positions 25-65 of SEQ ID NO:41.

[0101] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 binds to at least one, for example, at least two, at least three, at least four, or at least five amino acids in the amino acid sequence shown in SEQ ID NO:40. In particular, for example, when confirmed by alanine scanning, for example as described below and in Example 13, the binding of the antibody according to the present invention to human CD137 may depend on one or more of the following amino acid residues in SEQ ID NO:41, corresponding to F36, F53, T61, D63, and N83 in SEQ ID NO:30: Phe(F) at position 13, Phe(F) at position 30, Thr(T) at position 38, Asp(D) at position 40, and Asn(N) at position 60.

[0102] According to this embodiment, the binding of the antibody to mutant CD137 in which one or more amino acid residues at positions 13, 30, 38, 40, and 60 of SEQ ID NO:41 are substituted with alanine is reduced compared to wild-type CD137 having the amino acid sequence shown in SEQ ID NO:41. Preferably, the reduction in binding is determined when the z-score (magnitude of change) of the antibody is less than -1.5, and the z-score (magnitude of change) of the binding of the antibody to mutant CD137 is calculated as shown in Example 13.

[0103] Phe(F) at position 13 and / or Phe(F) at position 30 are not directly involved in antibody binding, but they can have a structural effect on the epitope. Therefore, the antibody according to the present invention may bind to the epitope on human CD137, and Thr(T) at position 38, Asp(D) at position 40, and / or Asn(N) at position 60 in SEQ ID NO:41 are directly involved in antibody binding.

[0104] In another embodiment, the binding of the antibody according to the present invention to human CD137 may depend on one or more of the following amino acid residues of SEQ ID NO:41 corresponding to the positions of SEQ ID NO:30: Leu(L) at position 1, Gln(Q) at position 2, Pro(P) at position 4, Gly(G) at position 11, Thr(T) at position 12, Asp(D) at position 15, and Gln(Q) at position 20, where Leu(L) at position 1, Gln(Q) at position 2, Pro(P) at position 4, Gly(G) at position 11, Thr(T) at position 12, Asp(D) at position 15, and Gln(Q) at position 20 of SEQ ID NO:41 correspond to L24, Q25, P27, G34, T35, D38, and Q43 of SEQ ID NO:30, respectively.

[0105] According to this embodiment, the binding of the antibody to mutant CD137 in which one or more amino acid residues at positions corresponding to positions 1, 2, 4, 11, 12, 15, and 20 of SEQ ID NO:41 are substituted with alanine is reduced compared to wild-type CD137 having the amino acid sequence shown in SEQ ID NO:41. Preferably, the reduction in binding is determined when the z-score (magnitude of change) of the antibody is less than -1.5, and the z-score (magnitude of change) of the binding of the antibody to mutant CD137 is calculated as shown in Example 13.

[0106] A method for comparing the binding of wild-type CD137 and alanine-substituted CD137 is: (i) The step of expressing wild-type CD137 and alanine-substituted CD137 in a suitable cell line, for example, HEK293 cells; (ii) A step of collecting the cells one day after transfection, and for each data point, a step of incubating a sample consisting of 100,000 cells together with the antibody according to the present invention labeled with a compound according to formula I (A488) in FACS buffer (phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.02% sodium azide) at room temperature for 30 minutes; (iii) Washing each sample with FACS buffer and subjecting the samples to analysis by flow cytometry, and determining the geometric mean of fluorescence intensity (gMFI) for antibody binding; and (iv) A step of standardizing the data for the binding strength of a non-crossblocking CD137-specific antibody, and a step of calculating the z-score (rate of change), as described in Example 13. It may include. TIFF0007836786000002.tif52128

[0107] As described in Example 13, the data is given by the following formula: Using TIFF0007836786000003.tif9128, the binding strength of a non-cross-blocking CD137-specific control antibody can be standardized, where "aa position" refers to the position where the mutation occurs to alanine or glycine.

[0108] The z-score is calculated using the following formula: It can be calculated according to TIFF0007836786000004.tif8128, where μ and σ are the mean and standard deviation of the standardized gMFI calculated from all variants.

[0109] In one embodiment of the present invention, the first antigen-binding region binds to the mutant human CD137 (Shuffle 4 / Boar) or its mature polypeptide shown in SEQ ID NO:34 to the same extent as the human CD137 or its mature polypeptide shown in SEQ ID NO:30, as measured by cell binding with cells transfected with the constructs shown in SEQ ID NO:34 and SEQ ID NO:30. The mutant human CD137 (Shuffle 4 / Boar) shown in SEQ ID NO:34 corresponds to a human CD137 amino acid sequence in which amino acids 89-114 are replaced with corresponding amino acids derived from Boar CD137. Therefore, in one embodiment of the present invention, the first antigen-binding region that binds to human CD137 does not bind to human CD137 epitopes that contain or require one or more amino acids located at positions 89-114 in SEQ ID:30.

[0110] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 competes for binding to human CD137 with an antigen-binding region including a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes the sequence shown in SEQ ID NO:8 and VL includes the sequence shown in SEQ ID NO:12.

[0111] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 competes for binding to human CD137 with an antigen-binding region comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes the sequence shown in SEQ ID NO:15 and VL includes the sequence shown in SEQ ID NO:16.

[0112] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 competes for binding to human CD137 with an antigen-binding region including a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH includes the sequence shown in SEQ ID NO:49 and VL includes the sequence shown in SEQ ID NO:53.

[0113] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 is a. A heavy chain variable region including the heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:11 or a sequence in which up to three amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified, and b. Light chain variable region including light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:14 or a sequence in which up to four amino acids, e.g., four amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified. It includes the heavy chain variable region and light chain variable region of an antibody that competes with an antibody for human CD137 binding.

[0114] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 is a. A heavy chain variable region including the heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:52 or a sequence in which up to three amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified in SEQ ID NO:52, and b. Light chain variable region including light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:55 or a sequence in which up to four amino acids, e.g., four amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified. It includes the heavy chain variable region and light chain variable region of an antibody that competes with an antibody for human CD137 binding.

[0115] In one aspect of the present invention, up to one amino acid, for example, one amino acid, is modified in the HCDR3 sequence. In one aspect of the present invention, up to two amino acids, for example, two amino acids, for example, one amino acid, are modified in the HCDR3 sequence. In one aspect, up to three amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid, are modified in the HCDR3 sequence. In one aspect of the present invention, up to one amino acid, for example, one amino acid, is modified in the LCDR3 sequence. In one aspect of the present invention, up to two amino acids, for example, two amino acids, for example, one amino acid, are modified in the LCDR3 sequence. In one aspect, up to three amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid, are modified in the LCDR3 sequence. In one aspect, up to four amino acids, for example, four amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid, are modified in the LCDR3 sequence.

[0116] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 is a. A heavy chain variable region including heavy chain complementarity determination region 1 (HCDR1) having the sequence shown in SEQ ID NO:9, heavy chain complementarity determination region 2 (HCDR2) having the sequence shown in SEQ ID NO:10, and heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:11, and b. Light chain variable region including light chain complementarity determination region 1 (LCDR1) having the sequence shown in SEQ ID NO:13, light chain complementarity determination region 2 (LCDR2) having GAS, and light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:14. The antibody contains the heavy chain variable region and light chain variable region of an antibody that competes with the antibody for human CD137 binding.

[0117] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 is a. A heavy chain variable region including heavy chain complementarity determination region 1 (HCDR1) having the sequence shown in SEQ ID NO:50, heavy chain complementarity determination region 2 (HCDR2) having the sequence shown in SEQ ID NO:51, and heavy chain complementarity determination region 3 (HCDR3) having the sequence shown in SEQ ID NO:52, and b. Light chain variable region including light chain complementarity determination region 1 (LCDR1) having the sequence shown in SEQ ID NO:54, light chain complementarity determination region 2 (LCDR2) having SAS, and light chain complementarity determination region 3 (LCDR3) having the sequence shown in SEQ ID NO:55. The antibody contains the heavy chain variable region and light chain variable region of an antibody that competes with the antibody for human CD137 binding.

[0118] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 binds to the same human CD137 epitope as the antibody containing the VH sequence shown in SEQ ID NO:15 and the VL sequence shown in SEQ ID NO:16.

[0119] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 binds to the same human CD137 epitope as the antibody containing the VH sequence shown in SEQ ID NO:49 and the VL sequence shown in SEQ ID NO:53.

[0120] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region and a light chain variable region of an antibody having specificity for CD137, comprising: a heavy chain variable region including HCDR3 having the sequence shown in SEQ ID NO:11 or a sequence in which up to three amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified in SEQ ID NO:11; and a light chain variable region including LCDR3 having the sequence shown in SEQ ID NO:14 or a sequence in which up to four amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified in SEQ ID NO:14.

[0121] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region and a light chain variable region of an antibody having specificity for CD137, comprising: a heavy chain variable region including HCDR3 having the sequence shown in SEQ ID NO:52 or a sequence in which up to three amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified in SEQ ID NO:52; and a light chain variable region including LCDR3 having the sequence shown in SEQ ID NO:55 or a sequence in which up to four amino acids, e.g., three amino acids, e.g., two amino acids, e.g., one amino acid, are modified in SEQ ID NO:55.

[0122] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) containing HCDR3 having the sequence shown in SEQ ID NO:11 or a sequence in which up to three amino acids, for example three amino acids, for example two amino acids, or for example one amino acid, are modified in SEQ ID NO:11. In one embodiment of the present invention, up to one amino acid is modified in the HCDR3 sequence. In one embodiment of the present invention, up to two amino acids are modified in the HCDR3 sequence. In one embodiment, up to three amino acids are modified in the HCDR3 sequence.

[0123] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) containing HCDR3 having the sequence shown in SEQ ID NO:52 or a sequence in which up to three amino acids, for example three amino acids, for example two amino acids, or for example one amino acid, are modified in SEQ ID NO:52. In one embodiment of the present invention, up to one amino acid is modified in the HCDR3 sequence. In one embodiment of the present invention, up to two amino acids are modified in the HCDR3 sequence. In one embodiment, up to three amino acids are modified in the HCDR3 sequence.

[0124] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR2 having the sequence shown in SEQ ID NO: 10 or a sequence in which up to 3 amino acids, for example 3 amino acids, for example 2 amino acids, for example 1 amino acid in SEQ ID NO: 10 are modified. In one aspect of the present invention, up to 1 amino acid in the HCDR2 sequence is modified. In one aspect of the present invention, up to 2 amino acids in the HCDR2 sequence are modified. In one aspect, up to 3 amino acids in the HCDR2 sequence are modified.

[0125] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR2 having the sequence shown in SEQ ID NO: 51 or a sequence in which up to 3 amino acids, for example 3 amino acids, for example 2 amino acids, for example 1 amino acid in SEQ ID NO: 51 are modified. In one aspect of the present invention, up to 1 amino acid in the HCDR2 sequence is modified. In one aspect of the present invention, up to 2 amino acids in the HCDR2 sequence are modified. In one aspect, up to 3 amino acids in the HCDR2 sequence are modified.

[0126] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR1 having the sequence shown in SEQ ID NO: 9 or a sequence in which up to 3 amino acids, for example 3 amino acids, for example 2 amino acids, for example 1 amino acid in SEQ ID NO: 9 are modified. In one aspect of the present invention, up to 1 amino acid in the HCDR1 sequence is modified. In one aspect of the present invention, up to 2 amino acids in the HCDR1 sequence are modified. In one aspect, up to 3 amino acids in the HCDR1 sequence are modified.

[0127] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR1 having the sequence shown in SEQ ID NO:50 or a sequence in which up to 3, for example 3, for example 2, for example 1 amino acid(s) in SEQ ID NO:50 is / are modified. In one aspect of the present invention, up to 1 amino acid in the HCDR1 sequence is modified. In one aspect of the present invention, up to 2 amino acids in the HCDR1 sequence are modified. In one aspect, up to 3 amino acids in the HCDR1 sequence are modified.

[0128] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR1 sequence, an HCDR2 sequence, and an HCDR3 sequence, and the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence each include the sequences shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and up to 3 amino acids in total are modified over the entire three HCDR sequences.

[0129] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR1 sequence, an HCDR2 sequence, and an HCDR3 sequence, and the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence each include the sequences shown in SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52, respectively, and up to 3 amino acids in total are modified over the entire three HCDR sequences.

[0130] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy-chain variable region (VH) that includes an HCDR1 sequence, an HCDR2 sequence, and an HCDR3 sequence, and the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence each include the sequences shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

[0131] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, wherein the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence include the sequences shown in SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52, respectively.

[0132] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising LCDR3 having the sequence shown in SEQ ID NO:14 or a sequence in which up to four amino acids, for example four amino acids, for example three amino acids, for example two amino acids, for example one amino acid, in SEQ ID NO:14 are modified.

[0133] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising LCDR3 having the sequence shown in SEQ ID NO:55 or a sequence in which up to four amino acids, for example four amino acids, for example three amino acids, for example two amino acids, for example one amino acid, in SEQ ID NO:55 are modified.

[0134] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising the sequence GAS, or an LCDR2 having a sequence in which up to two amino acids are modified in the GAS sequence. In one embodiment of the present invention, up to one amino acid is modified in the GAS sequence.

[0135] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) containing an LCDR2 having the sequence SAS, or a sequence in which up to two amino acids are modified in the SAS sequence. In one embodiment of the present invention, up to one amino acid is modified in the SAS sequence.

[0136] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising LCDR1 having the sequence shown in SEQ ID NO:13 or a sequence in which up to four amino acids, for example four amino acids, for example three amino acids, for example two amino acids, for example one amino acid, in SEQ ID NO:13 are modified.

[0137] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising LCDR1 having the sequence shown in SEQ ID NO:54 or a sequence in which up to four amino acids, for example four amino acids, for example three amino acids, for example two amino acids, for example one amino acid, in SEQ ID NO:54 are modified.

[0138] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 comprises a light chain variable region (VL) including an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO:13, the LCDR2 sequence includes the sequence shown as GAS, and the LCDR3 sequence includes the sequence shown in 14, and up to four amino acids in total are modified across the three LCDR sequences, for example, four amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid.

[0139] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO: 54, the LCDR2 sequence includes the sequence shown as SAS, and the LCDR3 sequence includes the sequence shown in 55, and up to four amino acids in total are modified across the three LCDR sequences, for example, four amino acids, for example, three amino acids, for example, two amino acids, for example, one amino acid.

[0140] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO:13, the LCDR2 sequence includes the sequence shown as GAS, and the LCDR3 sequence includes the sequence shown in 14.

[0141] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL) comprising an LCDR1 sequence, an LCDR2 sequence, and an LCDR3 sequence, wherein the LCDR1 sequence includes the sequence shown in SEQ ID NO: 54, the LCDR2 sequence includes the sequence shown as SAS, and the LCDR3 sequence includes the sequence shown in 55.

[0142] In a preferred embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a light chain variable region (VL) comprising the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence, wherein the HCDR1 sequence is the sequence shown in SEQ ID NO:9, the HCDR2 sequence is the sequence shown in SEQ ID NO:10, the HCDR3 sequence is the sequence shown in SEQ ID NO:11, the LCDR1 sequence is the sequence shown in SEQ ID NO:13, the LCDR2 sequence is the sequence shown as GAS, and the LCDR3 sequence is the sequence shown in 14.

[0143] In a preferred embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) containing the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a light chain variable region (VL) containing the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence, wherein the HCDR1 sequence is the sequence shown in SEQ ID NO: 50, the HCDR2 sequence is the sequence shown in SEQ ID NO: 51, and the HCDR3 sequence is the sequence shown in SEQ ID NO: 52, and the LCDR1 sequence is the sequence shown as SEQ ID NO: 54, the LCDR2 sequence is the sequence shown as SAS, and the LCDR3 sequence is the sequence shown in 55.

[0144] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:15. In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:8.

[0145] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) containing a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:49.

[0146] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a variable light chain region (VL) having a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence shown in SEQ ID NO: 16.

[0147] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a variable light chain region (VL) having a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence shown in SEQ ID NO: 12.

[0148] In one aspect of the present invention, the first antigen-binding region that binds to human CD137 includes a variable light chain region (VL) having a sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequence of the VL sequence shown in SEQ ID NO: 53.

[0149] Therefore, for example, the first antigen-binding region that can bind to human CD137 is a VH sequence having at least 70% amino acid sequence identity to the VH sequence shown in SEQ ID NO: 15, and a VL sequence having at least 70% amino acid sequence identity to the VL sequence shown in SEQ ID NO: 16, or a VH sequence having at least 75% amino acid sequence identity to the VH sequence shown in SEQ ID NO: 15, and a VL sequence having at least 75% amino acid sequence identity to the VL sequence shown in SEQ ID NO: 16, or A VH sequence having at least 80% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 80% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16, or A VH sequence having at least 85% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 85% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16, or A VH sequence having at least 90% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 90% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16, or A VH sequence having at least 95% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 95% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16, or A VH sequence having at least 97% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 97% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16, or A VH sequence having at least 99% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 99% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16, or A VH sequence having at least 100% amino acid sequence identity with the VH sequence shown in SEQ ID NO:15, and a VL sequence having at least 100% amino acid sequence identity with the VL sequence shown in SEQ ID NO:16. Includes.

[0150] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH), the VH includes the sequence shown in SEQ ID NO:15.

[0151] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL), the VL containing the sequence shown in SEQ ID NO:16.

[0152] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) and a variable region (VL), wherein the VH sequence includes the sequence shown in SEQ ID NO:15, and the VL sequence includes the sequence shown in SEQ ID NO:16.

[0153] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH), the VH includes the sequence shown in SEQ ID NO:8.

[0154] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL), the VL containing the sequence shown in SEQ ID NO:12.

[0155] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) and a variable region (VL), wherein the VH sequence includes the sequence shown in SEQ ID NO:8, and the VL sequence includes the sequence shown in SEQ ID NO:12.

[0156] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH), the VH includes the sequence shown in SEQ ID NO:49.

[0157] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a light chain variable region (VL), the VL includes the sequence shown in SEQ ID NO:53.

[0158] In one embodiment of the present invention, the first antigen-binding region that binds to human CD137 includes a heavy chain variable region (VH) and a variable region (VL), wherein the VH sequence includes the sequence shown in SEQ ID NO:49, and the VL sequence includes the sequence shown in SEQ ID NO:53.

[0159] Bispecific binding substances As described above, the binding substance according to the present invention includes a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. Therefore, the binding substance according to the present invention may be a multispecific binding substance, for example, a multispecific antibody or a bispecific antibody.

[0160] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR3 sequence shown in SEQ ID NO:11, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR3 sequence shown in SEQ ID NO:20.

[0161] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR2 sequence shown in SEQ ID NO:10, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR2 sequence shown in SEQ ID NO:19.

[0162] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:9, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18.

[0163] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. c. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR3 sequence shown in SEQ ID NO:52, and d. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR3 sequence shown in SEQ ID NO:20.

[0164] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. c. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR2 sequence shown in SEQ ID NO:51, and d. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR2 sequence shown in SEQ ID NO:19.

[0165] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:50, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18.

[0166] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:9, the HCDR2 sequence shown in SEQ ID NO:10, and the HCDR3 sequence shown in SEQ ID NO:11, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:19, and the HCDR3 sequence shown in SEQ ID NO:20.

[0167] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:50, the HCDR2 sequence shown in SEQ ID NO:51, and the HCDR3 sequence shown in SEQ ID NO:52, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:19, and the HCDR3 sequence shown in SEQ ID NO:20.

[0168] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the LCDR3 sequence shown in SEQ ID NO:14, and b. The second antigen-binding region includes a light chain variable region (VL) containing the LCDR3 sequence shown in SEQ ID NO:23.

[0169] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing LCDR2 having the sequence GAS, and b. The second antigen-binding region includes a light chain variable region (VL) containing LCDR2 having sequence DDN.

[0170] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:13, and b. The second antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22.

[0171] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. c. The first antigen-binding region includes a light chain variable region (VL) containing the LCDR3 sequence shown in SEQ ID NO:55, and d. The second antigen-binding region includes a light chain variable region (VL) containing the LCDR3 sequence shown in SEQ ID NO:23.

[0172] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. c. The first antigen-binding region includes a light chain variable region (VL) containing LCDR2 having sequence SAS, and d. The second antigen-binding region includes a light chain variable region (VL) containing LCDR2 with sequence DDN.

[0173] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. c. The first antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:54, and d. The second antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22.

[0174] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:13, the LCDR2 sequence shown as GAS, and the LCDR3 sequence shown in 14, and b. The second antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in 23.

[0175] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:54, the LCDR2 sequence shown as SAS, and the LCDR3 sequence shown in 55, and b. The second antigen-binding region includes a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in 23.

[0176] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:9, the HCDR2 sequence shown in SEQ ID NO:10, and the HCDR3 sequence shown in SEQ ID NO:11, as well as a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:13, the LCDR2 sequence shown as GAS, and the LCDR3 sequence shown in 14, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:19, and the HCDR3 sequence shown in SEQ ID NO:20, as well as a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in 23.

[0177] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:50, the HCDR2 sequence shown in SEQ ID NO:51, and the HCDR3 sequence shown in SEQ ID NO:52, and a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:54, the LCDR2 sequence shown as SAS, and the LCDR3 sequence shown in 55, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:19, and the HCDR3 sequence shown in SEQ ID NO:20, as well as a light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in 23.

[0178] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:8, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17.

[0179] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17.

[0180] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17.

[0181] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the sequence shown in SEQ ID NO:12, and b. The second antigen-binding region includes a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21.

[0182] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the sequence shown in SEQ ID NO:16, and b. The second antigen-binding region includes a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21.

[0183] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a light chain variable region (VL) containing the sequence shown in SEQ ID NO:53, and b. The second antigen-binding region includes a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21.

[0184] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:8, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:12, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21.

[0185] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:16, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21.

[0186] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1. a. The first antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:53, and b. The second antigen-binding region includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:21.

[0187] In a further embodiment of the present invention, the binding substance is a multispecific antibody, for example, a bispecific antibody.

[0188] In a preferred embodiment of the present invention, the binding substance is a bispecific antibody.

[0189] In one embodiment of the present invention, the binding substance takes the form of a full-length antibody or an antibody fragment.

[0190] In one embodiment of the present invention, the binding substance, particularly a multispecific antibody, for example, a bispecific antibody, comprises a first antigen-binding region and a second antigen-binding region, each of which comprises a heavy chain variable region (VH) and a light chain variable region (VL), preferably each of which comprises three CDR sequences, CDR1, CDR2, and CDR3, and four framework sequences, FR1, FR2, FR3, and FR4. Accordingly, the CDRs in the heavy chain variable region may be represented as HCDR1, HCDR2, and HCDR3, and the CDRs in the light chain variable region may be represented as LCDR1, LCDR2, and LCDR3. Furthermore, the framework sequences in the heavy chain variable region may be represented as HFR1, HFR2, HFR3, and HFR4, and the framework sequences in the light chain variable region may be represented as LFR1, LFR2, LFR3, and LFR4.

[0191] Accordingly, in one embodiment of the multispecific antibody of the present invention, for example, a bispecific antibody, the antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), the variable region comprising three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. In a more preferred embodiment of the bispecific antibody of the present invention, the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0192] In one embodiment of the present invention, the binding substance, in particular a multispecific antibody, for example a bispecific antibody, comprises a first antigen-binding region including a first heavy chain variable region (VH) and a first light chain variable region (VL), and a second antigen-binding region including a second heavy chain variable region (VH) and a second light chain variable region (VL).

[0193] In one embodiment of the present invention, the conjugate, in particular, the conjugate, taking the form of a multispecific antibody, such as a bispecific antibody, comprises a heavy chain variable region and a light chain variable region, each variable region comprising three complementarity-determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0194] In one embodiment of the present invention, the conjugate, in particular a conjugate in the form of a multispecific antibody, for example a bispecific antibody, includes the complementarity-determining region and the framework region arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0195] In one embodiment of the present invention, the conjugate, in particular a conjugate in the form of a multispecific antibody, for example a bispecific antibody, includes a heavy chain variable region in which the complementarity-determining region and the framework region are arranged from the amino terminus to the carboxyl terminus in the following order: HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4.

[0196] In one embodiment of the present invention, the conjugate, in particular a conjugate in the form of a multispecific antibody, for example a bispecific antibody, includes a light chain variable region in which the complementarity-determining region and the framework region are arranged from the amino terminus to the carboxyl terminus in the following order: LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4.

[0197] In one embodiment of the present invention, the binding substance comprises a polypeptide which is a heavy chain (HC). In one embodiment of the present invention, the heavy chain (HC) comprises a variable heavy chain region (VH) and a heavy chain constant region (CH).

[0198] In one embodiment of the present invention, the heavy chain steady region (CH) includes a steady region domain 1 region (CH1), a hinge region, a steady region domain 2 region (CH2), and a steady region domain 3 region (CH3).

[0199] In one embodiment of the present invention, the conjugate, in particular a conjugate taking the form of a multispecific antibody, for example a bispecific antibody, comprises (i) a polypeptide comprising the first heavy chain variable region (VH) and further comprising a first heavy chain constant region (CH), and (ii) a polypeptide comprising the second heavy chain variable region (VH) and further comprising a second heavy chain constant region (CH).

[0200] In one embodiment of the present invention, the conjugate, in particular a conjugate taking the form of a multispecific antibody, for example a bispecific antibody, comprises (i) a polypeptide comprising the first light chain variable region (VL) and further comprising a first light chain constant region (CL), and (ii) a polypeptide comprising the second light chain variable region (VL) and further comprising a second light chain constant region (CL).

[0201] In one embodiment of the present invention, the binding substance is an antibody comprising a first binding arm and a second binding arm, for example, a multispecific antibody, preferably a bispecific antibody. a. The first binding arm comprises (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL); and b. The second binding arm comprises (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH), and (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL).

[0202] In one embodiment of the present invention, the conjugate, in particular the conjugate, which takes the form of a multispecific antibody, for example a bispecific antibody, comprises a first heavy chain constant region (CH) and a second heavy chain constant region (CH), which preferably include one or more constant region domain 1 (CH1 region), hinge region, CH2 region, and CH3 region, preferably at least the hinge region, CH2 region, and CH3 region.

[0203] In one aspect of the present invention, the conjugate, in particular, the conjugate, which takes the form of a multispecific antibody, for example, a bispecific antibody, is an isotype conjugate selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one aspect of the present invention, the isotype is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

[0204] In one embodiment of the present invention, the first antigen-binding region is derived from a rabbit antibody. In one embodiment of the present invention, the first antigen-binding region is derived from a humanized antibody. In one embodiment of the present invention, the first binding arm is derived from a full-length antibody. In one embodiment of the present invention, the first binding arm is derived from a monoclonal antibody. In one embodiment of the present invention, the first binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In one embodiment of the present invention, the second antigen-binding region is derived from a rat antibody. In one embodiment of the present invention, the second antigen-binding region is human. In one embodiment of the present invention, the second antigen-binding region is derived from a humanized antibody. In one embodiment of the present invention, the second binding arm is derived from a full-length antibody. In one embodiment of the present invention, the second binding arm is derived from a monoclonal antibody. In one embodiment of the present invention, the second binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In one embodiment of the present invention, the first and second antigen-binding regions are derived from humanized antibodies. In one embodiment of the present invention, the first antigen-binding region and the second antigen-binding region are human antibodies. In one embodiment of the present invention, the first binding arm and the second binding arm are derived from a full-length antibody, for example, a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. In one embodiment of the present invention, the first binding arm and the second binding arm are derived from a monoclonal antibody.

[0205] In one embodiment of the present invention, the first antigen-binding region is derived from IgG1λ, and the second antigen-binding region is derived from IgG1κ.

[0206] The antibodies described herein include IgG1, IgG2, IgG3, and IgG4 antibodies, as well as combinations thereof, where the heavy chains are different isotypes and / or subclasses. In various embodiments, the antibody is an IgG1 antibody, and more specifically, an IgG1,κ or IgG1,λ isotype (i.e., IgG1,κ,λ), an IgG2a antibody (e.g., IgG2a,κ,λ), an IgG2b antibody (e.g., IgG2b,κ,λ), an IgG3 antibody (e.g., IgG3,κ,λ), or an IgG4 antibody (e.g., IgG4,κ,λ).

[0207] In one embodiment of the present invention, the binding substance is a multispecific binding substance, for example, a bispecific binding substance. In one embodiment of the present invention, the binding substance is an antibody (particularly a multispecific antibody, for example, a bispecific antibody), for example, a chimeric antibody or a humanized antibody or a human antibody. In one embodiment of the present invention, the binding substance takes the form of a full-length antibody or an antibody fragment. In one embodiment of the present invention, the first antigen-binding region is derived from a monoclonal antibody. In one embodiment of the present invention, the second antigen-binding region is derived from a monoclonal antibody. In one embodiment of the present invention, the first antigen-binding region is derived from a monoclonal antibody, and the second antigen-binding region is derived from a monoclonal antibody.

[0208] In one embodiment of the present invention, the binding substance is a full-length IgG1 antibody. In one embodiment of the present invention, the binding substance is a full-length human IgG1 antibody. In one embodiment of the present invention, the binding substance is a full-length human IgG1 antibody having one or more mutations in the constant region.

[0209] In one embodiment of the present invention, the conjugate is a chimeric antibody, a humanized antibody, or a human antibody. In an embodiment of the present invention in which the conjugate is a bispecific antibody, both halves may be human, humanized, or chimeric, or they may differ in terms of characteristics with respect to their sequence origin.

[0210] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region and a second antigen-binding region. a. The first antigen-binding region that binds to CD137 is derived from a chimeric antibody, and / or b. The second antigen-binding domain that binds to human PD-L1 is derived from a chimeric antibody.

[0211] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region and a second antigen-binding region. a. The first antigen-binding region that binds to CD137 is derived from a humanized antibody, and / or b. The second antigen-binding domain that binds to human PD-L1 is derived from a humanized antibody.

[0212] In one embodiment of the present invention, the binding substance comprises a first antigen-binding region and a second antigen-binding region. a. The first antigen-binding region that binds to human CD137 is derived from a human antibody, and / or b. The second antigen-binding domain that binds to human PD-L1 is derived from a human antibody.

[0213] In a preferred embodiment of the present invention, the binding substance comprises a first antigen-binding region and a second antigen-binding region. a. The first antigen-binding region that binds to human CD137 is derived from a humanized antibody, and / or b. The second antigen-binding domain that binds to human PD-L1 is derived from a human antibody.

[0214] In one embodiment of the present invention, the conjugate, in particular a conjugate in the form of a multispecific antibody, for example a bispecific antibody, comprises a first heavy chain constant region (CH) and a second heavy chain constant region (CH) including a CH3 region, wherein the two CH3 regions include asymmetric mutations.

[0215] In a preferred embodiment of the present invention, the conjugate, in particular, the conjugate, taking the form of a multispecific antibody, for example a bispecific antibody, comprises a first heavy chain constant region (CH) and a second heavy chain constant region (CH), each of which comprises at least a hinge region, a CH2 region, and a CH3 region, wherein in the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and in the second heavy chain, at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and the first and second heavy chains are not substituted at the same position.

[0216] Most preferably, (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain, and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0217] In one embodiment of the present invention, the conjugate is an antibody, for example, a multispecific antibody, preferably a bispecific antibody, which induces Fc-mediated effector function to a lower degree compared to another antibody containing the same first and second antigen-binding regions and two heavy chain constant regions (CH) including the human IgG1 hinge, CH2 region, and CH3 region. In one embodiment of the present invention, the first and second heavy chain constant regions are modified such that the antibody induces Fc-mediated effector function to a lower degree compared to an antibody that is identical except for containing an unmodified first and second heavy chain.

[0218] In one embodiment of the present invention, the effector function via Fc is measured by binding to the IgG Fc(Fcγ) receptor, by binding to C1q, or by induction of Fc-mediated FcR crosslinking.

[0219] In a preferred embodiment of the present invention, the effector function via Fc is measured by coupling with C1q.

[0220] In one embodiment of the present invention, the first and second heavy chain constant regions are modified to reduce the binding of C1q to the antibody compared to a wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and the C1q binding is preferably measured by ELISA.

[0221] In one embodiment of the present invention, in at least one of the first heavy chain constant region and the second heavy chain constant region, one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. In one embodiment of the present invention, the positions corresponding to L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E in the first heavy chain constant region and the second heavy chain constant region, respectively. In one embodiment of the present invention, the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A in the first heavy chain and the second heavy chain, respectively.

[0222] In a particularly preferred embodiment, the conjugate is a PD-L1×CD137 bispecific antibody comprising a first heavy chain constant region and a second heavy chain constant region, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0223] In one embodiment of the present invention, the binding substance induces and / or enhances the proliferation of T cells. In one embodiment of the present invention, the T cells are CD4 + T cells and / or CD8 + These are T cells.

[0224] In one embodiment of the present invention, the binding substance activates CD137 signaling only when the second antigen-binding domain is bound to PD-L1.

[0225] In one embodiment of the present invention, T cell proliferation is measured by co-culturing T cells expressing a specific T cell receptor (TCR) together with dendritic cells (DCs) that present the corresponding antigen recognized by the TCR on the major histocompatibility complex.

[0226] In one embodiment, the induction or enhancement of T cell proliferation is determined by an antigen-specific assay. In this antigen-specific assay, DCs are transfected with the claudin-6 antigen, and T cells are transfected with a TCR that recognizes the claudin-6-derived epitope presented on HLA-A2 on the DCs. This assay is described in Example 7.

[0227] The bispecific binding agent of the present invention may be able to mediate the increase of tumor-infiltrating lymphocytes (TILs) in ex vivo cultures of human tumor tissue. The increase in TILs may be 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, or 10 times or more. CD3 - CD56 + The increase in natural killer (NK) cells can be at least 10 times, for example, at least 20 times, at least 30 times, at least 40 times, or for example, at least 50 times. CD3 + CD8 + The increase in cytotoxic T lymphocytes (CTLs) can be at least twofold, at least threefold, at least fourfold, at least fivefold, at least sixfold, or, for example, at least sevenfold. Preferably, the increase in TILs was determined as an increase in TILs from human non-small cell lung cancer tissue specimens in response to incubation with bispecific binding agents at concentrations corresponding to 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL, for example, in response to incubation with a bispecific binding agent at a concentration corresponding to 0.1 μg / mL.

[0228] The increase in TIL is, (i) A step of providing a resected specimen of tumor tissue, for example, a fresh resected specimen, and a step of washing the specimen with hematopoietic cell culture medium. (ii) A step of cutting tumor tissue into fragments with a diameter of 1 to 2 mm, and a step of providing a sample containing two tumor tissue fragments. (iii) A step of incubating a sample with the bispecific binding agent of the present invention at 37°C and 5% CO2 for 72 hours in a tissue culture plate well containing a hematopoietic cell medium, such as Lonza®X-VIVO®15, containing 10% human serum albumin, an antibiotic, and Proleukin®S (recombinant human IL-2 analog; SEQ ID NO: 56), wherein if more than 25 TIL microclusters are observed in the sample, the cells in the sample are divided and moved into 6 samples or 6 wells in a tissue culture plate. (iv) A step of collecting TILs after the entire incubation period of 10 to 14 days, and a step of staining the TILs with labeled antibodies against human CD3, human CD4, human CD56, and human CD8, and a dye for staining non-living cells, such as aminoactimycin D, and (v) Steps to analyze each sample by flow cytometry. This can be determined in an assay method that includes the following:

[0229] The bispecific binding agent of the present invention is particularly effective in CD40 in the peripheral blood mononuclear cell (PBMC) population. + and CD8 + It may be possible to induce an increase in T cells, and the T cells are activated by incubation with an anti-CD3 antibody, for example clone UCHT1), preferably at a concentration of 0.03 to 0.1 μg / mL, for example, below the optimal level, and preferably incubated with a bispecific binding agent according to the present invention at a concentration corresponding to 0.2 μg / mL. In particular, a method for determining T cell increase is (i) A process to obtain PBMCs from the buffy coat of a healthy donor, for example, by isolating the Ficoll gradient, (ii) A step of labeling the PBMC with carboxyfluorescein succinimidyl ester (CFSE) dissolved in PBS, (iii) A step of providing a sample containing 75,000 CFSE-labeled PBMCs, and a step of incubating the sample in Iscove's Modified Dulbecco's Medium containing glutamine and human AB serum with anti-CD3 antibody, preferably at a concentration of 0.03 to 0.1 μg / mL which has been predetermined to induce T cell proliferation below the optimal level for each donor, and the bispecific conjugate of the present invention at a concentration of 0.2 μg / mL for 4 days at 37°C and 5% CO2. (iv) A step of staining PBMCs with labeled antibodies against human CD4, human CD8, and human CD56, and a dye for staining non-living cells, such as 7-aminoactimycin D, and (v) Various subpopulations (CD4) present in the sample + and CD8 + The process of analyzing CSFE of T cells by flow cytometry. It may include.

[0230] antibody type As described above, various antibody types have been described in the art. In principle, the conjugate of the present invention may be any isotype of antibody. The selection of isotype is typically determined by the requirements for a desired Fc-mediated effector function, e.g., ADCC induction, or an antibody lacking Fc-mediated effector function ("inert" antibody). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either the human light chain constant region, κ or λ, may be used. The effector function of the antibody of the present invention may be altered by isotype switching for various therapeutic uses, e.g., switching to IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. In one embodiment, both heavy chains of the antibody of the present invention are IgG1 isotype heavy chains, e.g., IgG1,κ. Optionally, the heavy chains may be modified in the hinge and / or CH3 region as described elsewhere in this specification.

[0231] Preferably, each antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each variable region comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. More preferably, the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0232] In one embodiment of the present invention, the conjugate is a full-length antibody, for example, a full-length IgG1 antibody. In another embodiment, the antibody is a full-length IgG4 antibody, preferably a full-length IgG4 antibody having a stabilizing hinge region. Modifications that stabilize the IgG4 hinge region, such as the S228P mutation in the core hinge, have been described in the Art. See, for example, Labrijn et al., 2009 Nat Biotechnol. 27(8):767-71.

[0233] In another embodiment of the present invention, the conjugate material includes an antibody fragment, for example, a monovalent fragment consisting of Fab' or Fab fragment, VL, VH, CL, and CH1 domains, a monovalent antibody as described in WO2007059782 (Genmab), an F(ab')2 fragment, an Fd fragment, an Fv fragment, an dAb fragment, a camelid or nanobody, or an isolated complementarity-determining region (CDR).

[0234] The binding substance of the present invention is preferably a human antibody, a humanized antibody, or a chimeric antibody. In embodiments in which the antibody is a bispecific antibody, both halves may be human, humanized, or chimeric. Alternatively, the halves may differ in their characteristics regarding the origin of their sequences.

[0235] For example, in one embodiment, the binding substance, such as a bispecific antibody, comprises two halves, each containing an antigen-binding region. (i) A halve containing an antigen-binding region capable of binding to human PD-L1 is a chimeric and / or (ii) A halve containing an antigen-binding region capable of binding to human CD137 is a chimeric entity if present.

[0236] For example, in another embodiment, a bispecific antibody comprises two halves, each containing an antigen-binding region. (i) The halves containing an antigen-binding region capable of binding to human PD-L1 are humanized, and / or (ii) If present, the halves containing an antigen-binding region capable of binding to human CD137 are humanized.

[0237] For example, in a further embodiment, a bispecific antibody comprises two halves, each containing an antigen-binding region. (i) The halves containing an antigen-binding region capable of binding to human PD-L1 are human, and / or (ii) A halogen containing an antigen-binding region capable of binding to human CD137 is human if present.

[0238] Therefore, for example, in one embodiment, the antigen-binding region capable of binding to human PD-L1 is humanized, and the antigen-binding region capable of binding to human CD137 is humanized if present.

[0239] In a different embodiment of the present invention, the antigen-binding region capable of binding to human PD-L1 is human, and the antigen-binding region capable of binding to human CD137 is human, if present.

[0240] In a further embodiment, the binding substance is a bispecific antibody comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD137, wherein the half containing the antigen-binding region capable of binding to human PD-L1 is human, humanized, or a chimeric entity, and the half containing the antigen-binding region capable of binding to human CD137 is humanized.

[0241] Preferably, the half-halves containing an antigen-binding region capable of binding to human PD-L1 are human, and the half-halves containing an antigen-binding region capable of binding to human CD137 are humanized.

[0242] Bispecific antibody type Many different types and uses of bispecific antibodies are known in the art and have been outlined by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97.

[0243] The bispecific antibodies according to the present invention are not limited to any specific bispecific type or method for generating a bispecific type.

[0244] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a single-chain antibody having specificity for two different epitopes via two scFvs linked in series by an extra peptide linker, for example; and (iii) a dual-variable-domain antibody (DVD-Ig) in which each light and heavy chain contains two variable domains in series via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig®) Molecule, In: Antibody Engineering, Springer Berlin). Heidelberg (2010): (iv) two chemically linked bispecific (Fab') fragments; (v) Tandab, a quadrivalent bispecific antibody formed by the fusion of two single-chain diabodies, each having two binding sites for a different target antigen; (vi) Flexibody, a polyvalent molecule formed by the combination of scFv and diabodies; (vii) a so-called "dock and lock" molecule based on the "dimerization-docking domain" in protein kinase A. Applying this to Fab yields a trivalent bispecific binding protein in which two identical Fab fragments are linked to one different Fab fragment; (viii) a so-called scorpion molecule, for example, a scorpion molecule in which two scFvs are fused to both ends of a human Fab arm; and (ix) diabodies.

[0245] In one embodiment of the present invention, the conjugate is a diabody or crossbody. In one embodiment, the conjugate is a bispecific antibody obtained via controlled Fab arm exchange (e.g., described in WO2011131746 (Genmab)).

[0246] Examples of different classes of binding materials according to the present invention include, but are not limited to, (i) IgG-like molecules having a complementary CH3 domain molecule that forces heterodimerization; (ii) recombinant IgG-like dual-targeting molecules, each containing a Fab fragment or portion of a Fab fragment of at least two different antibodies on both sides of the molecule; (iii) IgG fusion molecules in which a full-length IgG antibody is fused with an extra Fab fragment or portion of a Fab fragment; (iv) Fc fusion molecules in which a single-chain Fv molecule or a stabilized diabody is fused with a heavy chain constant domain, Fc region, or portion thereof; (v) Fab fusion molecules in which different Fab fragments are fused together and fused with a heavy chain constant domain, Fc region, or portion thereof; and (vi) ScFv-based and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused with each other or with another protein or carrier molecule fused with a heavy chain constant domain, Fc region, or portion thereof.

[0247] Examples of IgG-like molecules with complementary CH3 domain molecules include the Triomab / Quadroma molecule (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), the so-called Knob-into-Hole molecule (Genentech, WO9850431), CrossMAb (Roche, WO2011117329), and electrostatically-matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), and the LUZ-Y molecule (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body and PIG body molecules (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) molecule (EMD Serono, WO2007110205), Biclonics molecule (Merus, WO2013157953), FcΔAdp molecule (Regeneron, WO201015792), Bispecific IgG1 molecule and IgG2 molecule (Pfizer / Rinat, WO11143545), Azymetric scaffold molecule (Zymeworks / Merck, This includes, but is not limited to, WO2012058768, mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254), and DuoBody® molecules (Genmab, WO2011131746).

[0248] Examples of recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig molecules (WO2009058383), two-in-one antibodies (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), κλ bodies (NovImmune, WO2012023053), and CovX bodies (CovX / Pfizer; This includes, but is not limited to, Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17, 501-506.

[0249] Examples of IgG fusion molecules include the Dual Variable Domain (DVD)-Ig molecule (Abbott, US7,612,181), Dual domain double head antibody (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecificity molecule (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92), and BsAb molecule (Zymogenetics, WO2010111625), and the Hercules molecule (Biogen Idec, This includes, but is not limited to, US007951918, scFv fusion molecule (Novartis), scFv fusion molecule (Changzhou Adam Biotech Inc, CN 102250246), and TvAb molecule (Roche, WO2012025525, WO2012025530).

[0250] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusion (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), the Scorpion molecule (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecule (MacroGenics, WO2008157379, WO2010080538), and the dual (ScFv)2-Fab molecule (National Research Center for Antibody Medicine-China).

[0251] Examples of Fab-fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86.), dual-action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), divalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7.), and Fab-Fv molecules (UCB-Celltech, WO2009040562A1).

[0252] Examples of ScFv antibodies, diabody-based antibodies, and domain antibodies include bispecific T cell engager (BiTE) molecules (Micromet, WO2005061547), tandem diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66., dual affinity retargeting technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), and human serum albumin ScFv fusion (Merrimack, This includes, but is not limited to, WO2010059315, combody molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75.), dual-targeting molecules (Ablynx, Hmila et al., FASEB J. 2010), and dual-targeting heavy chain only domain antibodies.

[0253] In one aspect, the bispecific antibody of the present invention comprises a first Fc sequence containing a first CH3 region and a second Fc sequence containing a second CH3 region, wherein the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between the first and second CH3 regions. Further details on these interactions and how they can be realized are shown in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0254] As further described herein, the stable bispecific antibody PD-L1 × CD137 antibody contains a small number of conservative asymmetric mutations in the CH3 region and can be obtained in high yield using a specific method based on one homodimeric PD-L1 antibody and one homodimeric CD137 antibody. Asymmetric mutation means that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at non-identical positions.

[0255] In one embodiment, the bispecific antibody of the present invention, as defined in any embodiment disclosed herein, comprises a first CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409 in the human IgG1 heavy chain, wherein the first CH3 region and the second CH3 region are not substituted at the same position.

[0256] In one embodiment, the bispecific antibody of the present invention, as defined in any embodiment disclosed herein, comprises a first CH3 region having an amino acid substitution at position 366 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407, and 409 in the human IgG1 heavy chain. In one embodiment, the amino acid at position 366 in the human IgG1 heavy chain is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

[0257] In one embodiment, the bispecific antibody of the present invention as defined in any embodiment disclosed herein comprises a first CH3 region having an amino acid substitution at position 368 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 370, 399, 405, 407, and 409 in the human IgG1 heavy chain.

[0258] In one embodiment, the bispecific antibody of the present invention as defined in any embodiment disclosed herein comprises a first CH3 region having an amino acid substitution at position 370 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407, and 409 in the human IgG1 heavy chain.

[0259] In one embodiment, the bispecific antibody of the present invention, as defined in any embodiment disclosed herein, comprises a first CH3 region having an amino acid substitution at position 399 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407, and 409 in the human IgG1 heavy chain.

[0260] In one embodiment, the bispecific antibody of the present invention, as defined in any embodiment disclosed herein, comprises a first CH3 region having an amino acid substitution at position 405 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407, and 409 in the human IgG1 heavy chain.

[0261] In one embodiment, the bispecific antibody of the present invention, as defined in any embodiment disclosed herein, comprises a first CH3 region having an amino acid substitution at position 407 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 409 in the human IgG1 heavy chain.

[0262] In one embodiment, the bispecific antibody of the present invention, as defined in any embodiment disclosed herein, comprises a first CH3 region having an amino acid substitution at position 409 in the human IgG1 heavy chain, and a second CH3 region having an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407 in the human IgG1 heavy chain.

[0263] Accordingly, in one embodiment, the bispecific antibody of the present invention as defined in any embodiment disclosed herein comprises sequences of a first CH3 region and a second CH3 region containing asymmetric mutations, i.e., mutations at different positions in two CH3 regions, for example, a mutation at position 405 in one CH3 region and a mutation at position 409 in the other CH3 region.

[0264] In one embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, and 407. In one such embodiment, the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has at position 405 an amino acid other than Phe, for example, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu. In a further embodiment, the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has at position 405 an amino acid other than Phe, Arg, or Gly, for example, Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys.

[0265] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region comprises an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, and comprises Lys at position 409. In a further embodiment, the first CH3 region includes Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region includes an amino acid other than Phe, Arg, or Gly at position 405, for example, Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and includes Lys at position 409.

[0266] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, such as Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region comprises Leu at position 405 and Lys at position 409. In a further embodiment, the first CH3 region contains Phe at position 405 and Arg at position 409, and the second CH3 region contains an amino acid other than Phe, Arg, or Gly at position 405, such as Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and contains Lys at position 409. In another embodiment, the first CH3 region contains Phe at position 405 and Arg at position 409, and the second CH3 region contains Leu at position 405 and Lys at position 409.

[0267] In a further embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises at position 409 an amino acid other than Lys, Leu, or Met, such as Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region comprises Lys at position 409, Thr at position 370, and Leu at position 405. In a further embodiment, the first CH3 region comprises Arg at position 409, and the second CH3 region comprises Lys at position 409, Thr at position 370, and Leu at position 405.

[0268] In further embodiments, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises Lys at position 370, Phe at position 405, and Arg at position 409, and the second CH3 region comprises Lys at position 409, Thr at position 370, and Leu at position 405.

[0269] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region comprises Lys at position 409 and (a) comprises Ile at position 350 and Leu at position 405, or (b) comprises Thr at position 370 and Leu at position 405.

[0270] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises Arg at position 409, and the second CH3 region comprises Lys at position 409 and (a) comprises Ile at position 350 and Leu at position 405, or (b) comprises Thr at position 370 and Leu at position 405.

[0271] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises Thr at position 350, Lys at position 370, Phe at position 405, and Arg at position 409, and the second CH3 region comprises Lys at position 409 and (a) comprises Ile at position 350 and Leu at position 405, or (b) comprises Thr at position 370 and Leu at position 405.

[0272] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region comprises Thr at position 350, Lys at position 370, Phe at position 405, and Arg at position 409, and the second CH3 region comprises Ile at position 350, Thr at position 370, Leu at position 405, and Lys at position 409.

[0273] In one embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405, for example, an amino acid other than Phe, Arg, or Gly at position 405, or the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, and the second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0274] In one embodiment, the bispecific antibody of the present invention as defined in any embodiment disclosed herein comprises a first CH3 region having an amino acid other than Lys, Leu, or Met at position 409, and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0275] In one embodiment, the bispecific antibody of the present invention as defined in any embodiment disclosed herein comprises a first CH3 region having Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409.

[0276] In one embodiment of the present invention, a bispecific antibody as defined in any embodiment disclosed herein comprises a first CH3 region having Tyr at position 407 and Arg at position 409, and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409.

[0277] In another embodiment of the present invention, the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has at position 407 an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr, for example, Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys. In another embodiment of the present invention, the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has at position 407 Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp.

[0278] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has at position 407 Gly, Leu, Met, Asn, or Trp.

[0279] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, and has Lys at position 409.

[0280] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, such as Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409.

[0281] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, such as Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has Gly, Leu, Met, Asn, or Trp at position 407 and Lys at 409.

[0282] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has Tyr at position 407 and Arg at position 409, and the second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, for example, Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, and has Lys at position 409.

[0283] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has Tyr at position 407 and Arg at position 409, and the second CH3 region has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409.

[0284] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has Tyr at position 407 and Arg at position 409, and the second CH3 region has Gly, Leu, Met, Asn, or Trp at position 407 and Lys at position 409.

[0285] In another embodiment, the bispecific antibody of the present invention is defined in any embodiment disclosed herein, wherein the first CH3 region has at position 409 an amino acid other than Lys, Leu, or Met, for example, Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region is (i) Having an amino acid other than Phe, Leu, and Met at position 368, for example, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, or (ii) Having a Trp at position 370, or (iii) Having an amino acid other than Asp, Cys, Pro, Glu, or Gln at position 399, for example, Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys, or (iv) Having an amino acid other than Lys, Arg, Ser, Thr, or Trp at position 366, for example, Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys.

[0286] In one embodiment, the first CH3 region has Arg, Ala, His, or Gly at position 409, and the second CH3 region is (i) Position 368 has Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp, or (ii) Having a Trp at position 370, or (iii) having Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg, or Tyr at position 399, (iv) Position 366 has Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr.

[0287] In one embodiment, the first CH3 region has Arg at position 409, and the second CH3 region is (i) Position 368 has Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp, or (ii) Having a Trp at position 370, or (iii) Position 399 has Phe, His, Lys, Arg, or Tyr, or (iv) Position 366 contains Ala, Asp, Glu, His, Asn, Val, and Gln.

[0288] In a preferred embodiment of the present invention, the bispecific antibody comprises a first heavy chain and a second heavy chain, each of which comprises at least a hinge region, a CH2 region, and a CH3 region, wherein (i) in the first heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L, and in the second heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R, or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain is R, and in the second heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain is L.

[0289] In addition to the amino acid substitutions described above, the first and second heavy chains may further contain amino acid substitutions, deletions, or insertions compared to the wild-type heavy chain sequence.

[0290] In one embodiment of the present invention, neither the first Fc sequence nor the second Fc sequence contains a Cys-Pro-Ser-Cys sequence in the (core) hinge region.

[0291] In a further embodiment of the present invention, both the first Fc sequence and the second Fc sequence contain a Cys-Pro-Pro-Cys sequence in the (core) hinge region.

[0292] Method for preparing bispecific antibodies In the preparation of the bispecific antibodies of the present invention, conventional methods such as hybrid hybridomas and chemical bonding (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649) can be used. When two antibodies consisting of different heavy and light chains are co-expressed in host cells, a mixture of possible antibody products is produced in addition to the desired bispecific antibody. The desired bispecific antibody can then be isolated, for example, by affinity chromatography or a similar method.

[0293] When different antibody constructs are expressed simultaneously, strategies that favor the formation of functionally bispecific products can be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). When rat hybridomas producing different antibodies are fused with mouse hybridomas, a limited number of heterodimeric proteins are produced due to species-specific preferential heavy / light chain pair formation. Another strategy that promotes heterodimerization over homodimerization is the "knob-into-hole" strategy. In this strategy, a protrusion is introduced on the first heavy chain polypeptide, and a corresponding cavity on the second heavy chain polypeptide, so that the protrusion can be positioned within the cavity at the interface of these two heavy chains, with the aim of promoting heterodimerization and preventing homodimerization. The "protrusion" is constructed by exchanging a smaller amino acid side chain originating from the interface of the first polypeptide with a larger side chain. A compensatory "cavity" of the same or similar size as the protrusion is created at the interface of the second polypeptide by exchanging a larger amino acid side chain for a smaller amino acid side chain (U.S. Patent No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies that favor heterodimerization when different antibody domains are co-expressed in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are exchanged with charged amino acids so that homodimerization is electrostatically unfavorable and heterodimerization is electrostatically favorable. WO2007110205 (Merck) describes yet another strategy in which differences between IgA and IgG CH3 domains are utilized to promote heterodimerization.

[0294] Another in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab), where bispecific antibodies are formed by a "Fab arm" or "half-arm" exchange (exchange of the heavy chain and the attached light chain) between two monospecific IgG4 antibodies or IgG4-like antibodies incubated under reducing conditions. The resulting product is a bispecific antibody having two Fab arms that may contain different sequences.

[0295] A preferred method for preparing a bispecific PD-L1 × CD137 antibody is: (a) A step of providing a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region; (b) A step of providing a second antibody comprising a second Fc region, wherein the Fc region comprises a second CH3 region, the first antibody is a CD137 antibody and the second antibody is a PD-L1 antibody, or vice versa, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction between the first CH3 region and the second CH3 region; (c) The step of incubating the first antibody together with the second antibody under reducing conditions; and (d) Steps to obtain the bispecific PD-L1×CD137 antibody. This includes the methods described in WO2011131746 and WO2013060867 (Genmab), including the method described in WO2011131746 and WO2013060867 (Genmab).

[0296] Similarly, (a) A step of culturing host cells that produce a first antibody having an antigen-binding region capable of binding to human CD137 as defined herein, and purifying the first antibody from the culture; (b) A step of culturing host cells that produce a second antibody having an antigen-binding domain capable of binding to human PD-L1 as defined herein, and purifying the second antibody from the culture; (c) the step of incubating the first antibody together with the second antibody under conditions of sufficient reducing to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization; and (d) Steps to obtain the bispecific antibody A method for producing antibodies according to the present invention is provided, including the following.

[0297] In one embodiment of the present invention, the first antibody and the second antibody are incubated under conditions of sufficient reducing to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization, and the heterodimer interaction between the first antibody and the second antibody in the resulting heterodimer antibody is such that no Fab arm exchange occurs after 24 hours at 37°C with 0.5 mM GSH.

[0298] Although not limited to theory, in step (c), the heavy chain disulfide bond in the hinge region of the parent antibody is reduced, and the resulting cysteine ​​can then form an inter-heavy chain disulfide bond with a cysteine ​​residue of another parent antibody molecule (which has different specificity from the beginning). In one embodiment of this method, the reducing conditions in step (c) include the addition of a reducing agent, for example, selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step (c) includes restoring the conditions to non-reducing or less reducing conditions, for example by removing the reducing agent, for example by desalting.

[0299] For this method, either the CD137 antibody or the PD-L1 antibody can be used, including first and second CD137 antibodies and PD-L1 antibodies, each containing a first Fc region and / or a second Fc region. Examples of such first and second Fc regions may include any of the above, including combinations of such first and second Fc regions. In a particular embodiment, the first and second CD137 antibodies and PD-L1 antibodies may each be selected to obtain a bispecific antibody as described herein.

[0300] In one embodiment of this method, the first antibody and / or the second antibody are full-length antibodies.

[0301] The Fc regions of the first and second antibodies may include, but are not limited to, any isotype of IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of both the first and second antibodies are Fc regions of the IgG1 isotype. In another embodiment, one of the Fc regions of the antibodies is an Fc region of the IgG1 isotype, and the other is an Fc region of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody contains the Fc sequences of IgG1 and IgG4, and therefore may have interesting intermediate properties with respect to the activation of effector function.

[0302] In a further embodiment, one of the antibody starting proteins is engineered not to bind to protein A, so that the heterodimer protein can be separated from the homodimer starting protein by passing the product over a protein A column.

[0303] As described above, the sequences of the first and second CH3 regions of the homodimer-starting antibody are different, and the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between the first and second CH3 regions. Further details on these interactions and how they are achieved are shown in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference in their entirety.

[0304] In particular, stable bispecific PD-L1 × CD137 antibodies can be obtained in high yield using the above-described method of the present invention, based on two homodimer starting antibodies that bind to CD137 and PD-L1, respectively, and contain a very small number of conservative asymmetric mutations in the CH3 region. Asymmetric mutations mean that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at non-identical positions.

[0305] The bispecific antibodies of the present invention may also be obtained by co-expressing constructs encoding a first polypeptide and a second polypeptide in a single cell. Therefore, in a further aspect, the present invention may be obtained by (a) A step of providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc sequence and a first antigen-binding region of a first antibody heavy chain, wherein the first Fc sequence comprises a first CH3 region, (b) A step of providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc sequence and a second antigen-binding region of a second antibody heavy chain, wherein the second Fc sequence comprises a second CH3 region, The sequences of the first CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is stronger than the homodimer interaction between the first CH3 region and the second CH3 region, respectively, wherein the first homodimer protein has an amino acid other than Lys, Leu, or Met at position 409, and the second homodimer protein has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, and 407. Optionally, the first nucleic acid construct and the second nucleic acid construct encode the light chain sequences of the first antibody and the second antibody. (c) A step of simultaneously expressing the first nucleic acid construct and the second nucleic acid construct in a host cell, (d) Steps to obtain the heterodimer protein from the cell culture. This relates to a method for producing bispecific antibodies, including [specific antibody type].

[0306] Materials and methods for producing antibodies of the present invention In a further aspect, the present invention relates to materials and methods for recombinant antibody production according to the present invention. Suitable expression vectors, including promoters and enhancers, and suitable host cells for antibody production are well known in the art.

[0307] Therefore, in one situation, (i) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to human PD-L1, as defined herein, and / or (ii) A nucleic acid sequence encoding an antibody light chain sequence containing an antigen-binding region capable of binding to human PD-L1, as defined herein. A nucleic acid construct including the following is provided.

[0308] In one embodiment, the nucleic acid construct is (i) a nucleic acid sequence encoding a heavy chain sequence of an antibody having an antigen-binding region capable of binding to human CD137, as defined herein, and (ii) A nucleic acid sequence encoding a light chain sequence of an antibody that includes an antigen-binding region capable of binding to human CD137, as defined herein. It also includes.

[0309] In a further aspect, the present invention relates to an expression vector comprising a nucleic acid construct as defined above herein.

[0310] In another aspect, the present invention relates to nucleic acids encoding a binding substance or its polypeptide chain as described in any aspect or embodiment described herein. In another aspect, the present invention relates to an expression vector comprising a nucleic acid.

[0311] In the context of the present invention, the expression vector may be any suitable vector, including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression regulatory elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the PD-L1 antibody-coding nucleic acid or CD137 antibody-coding nucleic acid may be, for example, a naked DNA vector or RNA vector containing a linear expression element (e.g., described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compressed nucleic acid vector (e.g., described in US6,077,835 and / or WO00 / 70087), a plasmid vector, e.g., pBR322, pUC19 / 18, or pUC118 / 119, a "midge" minimal-size nucleic acid vector (e.g., described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, e.g., a construct precipitated with Ca3(P04)2 (e.g., WO200046147, Benvenisty and Reshef, PNAS USA 83). These are included in 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, US5,589,466 and US5,973,972).

[0312] In one embodiment, the vector is suitable for the expression of PD-L1 antibodies and / or CD137 antibodies in bacterial cells. Examples of such vectors include expression vectors, e.g., BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem). 264This includes 5503-5509 (1989), and pET vectors (Novagen, Madison WI, etc.).

[0313] The expression vector may be any vector suitable for expression in a yeast system, either similarly or alternatively. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inductive promoters, such as α factor, alcohol oxidase, and PGH (F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153 (For an overview, see 516-544 (1987)).

[0314] The expression vector may also be a vector suitable for expression in mammalian cells, for example, a vector containing glutamine synthase as a selection marker, e.g., the vector described in Bebbington (1992) Biotechnology (NY) 10:169-175.

[0315] Nucleic acids and / or vectors may also include nucleic acid sequences encoding secretory / localization sequences. These secretory / localization sequences can target polypeptides, such as nascent polypeptide chains, into the pericellular lumen or into the cell culture medium. Such sequences are known in the art and include secretory leaders or signal peptides.

[0316] The expression vector may include, and may be conjugated to, any suitable promoter, enhancer, and other expression-enhancing elements. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer, as well as RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, origins of replication for plasmid products in Escherichia coli (E. coli), antibiotic resistance genes as selection markers, and / or convenient cloning sites (e.g., polylinkers). Nucleic acids may also include inductive promoters, such as CMV IE, in opposition to constitutive promoters.

[0317] In one embodiment, the PD-L1 antibody-coding expression vector and / or the CD137 antibody-coding expression vector may be placed in a host cell or host animal via a viral vector, and / or delivered to a host cell or host animal.

[0318] In a further aspect, the present invention relates to a host cell comprising one or more of the nucleic acid constructs or expression vectors specified herein.

[0319] Accordingly, the present invention also relates to recombinant eukaryotic host cells or prokaryotic host cells, such as transfectomas, that produce the antibodies of the present invention.

[0320] Examples of host cells include yeast, bacterial cells, plant cells, and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NS0 cells, or lymphocytes. The preferred host cell is CHO-K1 cells.

[0321] In one embodiment of the present invention, the cells are mammalian cells, for example, Chinese hamster ovary cells.

[0322] For example, in one embodiment, the host cell may contain a first nucleic acid construct and a second nucleic acid construct stably integrated into the cellular genome. In another embodiment, the present invention provides a cell containing a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which includes the first nucleic acid construct and the second nucleic acid construct specified above.

[0323] In a further aspect, the present invention relates to a hybridoma that produces a PD-L1 antibody as defined herein.

[0324] Fc area In some embodiments of the present invention, the binding substance according to the present invention includes an Fc region consisting of two heavy chain Fc sequences in addition to the antigen-binding region.

[0325] The first Fc sequence and the second Fc sequence may each be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, each of the first Fc sequence and the second Fc sequence may be of the IgG4 isotype, or derived therefrom, and may optionally have one or more mutations or modifications. In another embodiment, each of the first Fc sequence and the second Fc sequence may be of the IgG1 isotype, or derived therefrom, and may optionally have one or more mutations or modifications. In another embodiment, one of the Fc sequences is an IgG1 isotype Fc sequence and the other is an IgG4 isotype Fc sequence, or derived from each of these isotypes, and may optionally have one or more mutations or modifications.

[0326] In one embodiment of the present invention, one or both Fc sequences lack effector function. For example, the Fc sequence may be an IgG4 isotype Fc sequence, or a non-IgG4 type Fc sequence, such as an IgG1, IgG2, or IgG3 Fc sequence, which is mutated to reduce or even eliminate its effector function, such as its ability to mediate ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177 (2):1129-1138 (2006) and Hezareh M, J Virol.; 75 This is described in (24):12161-12168 (2001). In another embodiment, one or both Fc sequences contain an IgG1 wild-type sequence.

[0327] The antibodies according to the present invention may include modifications to the Fc region. When an antibody includes such modifications, it may become an inactive or deactivated antibody. As used herein, the terms “inactive,” “inactive,” or “deactivated” mean at least an Fc region that cannot bind to any Fcγ receptor, cannot induce Fc-mediated FcR crosslinking, or cannot induce FcR-mediated crosslinking of a target antigen via two Fc regions of an individual antibody, or cannot bind to C1q. The inactivity of the Fc region of an antibody, for example, a humanized or chimeric CD137 or PD-L1 antibody, is advantageously tested using a monospecific type of the antibody.

[0328] For the development of therapeutic antibodies, several variants of the antibody's Fc region can be constructed to be inactive in the interaction between the Fcγ (gamma) receptor and C1q. Examples of such variants are described herein.

[0329] Accordingly, in one embodiment of the antibody of the present invention, the antibody comprises a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified to induce Fc-mediated effector function to a lesser extent than an antibody that is identical except for containing an unmodified first heavy chain and a second heavy chain. The Fc-mediated effector function may be measured by determination by binding to the Fcγ receptor, by binding to C1q, or by induction of Fc-mediated FcR crosslinking.

[0330] In another such embodiment, the heavy chain constant sequence and the light chain constant sequence are modified such that the binding of C1q to the antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the unmodified antibody, and the C1q binding is measured by ELISA.

[0331] Therefore, amino acids within the Fc region, which play a central role in the interaction with the C1q and Fcγ receptors, can be modified.

[0332] For example, examples of amino acid positions that can be modified in IgG1 isotype antibodies include positions L234, L235, and P331. Combinations of these, such as L234F / L235E / P331S, can significantly reduce binding to human CD64, CD32, CD16, and C1q.

[0333] Therefore, in one embodiment, the amino acids at at least one position corresponding to L234, L235, and P331 may be A, A, and S, respectively (Xu et al., 2000, Cell Immunol. 200(1):16-26; Oganesyan et al., 2008, Acta Cryst.(D64):700-4). Also, the L234F and L235E amino acid substitutions can yield an Fc region in which interaction with the Fcγ receptor and C1q is suppressed (Canfield et al., 1991, J. Exp. Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40). Therefore, in one embodiment, the amino acids at the positions corresponding to L234 and L235 may be F and E, respectively. The D265A amino acid substitution can reduce binding to all Fcγ receptors and inhibit ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604). Therefore, in one embodiment, the amino acid at the position corresponding to D265 may be A. Binding to C1q can be suppressed by mutating positions D270, K322, P329, and P331. Mutations to these positions to D270A, K322A, P329A, or P331A can make the antibody CDC-deficient (Idusogie EE, et al., 2000, J Immunol. 164: 4178-84). Therefore, in one embodiment, the amino acids at at least one position corresponding to D270, K322, P329, and P331 may be A, A, A, and A, respectively.

[0334] An alternative approach to minimizing the interaction between the Fc region and the Fcγ receptor and C1q is to remove the glycosylation site of the antibody. Mutation of position N297 to, for example, Q, A, or E removes a glycosylation site that is significant for IgG-Fcγ receptor interaction. Thus, in one embodiment, the amino acid at the position corresponding to N297 may be G, Q, A, or E (Leabman et al., 2013, MAbs; 5(6):896-903). Another alternative approach to minimizing the interaction between the Fc region and the Fcγ receptor may be obtained by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (Shields et al., 2001, J. Biol. Chem. (276):6591-604).

[0335] Alternatively, while human IgG2 and IgG4 subclasses are thought to have impaired interactions with C1q and Fcγ receptors in nature, interactions with the Fcγ receptor have been reported (Parren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725). In both isotypes, mutations that suppress these residual interactions can reduce undesirable side effects associated with FcR binding. In the case of IgG2, these include L234A and G237A, and in the case of IgG4, they include L235E. Therefore, in one embodiment, the amino acids at the positions corresponding to L234 and G237 in the human IgG2 heavy chain may be A and A, respectively. In one embodiment, the amino acid at the position corresponding to L235 in the human IgG4 heavy chain may be E.

[0336] Other approaches to further minimize the interaction between IgG2 antibodies and the Fcγ receptor and C1q include those described in WO2011066501 and Lightle, S., et al., 2010, Protein Science (19):753-62.

[0337] The hinge region of the aforementioned antibody can also be important for its interaction with the Fcγ receptor and complement (Brekke et al., 2006, J Immunol 177:1129-1138; Dall'Acqua WF, et al., 2006, J Immunol 177:1129-1138). Therefore, mutations within or deletions within the hinge region may affect the effector function of the antibody.

[0338] Accordingly, in one embodiment, the antibody comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, wherein in at least one of the first immunoglobulin heavy chain and the second immunoglobulin heavy chain, one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0339] In one embodiment, in both the first and second heavy chains, one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0340] In one embodiment of the present invention, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is not D.

[0341] Accordingly, in one aspect of the present invention, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in the human IgG1 heavy chain is selected from the group consisting of A and E.

[0342] In a further embodiment of the present invention, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are not L and L, respectively.

[0343] In a particular embodiment of the present invention, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

[0344] In one embodiment of the present invention, in both the first and second heavy chains, the amino acids at positions L234 and L235 in the human IgG1 heavy chain are F and E, respectively.

[0345] In a particular embodiment of the present invention, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0346] In a particularly preferred embodiment of the present invention, in both the first and second heavy chains, the amino acids at positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively.

[0347] In a more particularly preferred embodiment of the present invention, the conjugate is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering of both the first and second heavy chains are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0348] In a more particularly preferred embodiment of the present invention, the conjugate is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering of both the first and second heavy chains are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0349] Antibody variants having the combination of three amino acid substitutions L234F, L235E, and D265A, and further possessing the K409R or F405L mutation, are named in this specification with the suffix "FEAR" or "FEAL," respectively.

[0350] In a preferred embodiment, the bispecific antibody of the present invention is (i) Half-halves of an antibody derived from IgG1-CD137-FEAL, and half-halves of an antibody derived from IgG1-PDL1-547-FEAR, or (ii) Half-halves of antibodies derived from IgG1-CD137-FEAR, and half-halves of antibodies derived from IgG1-PD-L1-547-FEAL Includes.

[0351] In a further embodiment of the present invention, to manipulate the serum half-life of a bispecific antibody, one or both antibody-forming moieties of the bispecific antibody are manipulated to reduce or increase binding to the embryonic Fc receptor (FcRn). Techniques for increasing or decreasing serum half-life are well known in the art. See, for example, Dall'Acqua et al. 2006, J. Biol. Chem., 281:23514-24; Hinton et al. 2006, J. Immunol., 176:346-56; and Zalevsky et al. 2010 Nat. Biotechnol., 28:157-9.

[0352] combination In a further aspect, the present invention provides antibodies conjugated or linked to one or more therapeutic moieties, such as cytokines, immunosuppressants, immunostimulatory molecules, and / or radioisotopes. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates.” Immunoconjugates comprising one or more cytotoxins are referred to as “immunotoxins.”

[0353] In one embodiment, the first Fc sequence and / or the second Fc sequence are conjugated with a drug or prodrug, or contain an acceptor group of the drug or prodrug. Such an acceptor group may be, for example, a non-natural amino acid.

[0354] composition In one aspect, the present invention relates to a composition comprising a conjugate (e.g., a bispecific antibody), nucleic acid, expression vector, or cell according to any one of the embodiments or aspects disclosed herein. In one embodiment of the present invention, the composition is a pharmaceutical composition. In one embodiment of the present invention, the composition further comprises an acceptable pharmaceutical carrier and / or excipient.

[0355] In a further aspect, the present invention relates to a pharmaceutical composition comprising a conjugate (e.g., a multispecific antibody, e.g., a bispecific antibody), nucleic acid, expression vector, or host cell according to any one of the embodiments disclosed herein, and a pharmaceutically acceptable carrier.

[0356] The pharmaceutical composition of the present invention may contain one type of binding agent of the present invention (for example, one type of multispecific antibody, preferably a bispecific antibody), or a combination of different binding agents of the present invention (for example, multispecific antibodies, for example, bispecific antibodies).

[0357] The pharmaceutical composition may be formulated according to conventional techniques, for example, the techniques disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutical composition of the present invention may include, for example, diluents, expanders, salts, buffers, surfactants (e.g., nonionic surfactants, e.g., Tween-20 or Tween-80), stabilizers (e.g., sugar- or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition.

[0358] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, antioxidants and absorption retardants, etc., that are physiologically compatible with the binding substances of the present invention (e.g., multispecific, e.g., bispecific antibodies), nucleic acids, expression vectors, or host cells. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters, e.g., ethyl oleate, and / or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. Appropriate fluidity can be maintained, for example, by using coating materials, e.g., lecithin; in the case of dispersions, by maintaining the required particle size; and by using surfactants.

[0359] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, for example: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; (2) oil-soluble antioxidants, such as palmitate ascorbic acid, butylhydroxyanisole, butylhydroxytoluene, lecithin, propyl gallate, α-tocopherol; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0360] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, glycerol, or sodium chloride.

[0361] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for a selected route of administration, such as preservatives, humectants, emulsifiers, dispersants, or buffers, which can enhance the shelf life or efficacy of the pharmaceutical composition. The conjugates of the present invention (e.g., multispecific, e.g., bispecific antibodies) may be prepared using sustained-release formulations comprising a carrier that protects the conjugates to prevent rapid release, such as a graft, a transdermal patch, and a delivery system encapsulated in microcapsules. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or polylactic acid wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art.

[0362] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent, along with, if necessary, one or a combination of the components listed above, and then performing sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components, such as those listed above. For sterile powders used to prepare sterile injectable solutions, examples of preparation methods include vacuum drying and freeze-drying, which produce a powder of the active component and any further desirable components from a pre-filtered sterile solution of the active component + any further desirable components.

[0363] The actual dose level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient. The selected dose level depends on various pharmacokinetic factors, including the activity of the particular composition of the present invention or its amide used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, overall health, and medical history of the patient being treated, as well as similar factors well known in the medical field.

[0364] The pharmaceutical composition may be administered by any suitable route and method. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, “administered parenterally” means a method of administration other than enteral and topical administration, usually by injection, and includes injection and infusion into the epidermis, intravenous, intramuscular, intraarterial, subarachnoid space, sac, orbital, cardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal regions.

[0365] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0366] use In one aspect, the present invention relates to a conjugated substance for pharmaceutical use according to any one of the embodiments disclosed herein, or to a nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein.

[0367] In a further aspect, the present invention relates to a conjugate substance according to any one of the embodiments disclosed herein, or a nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein, for use in the treatment of a disease, such as cancer.

[0368] In a further aspect, the present invention relates to a method for treating a disease, comprising the step of administering to a subject in need an effective amount of a conjugate or nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein, according to any one of the embodiments disclosed herein.

[0369] In particular, the binding substance according to the present invention may be especially useful in therapeutic situations where specific targeting and T-cell-mediated elimination of PD-L1-expressing cells are desirable, and may be more efficient than conventional anti-PD-L1 antibodies in certain specific indications and situations.

[0370] The conjugates of the present invention also have further utility in the therapy and diagnosis of various PD-L1-related diseases. For example, the conjugates (particularly antibodies) can be used in vivo or in vitro to induce one or more of the following biological activities: inhibition of growth and / or differentiation of PD-L1-expressing cells; death of PD-L1-expressing cells; mediation of phagocytosis or ADCC of PD-L1-expressing cells in the presence of human effector cells; mediation of CDC of PD-L1-expressing cells in the presence of complement; mediation of apoptosis of PD-L1-expressing cells; and / or induction of translocation to lipid rafts when bound to PD-L1. In another aspect, the present invention relates to a method for treating a disease, comprising the step of administering to a subject requiring it a conjugate, nucleic acid, expression vector, cell, or composition described in any embodiment disclosed herein. In one aspect of the present invention, the method relates to the treatment of a disease which is cancer.

[0371] In one aspect, the present invention relates to a conjugate substance, or a nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein, for use in the treatment of cancer, according to any one of the embodiments disclosed herein.

[0372] In a further aspect, the present invention relates to a conjugate substance according to any one of the embodiments disclosed herein, or a nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein, for use in the treatment of cancerous diseases characterized by the presence of solid tumors.

[0373] In one embodiment of the present invention, the binding substances, nucleic acids, expression vectors, cells, or compositions disclosed herein are for use in the treatment of cancer or methods for treating cancer, wherein cancer is characterized by the presence of a solid tumor or is selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer.

[0374] In a further aspect, the present invention relates to a conjugate substance according to any one of the embodiments disclosed herein, or a nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein, for use in the treatment of cancers selected from the group consisting of melanoma, ovarian cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcomas, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, ovarian cancer, endometriotic cancer, prostate cancer, penile cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, Merkel cell carcinoma, and mesothelioma.

[0375] In a particular aspect, lung cancer is non-small cell lung cancer (NSCLC).

[0376] In a further aspect, the present invention relates to the use of a conjugate in any one of the embodiments disclosed herein, or a nucleic acid, expression vector, host cell, or pharmaceutical composition disclosed herein, for the manufacture of a pharmaceutical, such as a pharmacopoeia for treating cancer, for example, a cancerous disease characterized by the presence of a solid tumor, or a pharmacopoeia selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer.

[0377] In one embodiment of the present invention, the methods or uses of any embodiment disclosed herein include combinations with one or more further therapeutic substances, such as chemotherapeutic agents.

[0378] In one aspect, the present invention is a. A step of culturing host cells that produce a first antibody containing an antigen-binding region that binds to human CD137, and optionally purifying the first antibody from the culture; b. A step of culturing host cells that produce a second antibody containing an antigen-binding domain that binds to human PD-L1, and optionally purifying the second antibody from the culture; c. The step of incubating the first antibody together with the second antibody under conditions of sufficient reducing to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization; and d. Steps to obtain the CD137×PD-L1 bispecific antibody. The present invention relates to a method for producing a bispecific antibody as described in any embodiment disclosed herein, including the present invention.

[0379] The present invention also relates to a method for inhibiting the growth and / or proliferation of one or more PD-L1-expressing tumor cells, and / or a method for inducing the death and / or elimination of one or more PD-L1-expressing tumor cells, comprising the step of administering a conjugate of the present invention (e.g., a bispecific antibody) or a composition of the present invention to an individual in need thereof.

[0380] The present invention also, (a) A step of selecting subjects suffering from cancer, including PD-L1 expressing tumor cells, and (b) A step of administering the conjugate substance of the present invention (e.g., a bispecific antibody) or the pharmaceutical composition of the present invention to a target. This also relates to methods for treating cancer, including [mention specific methods].

[0381] The aforementioned treatment methods and administration regimens are adjusted to produce the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over a period of time, with the dose being proportionally reduced or increased as indicated by the severity of the treatment situation. Parenteral compositions may be formulated in unit dosage forms for ease of administration and uniformity of dosing.

[0382] The effective dosage and administration regimen of the aforementioned conjugate substance will depend on the disease or condition to be treated and can be determined by those skilled in the art. An exemplary and non-limiting range of therapeutically effective amounts of the compounds of the present invention is about 0.001 to 10 mg / kg, for example, about 0.001 to 5 mg / kg, for example, about 0.001 to 2 mg / kg, for example, about 0.001 to 1 mg / kg, for example, about 0.001 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, or about 10 mg / kg. Another exemplary and non-limiting range of therapeutically effective amounts of the conjugate substance of the present invention (e.g., bispecific antibody) is about 0.1 to 100 mg / kg, for example, about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg / kg, for example, about 0.1 to 10 mg / kg, for example, about 0.5 mg / kg, for example, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg.

[0383] A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the conjugate (e.g., bispecific antibody) used in the pharmaceutical composition at a level lower than the dose required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. Generally, a suitable daily dose of the conjugate (e.g., bispecific antibody) of the present invention is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Administration may be, for example, parenteral, intravenous, intramuscular, or subcutaneous. In one embodiment, the conjugate (e.g., bispecific antibody) is mg / m² 2The drug may be administered by infusion in weekly doses calculated in units. Such doses may be based on the mg / kg doses shown above, for example, according to the ratio (mg / kg) × 70:1.8. Such administrations may be repeated, for example, 1 to 8 times, or 3 to 5 times. Administration may be carried out by continuous infusions over a period of 2 to 24 hours, for example, 2 to 12 hours. In one embodiment, in order to reduce toxic side effects, the conjugate (e.g., bispecific antibody) may be administered by slow, continuous infusions over a long period, for example, more than 24 hours.

[0384] In one embodiment, the conjugate may be administered in weekly doses calculated as a fixed dose, for example, 4 to 6 times, up to 8 times if given once a week. Such a regimen may be repeated once or more times as needed, for example, after 6 or 12 months. Such a fixed dose may be based on the mg / kg dose indicated above, using a body weight estimate of 70 kg. The dose may be determined or adjusted by measuring the blood volume of the conjugate of the present invention (e.g., a bispecific antibody) at the time of administration, for example, by taking a biological sample and using an anti-idiotype antibody that targets the PD-L1 antigen-binding region of the antibody of the present invention.

[0385] In one embodiment, the binding substance may be administered as maintenance therapy, for example, once a week for six months or more.

[0386] The conjugate may also be administered prophylactically to reduce the risk of developing cancer, to delay the onset of events in cancer progression, and / or to reduce the risk of recurrence if the cancer is in remission.

[0387] The conjugate substance of the present invention may also be administered in combination therapy, that is, in combination with other therapeutic substances related to the disease or condition to be treated. Accordingly, in one embodiment, a pharmaceutical product containing the conjugate substance (e.g., a bispecific antibody) is intended for use in combination with one or more further therapeutic substances, such as cytotoxic agents, chemotherapeutic agents, or anti-angiogenic agents.

[0388] In one aspect, the present invention relates to an anti-idiotype antibody that binds to a first antigen-binding region and / or a second antigen-binding region as defined in any one of the embodiments disclosed herein.

[0389] In a further aspect, the present invention relates to an anti-idiotype antibody that binds to a CD137 binding region as defined in any one of the embodiments disclosed herein. In a further aspect, the present invention relates to an anti-idiotype antibody that binds to a PD-L1 binding region as defined in any one of the embodiments disclosed herein.

[0390] The present invention is further illustrated by the following embodiments, which should not be construed as limiting the scope of the invention.

[0391] array (Table 1) TIFF0007836786000005.tif132170TIFF0007836786000006.tif229170TIFF0007836786000007.tif227170TIFF0007836786 000008.tif243170TIFF0007836786000009.tif208170TIFF0007836786000010.tif229170TIFF0007836786000011.tif66170 [Examples]

[0392] Example 1: Preparation of CD137 antibody Antibodies CD137-005 and CD137-009 were prepared as described in Example 1 of WO2016 / 110584. In short, rabbits were immunized with a protein mixture containing human CD137-Fc fusion protein. Single B cells were sorted from blood and screened by ELISA and flow cytometry for the production of CD137-specific antibodies. RNA was extracted from screening-positive B cells and sequenced. The variable regions of the heavy and light chains were gene-synthesized and cloned into human IgG1κ or human IgG1λ expression vectors containing human IgG1 heavy chains with the following amino acid mutations: L234F, L235E, D265A, and F405L(FEAL) or F405L(FEAL). The amino acid position numbers follow EU numbering (corresponding to SEQ ID NO: 25). The variable region sequences of the chimeric CD137 antibody (CD137-009) are shown in the sequence listings SEQ ID NO:8 and SEQ ID NO:12 in this specification.

[0393] Example 2: Humanization of rabbit (chimeric) CD137 antibody Humanized antibody sequences from rabbit anti-CD137-009 were generated at Antitope (Cambridge, UK). The humanized antibody sequences were constructed using germline humanization (CDR grafting) technology. Humanized V-region genes were designed based on human germline sequences with the closest homology to the VH and Vκ amino acid sequences of the rabbit antibody. A series of seven VH and three Vκ(VL) germline humanized V-region genes were designed. Structural models of non-human parental antibody V-regions were created using Swiss PDB and analyzed to identify amino acids in the V-region framework that may be important for antibody binding properties. These amino acids were considered for incorporation into antibodies grafted with one or more variant CDRs. The germline sequences used as the basis for humanization design are shown in Table 2.

[0394] (Table 2) The best-matching sequences of human germline V and J segments TIFF0007836786000012.tif46170

[0395] Next, the variant sequence with the lowest incidence of potential T cell epitopes was selected using in silico technology, iTope® and TCED® (T Cell Epitope Database), which are under Antitope's intellectual property rights (Perry, LCA, Jones, TD and Baker, MP New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9 (6): 385-396; Bryson, CJ, Jones, TD and Baker, MP Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24 (1):1-8). Finally, the nucleotide sequences of the designed variants were codon-optimized.

[0396] The variable region sequences of the humanized CD137 antibody (CD137-009-HC7LC2) are shown in sequence ID NO:15 and SEQ ID NO:16 in this specification.

[0397] Example 3: DNA shuffling between wild boar CD137 or elephant CD137 and human CD137 to determine the domains important for CD137 antibody binding. To determine the domains crucial for the binding of the CD137 antibody to human CD137, DNA shuffling was performed between human CD137 and boar CD137 (boar (sus sculfa); XP_005665023) or between human CD137 and African elephant CD137 (African elephant (loxodonta africana); XP_003413533). Shuffle constructs were prepared from the DNA encoding human CD137 by exchanging the human domain with the boar (shuffle constructs 1-4, 6) domain or the elephant (shuffle construct 5) domain. The amino acid sequences of the shuffle constructs are shown in Table 1.

[0398] If a domain in human CD137 is important for the binding of an anti-CD137 antibody, then if this domain is replaced with a wild boar domain or an African elephant domain, the binding will be lost.

[0399] The homology between human CD137 and boar CD137, and between human CD137 and African elephant CD137, is 70.2% and 74.5%, respectively. The requirement for selecting these two species was that the domains of interest in African elephants and boars were sufficiently different from those in human CD137, resulting in binding loss while preserving the significant structural interactions necessary to minimize the risk of misfolding or loss of expression. Figure 1 shows the sequence alignments of human CD137, boar CD137, and African elephant CD137. Figure 2 shows constructs of human CD137 containing the boar CD137 domain or the African elephant CD137 domain, as shown.

[0400] 3 x 10 6Each HEK293T-17 cell was seeded into a T75 culture flask (Greiner Bio-One, catalog number 658175) containing 20 mL of RPMI 1640 GlutaMAX medium with 10% FCS (Biochrom, catalog number S0115). After O / N incubation, the cells were transiently transfected with an expression vector encoding a shuffle construct or boar CD137, African elephant CD137, or human CD137 downstream of a constitutively active human elongation factor-1α (EF-1α) promoter using TransIT®-LT1 transfection reagent, Mirus Bio (VWR International, catalog number 731-0029) according to the manufacturer's instructions. The following day, cells were collected using 1.5 mL Accutase (Sigma Aldrich, catalog number A6964) (incubated at 37°C for 5 minutes), and flow cytometry was performed essentially as described above to measure the surface expression of the shuffle constructs as well as human CD137, African elephant CD137, and wild boar CD137, and to measure the binding of antibody clones to the various shuffle constructs. To measure the cell surface expression of the constructs, transduced cells were incubated with 1 μg / mL goat polyclonal anti-human CD137 (R&D Systems, catalog number AF838) in FACS buffer (4°C, 20 minutes), and then incubated with APC-labeled anti-goat IgG (H+L) (R&D Systems, catalog number F0108) (4°C, 20 minutes). The binding of various CD137 antibody clones to shuffle construct-expressing cells was measured by incubating transduced cells with 1 μg / mL of antibody clone, followed by incubation with APC-labeled AffiniPureF(ab')2 fragment (1:50 final dilution; Jackson, catalog no. 109-136-127).

[0401] All CD137 shuffle constructs, as well as human CD137, African elephant CD137, and wild boar CD137, were expressed on the cell surface. Expression levels were similar (Figure 3).

[0402] Figure 4 shows that CD137-009 exhibited loss of binding to African elephant CD137 and wild boar CD137. CD137-009 also exhibited loss of binding to shuffle construct 5 compared to its binding to human CD137.

[0403] Example 4: Preparation of PD-L1 antibody Immunization and hybridoma production were performed at Aldevron GmbH (Freiburg, Germany). The cDNA encoding amino acids 19-238 of human PD-L1 was cloned into an expression plasmid under Aldevron's intellectual property rights. The antibody PD-L1-547 was produced by immunizing OmniRat animals (transgenic rats expressing a diverse antibody repertoire with a complete human idiotype; Ligand Pharmaceuticals Inc., San Diego, USA) using intradermal application of human PD-L1 cDNA-coated gold particles with a handheld particle bombardment device ("gene gun"). Serum samples were collected after a series of immunizations and tested by flow cytometry against HEK cells transiently transfected with the expression plasmid to express human PD-L1. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA derived from hybridomas producing PD-L1-specific antibodies was extracted and sequenced. The variable regions of the heavy and light chains (SEQ ID NO: 17 and 21) were synthesized and cloned into a human IgG1λ expression vector containing a human IgG1 heavy chain with the following amino acid mutations: L234F, L235E, D265A, and K409R (FEAR). The amino acid position numbers follow EU numbering (corresponding to SEQ ID NO: 24).

[0404] Example 5: Production of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific IgG1 antibodies were prepared by Fab arm exchange under controlled reducing conditions. The basis of this method is the use of complementary CH3 domains that promote heterodimerization under specific assay conditions, as described in WO2011 / 131746. F405L and K409R (EU numbering) mutations were introduced into the relevant antibodies to generate antibody pairs with complementary CH3 domains.

[0405] To produce bispecific antibodies, two complementary antibodies, each with a final concentration of 0.5 mg / mL, were incubated in 100 μL of PBS with 75 mM 2-mercaptoethylamine-HCl (2-MEA) at 31°C for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol. Bispecific antibodies were produced by combining the following antibodies from Examples 1 and 4. - CD137-009-FEAL antibody combined with PD-L1-547-FEAR antibody, - PD-L1-547-FEAL antibody combined with CD137-009-FEAR, - PD-L1-547-FEAL antibody combined with CD137-009-HC7LC2-FEAR antibody, - Using the gp120-specific antibody b12 (Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23) as the first arm, a b12-FEAL antibody is formed by combining it with PD-L1-547-FEAR antibody, CD137-009-FEAR, or CD137-009-HC7LC2-FEAR antibody. - PD-L1-547-FEAL or CD137-009-FEAL and b12-FEAR antibody.

[0406] Example 6: Effect of PD-L1 antibody on PD-1 / PD-L1 interaction The effect of a monovalent PD-L1b12-FEAL×PD-L1-547-FEAR antibody on the interaction between PD-1 and PD-L1 was measured using a PD-1 / PD-L1 inhibitory bioassay developed by Promega (Madison, USA). This bioluminescence cell assay consists of two genetically modified cell lines: PD-1 effector cells, which are Jarcut T cells expressing human PD-1 and a luciferase reporter activated by an NFAT response element (NFAT-RE); and PD-L1 aAPC / CHO-K1 cells, which are CHO-K1 cells expressing human PD-L1 and a modified cell surface protein designed to activate cognitive TCRs in an antigen-independent manner. When the two cell types are cultured together, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-RE-mediated luminescence. When an antibody that blocks the PD-1 / PD-L1 interaction is added, an inhibitory signal is released, leading to TCR activation and luminescence via NFAT-RE.

[0407] PD-L1 aAPC / CHO-K1 cells (Promega, catalog number J109A) were thawed according to the manufacturer's protocol, resuspended in Ham's F12 medium (Promega, catalog number J123A) containing 10% fetal bovine serum (FBS; Promega, catalog number J121A), and plated in 96-well flat-bottom culture plates (CulturPlate-96, Perkin Elmer, catalog number 6005680). The plates were incubated at 37°C in 5% CO2 for 16 hours. The supernatant was removed, and serially diluted antibodies (final concentrations of 5-0.001 μg / mL; 4-fold dilution in RPMI1640 [Lonza, catalog number BE12-115F] containing 1% fetal bovine serum [FBS; Promega, catalog number J121A]) were added. PD-1 effector cells (Promega, catalog number J115A; thawed according to the manufacturer's protocol and resuspended in RPMI / 1% FBS) were added. The plate was incubated at 5% CO2, 37°C for 6 hours. After equilibration to room temperature, 40 μl of Bio-Glo reagent (Bio-Glo luciferase assay substrate [Promega catalog number G720B] reconstituted with Bio-Glo luciferase assay buffer [Promega, catalog number G7198] according to the manufacturer's protocol) was added to each well. The plate was incubated at room temperature for 5-10 minutes, and luminescence was measured using an EnVision Multilabel Reader (PerkinElmer). The effect on PD1-PD-L1 interaction compared to the control (no antibody added) was calculated as follows. Induction ratio = RLU (Induction - Background) / RLU (Control without antibody - Background) RLU stands for relative light unit.

[0408] Figure 5 shows that the monovalent antibody b12-FEAL×PD-L1-547-FEAR efficiently inhibited the PD1-PD-L1 interaction.

[0409] Example 7: Antigen-specific CD8 to measure the effect of bispecific antibodies binding to PD-L1 and CD137 + T-cell proliferation assay A schematic diagram of the predicted mechanism of action of the CD137×PD-L1 bispecific antibody is shown in Figure 6.

[0410] To measure the induction of T cell proliferation by bispecific antibodies targeting PD-L1 and CD137 in antigen-specific assays, dendritic cells (DCs) were transfected with claudin-6 in vitro transcription RNA (IVT-RNA). T cells were transfected with PD-1 IVT-RNA and a claudin-6-specific HLA-A2-restricted T cell receptor (TCR) to express the claudin-6 antigen. This TCR can recognize claudin-6-derived epitopes presented on HLA-A2 on DCs. The CD137 × PD-L1 bispecific antibody cross-links PD-L1 endogenously expressed on monocyte-derived dendritic cells or tumor cells with CD137 on T cells, inhibiting inhibitory PD-1 / PD-L1 interactions while simultaneously clustering CD137, resulting in T cell proliferation. When CD137 receptors expressed on T cells cluster, the CD137 receptors are activated, thereby delivering co-stimulatory signals to T cells.

[0411] HLA-A2 + Peripheral blood mononuclear cells (PBMCs) were obtained from a healthy donor (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs using magnetically activated cell sorting (MACS) technology with anti-CD14 microbeads (Miltenyi; catalog number 130-050-201) according to the manufacturer's instructions. Peripheral blood lymphocytes (PBL, CD14-negative fraction) were frozen for future T cell isolation. 1 × 10⁶ cells were frozen to differentiate them into immature dendritic cells (iDCs). 6Monocytes / ml were cultured for 5 days in RPMI GlutaMAX (Life technologies GmbH, catalog number 61870-044) containing 5% human AB serum (Sigma-Aldrich Chemie GmbH, catalog number H4522-100ML), sodium pyruvate (Life technologies GmbH, catalog number 11360-039), non-essential amino acids (Life technologies GmbH, catalog number 11140-035), 100 IU / mL penicillin-streptomycin (Life technologies GmbH, catalog number 15140-122), 1000 IU / mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi, catalog number 130-093-868), and 1000 IU / mL interleukin-4 (IL-4; Miltenyi, catalog number 130-093-924). Half of the culture medium was replaced with fresh medium once during these 5 days. iDCs were collected by gathering non-adherent cells, and adherent cells were detached by incubation with PBS containing 2 mM EDTA at 37°C for 10 minutes. After washing, the iDCs were frozen in RPMI GlutaMAX containing 10% v / v DMSO (AppliChem GmbH, catalog no. A3672,0050) + 50% v / v human AB serum for future antigen-specific T cell assays.

[0412] antigen-specific CD8 + Frozen PBLs and iDCs from the same donor were thawed one day before initiating the T cell proliferation assay. CD8 +T cells were isolated from PBLs according to the manufacturer's instructions using MACS technology with anti-CD8 microbeads (Miltenyi, catalog number 130-045-201). Using a BTX ECM® 830 Electroporation System (BTX; 500V, 1×3ms pulse), approximately 10-15×10⁶ T cells were isolated in a 4mm electroporation cuvette (VWR International GmbH, catalog number 732-0023) containing 250μL of X-Vivo15 (Biozym Scientific GmbH, catalog number 881026). 6 CD8 + T cells were electroporated with 10 μg of α-chain coding in vitro translation (IVT)-RNA and 10 μg of β-chain coding IVT-RNA (HLA-A2 restricted; described in WO2015150327A1) of claudin-6 specific mouse TCR, and 10 μg of PD-1 coding IVT-RNA. Immediately after electroporation, the cells were transferred to fresh IMDM medium (Life Technologies GmbH, catalog no. 12440-061) supplemented with 5% human AB serum and rested at 37°C and 5% CO2 for at least 1 hour. The T cells were labeled with 1.6 μM carboxyfluorescein succinimimidyl ester (CFSE; Invitrogen, catalog no. C34564) dissolved in PBS according to the manufacturer's instructions and incubated over-the-night in IMDM medium supplemented with 5% human AB serum.

[0413] In 250 μL of X-Vivo15 medium, use the electroporation system as described above (300 V, 1 × 12 ms pulse) to obtain 5 × 10 6 Up to thawed iDCs were electroporated with 5 μg of full-length claudin-6 encoded IVT-RNA and incubated overnight in IMDM medium supplemented with 5% human AB serum.

[0414] The following day, cells were collected. Cell surface expression of claudin-6 and PD-L1 on DCs, and cell surface expression of TCR and PD-1 on T cells were checked by flow cytometry. DCs were stained with Alexa647-conjugated CLDN6-specific antibody (non-commercial; manufactured in-house) and anti-human CD274 antibody (PD-L1, eBioscienes, catalog no. 12-5983), and T cells were stained with anti-mouse TCR β-chain antibody (Becton Dickinson GmbH, catalog no. 553174) and anti-human CD279 antibody (PD-1, eBioscienes, catalog no. 17-2799). 5,000 electroporated DCs were incubated with 50,000 electroporated CFSE-labeled T cells in a 96-well round-bottom plate containing IMDM GlutaMAX with 5% human AB serum, in the presence of bispecific or control antibodies. T cell proliferation was measured by flow cytometry after 5 days. A detailed analysis of T cell proliferation based on CFSE peaks indicating cell division was performed using FlowJo software. In the results, "Divided Cells %" indicates the percentage of cells that divided, and "Proliferation Index" indicates the average number of divisions for the divided cells.

[0415] The monovalent PD-L1 control antibody, b12-FEAL×PD-L1-547-FEAR, with one unrelated binding arm, enhances T cell proliferation to some extent compared to incubation with b12 (as normal IgG1), while the bispecific antibody CD137-009-FEAL×PD-L1-547-FEAR is potent for CD8 + This induced T cell proliferation (Figure 7). This was reflected by an increase in the percentage of dividing cells (Figure 7B and D left panel) and an increase in the proliferation index (Figure 7B and D right panel).

[0416] Furthermore, this assay method uses the EC of CD137-009-FEAL×PD-L1-547-FEAR 50The values ​​were determined. Towards this target, bispecific antibodies were analyzed in 3-fold serial dilutions from 1 to 0.00015 μg / mL (Figure 8). The percentage of dividing cells and the proliferation index were determined using FlowJo software. The curves were analyzed by nonlinear regression (sigmoid dose-response with gradient changes) using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The EC of antigen-specific T cell proliferation induction by CD137-009-FEAL × PD-L1-547-FEAR 50 The values ​​were 0.003492 μg / mL for "dividing cells %" and 0.005388 μg / mL for "proliferation index".

[0417] Example 8: Comparison of bispecific antibodies targeting PD-L1 and CD137 with combinations of two monovalent CD137 antibodies and PD-L1 antibodies or combinations of two parent antibodies (PD-L1-547 + CD137-009) in an antigen-specific T cell assay using an active PD-1 / PD-L1 axis. To measure the induction of T cell proliferation by bispecific antibodies targeting PD-L1 and CD137, an antigen-specific T cell proliferation assay using an active PD-1 / PD-L1 axis was performed (a general assay setup similar to Example 7). Briefly, 5,000 DCs electroporated with claudin-6-IVT-RNA were incubated with 50,000 CFSE-labeled T cells electroporated with claudin-6-specific TCR and PD1-IVT-RNA in a 96-well round-bottom plate containing IMDM GlutaMAX with 5% human AB serum, in the presence of either a bispecific antibody or a control antibody. T cell proliferation was measured by flow cytometry after 5 days. Detailed analysis of T cell proliferation based on CFSE peaks indicating cell division was performed using FlowJo software. In the results, "Divided Cells %" indicates the percentage of divided cells, and "Proliferation Index" indicates the average number of divisions of divided cells.

[0418] Neither CD137-009-FEAL×b12-FEAR, a monovalent CD137 control antibody with one unrelated binding arm, nor the corresponding bivalent parent antibody CD137-009 affected T cell proliferation compared to IgG1-b12. In contrast, incubation with monovalent PD-L1 control antibody and bivalent parent antibodies (b12-FEAL×PD-L1-547-FEAR and PD-L1-547, respectively) moderately enhanced T cell proliferation compared to incubation with IgG1-b12 control antibody. Incubation with the combined monovalent control antibody (CD137-009-FEAL×b12-FEAR+b12-FEAL×PD-L1-547-FEAR) and the combined corresponding parent antibody (CD137-009+PD-L1-547) allowed for the detection of comparable levels of T cell proliferation. In contrast, the bispecific antibody CD137-009-FEAL×PD-L1-547-FEAR is potent against CD8 + It induced T cell proliferation, which was superior to both combined controls (monovalent and bivalent) (Figure 9). This was reflected by an increase in dividing cell percentage (Figure 9B) and an increase in the proliferation index (Figure 9C).

[0419] Example 9: Ex vivo TIL augmentation assay to evaluate the effect of CD137 × PD-L1 bispecific antibody on tumor-infiltrating lymphocytes To evaluate the effect of CD137-009-FEAL×PD-L1-547-FEAR on tumor-infiltrating lymphocytes (TILs), ex vivo cultures of human tumor tissue were performed as follows: Fresh human tumor tissue resection specimens were washed three times using a spatula or serological pipette by transferring isolated tumor masses from one well of a 6-well plate (Fisher Scientific catalog no. 10110151) containing washing medium to the next well. The washing medium consisted of X-VIVO 15 (Biozym, catalog no. 881024) supplemented with 1% Pen / Strep (Thermo Fisher, catalog no. 15140-122) and 1% Fungizone (Thermo Fisher, catalog no. 15290-026). Next, the tumors were dissected with a surgical knife (Braun / Roth, catalog no. 5518091 BA223) and cut into fragments approximately 1-2 mm in diameter. Each of the two fragments was placed in one well of a 24-well plate (VWR international, catalog number 701605) containing 1 mL of TIL medium (X-VIVO 15, containing 10% human serum albumin (HSA, CSL Behring, catalog number PZN-6446518), 1% Pen / Strep, and 1% Fungizone, with 10 U / mL IL-2 (Proleukin® S, Novartis Pharma, catalog number 02238131)). CD137-009-FEAL×PD-L1-547-FEAR was added at the indicated final concentration. The culture plate was incubated at 37°C and 5% CO2. After 72 hours, 1 mL of fresh TIL medium containing the indicated concentration of bispecific antibody was added to each well. The wells were monitored by microscope every other day for the formation of TIL clusters. If more than 25 TIL microclusters were detected in each well, the wells were transferred one by one. To divide the TIL cultures, the cells in the wells of a 24-well plate were resuspended in 2 mL of medium and transferred to the wells of a 6-well plate. In addition, 2 mL of TIL medium was added to each well.

[0420] After a total culture period of 10–14 days, TILs were collected and analyzed by flow cytometry. Cells were stained with the following reagents. All reagents were diluted 1:50 with staining buffer (D-PBS containing 5% FCS and 5 mM EDTA), anti-human CD4-FITC (Miltenyi Biotec, catalog no. 130-080-501), anti-human CD3-PE-Cy7 (BD Pharmingen, catalog no. 563423), 7-aminoactimycin D (7-AAD, Beckman Coulter, catalog no. A07704), anti-human CD56-APC (eBioscience, catalog no. 17-0567-42), and anti-human CD8-PE (TONBO, catalog no. 50-0088). To quantitatively compare the cells obtained between different treatment groups, the cell pellets were resuspended after a final wash in FACS buffer with BD® CompBeads (BD biosciences, catalog no. 51-90-9001291). Flow cytometry analysis was performed using a BD FACSCanto® II flow cytometer (Becton Dickinson), and the obtained data were analyzed using FlowJo 7.6.5 software. By standardizing the obtained 7AAD-negative cell fraction relative to the number of beads obtained, the relative number of biotillaries (TILs) and CD3 levels per 1,000 beads related to the corresponding wells in a 6-well plate were determined. + CD8 + T cell count, CD3 + CD4 + T cell count and CD3 - CD56 + The number of NK cells was calculated.

[0421] Figure 10 shows the analysis of TIL enlargement from human non-small cell lung cancer tissue samples. Here, CD137-009-FEAL×PD-L1-547-FEAR was added at the following concentrations: 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL. Tissue samples from the same patients without antibody addition served as negative controls. After 10 days of culture, TILs were collected and analyzed by flow cytometry. Five samples (from the first five wells) were measured for each antibody concentration derived from different wells of a 24-well plate. In all samples cultured with the bispecific antibody, the number of live TILs increased significantly compared to the control sample without the antibody. Overall, a 10-fold increase in live TILs was observed when 0.1 μg / mL CD137-009-FEAL×PD-L1-547-FEAR was added to the culture (Figure 10A). CD3 + CD4 + While T helper cells increased only slightly (Figure 10C; 2.8-fold increase), in contrast, CD3 - CD56 + The most significant increase in TIL was observed in NK cells (Figure 10D; up to 64-fold increase compared to the control). CD3 + CD8 + A potent effect on cytotoxic T lymphocytes (CTLs) was also observed (Figure 10B; a 7.4-fold increase compared to the control).

[0422] Example 10: Effect of a surrogate bispecific mouse antibody that binds to mPD-L1 and mCD137 on ovalbumin-specific T cell proliferation in C57BL / 6 mice after OT-I CD8+ adoptive T cell transfer. Surrogate mouse bispecific antibodies mCD137-3H3×mPD-L1-MPDL3280A, mCD137-3H3×b12, and mPD-L1-MPDL3280A×b12 were prepared using a method for producing mouse bispecific antibodies based on controlled Fab arm exchange (Labrijn et al, 2017 Sci Rep. 7(1): 2476 and WO2016097300).

[0423] A monoclonal antibody 3H3 constituting mouse 4-1BB was obtained from BioXcell (catalog number BE0239), and the protein was sequenced using ProtTech. The identified cDNA sequence was predicted using a method protected by intellectual property rights. The variable regions of the heavy and light chains were gene-synthesized and cloned into a mouse IgG2a expression vector containing the mouse IgG2a constant region with the following amino acid mutations: L234A, L235A, F405L, and R411T. Similarly, the b12 variable region was cloned into this expression vector.

[0424] The antibody MPDL3280A (with its heavy chain and light chain variable sequences shown in SEQ ID NO: 57 and 58, respectively) is said to bind to both human and mouse PD-L1. The heavy and light chain variable regions of this antibody were cloned into a mouse IgG2a expression vector containing the mouse IgG2a constant region, which includes the following amino acid mutations: L234A, L235A, T370K, and K409R.

[0425] Under the controlled reducing conditions described above, bispecific mouse antibodies (essentially rat-human-mouse chimeric) were produced by Fab arm exchange.

[0426] Female C57BL / 6JOlaHsd mice (Envigo RMS GmbH, Rossdorf, Germany), 6–8 weeks old and weighing 17–24 g, were acclimatized to the animal facility for at least 6 days prior to trial registration. These mice were used as recipients. Homozygous female or male C57BL / 6 Thy1.1 × C57BL / 6J OT-1 mice, homozygous for both OT-1 and Thy1.1 alleles, were bred in-house (heteronautically from C57BL / 6-Tg(TcraTcrb)1100Mjb / Crl and B6.PL-Thy1a / CyJ mice) and used as donors. Mice were free to access food (ssniff MZ autoclavable Soest, Germany) and sterile water and were housed in a 12-hour light / dark cycle at 22°C ± 2°C and 55% ± 15% relative humidity.

[0427] On the first day of the experiment, C57BL / 6 Thy1.1×C57BL / 6J OT-1 donor mice were sacrificed and their spleens were isolated. The spleens were mechanically dissociated, and the erythrocytes were lysed by resuspending the splenocyte pellet in erythrocyte lysis buffer (8.25 g / L NH4Cl, 1 g / L KHCO3, 0.1 mM EDTA, pH 7). Subsequently, the splenocytes were washed with Dulbecco's PBS (DPBS) and CD8 + T cells were isolated using CD8a (Ly-2) microbeads combined with autoMACS Pro Separator (both Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) in mice. + / OT-1 + / Thy1.1 + T cells (2.5~5×10 5 Cells were injected into the posterior orbit of each C57BL / 6JOlaHsd recipient mouse in a total volume of 200 μL. The day after adoptive cell transfer, the posterior orbit of the recipient mice was "vaccinated" with 100 μg ovalbumin / 200 μL PBS as an antigen stimulus. Six hours later, the posterior orbit of the mice was treated with the respective bispecific antibodies. Specifically, 100 μg or 20 μg of mCD137-3H3×mPD-L1-MPDL3280A, mCD137-3H3×b12, or mPD-L1-MPDL3280A×b12 antibody was injected per mouse. An injection of ordinary PBS was used as a baseline reference, and untreated animals (mice given only donor cells) were used as negative controls. Six days later, 100 μL of blood was collected via the postorbital pathway and analyzed using a BD FACSCanto II cytometer (Becton Dickinson GmbH) with V500 rat anti-mouse CD45 (Becton Dickinson GmbH, catalog number 561487), FITC rat anti-mouse CD8a (Life Technologies, catalog number MCD0801), and Alexa Fluor 647 anti-rat CD90 / mouse CD90.1 (BioLegend Europe, catalog number 202508) antibodies.+ CD8 + We analyzed whether T cells were present. Thy1.1(CD90.1)-positive cells were used as a substitute for OT-1 specific T cells.

[0428] Figure 11A is a schematic diagram of the OT-1 adoptive T cell transfer assay. B shows Thy1.1 as measured by flow cytometry. + CD8 + The analysis of T cell frequency is shown. n=5 mice were used for each bispecific antibody treatment modality. Ovalbumin antigen stimulation alone detected a significant increase in Thy1.1+CD8+ T cell frequency compared to untreated animals. Interestingly, both mCD137-3H3-×b12 and mPD-L1-MPDL3280A×b12, monovalent control antibodies with one unrelated b12 binding arm, failed to increase ovalbumin-specific OT-1 T cell enlargement compared to animals treated with ovalbumin alone. In contrast, the bispecific antibody mCD137-3H3×mPD-L1-MPDL3280A increased CD8+ frequency by 10–20% at the tested dose levels (20 μg and 100 μg of antibody). + / OT-1 + / Thy1.1 + We were able to induce potent OT-1 T cell proliferation, which leads to a higher T cell frequency (percentage of the total T cell population).

[0429] Example 11: Effect of a surrogate bispecific mouse antibody that binds to mPD-L1 and mCD137 on tumor growth in a subcutaneous syngeneic CT26 mouse tumor model. Female BALB / c Rj mice (Janvier, Genest-St.-Isle, France), 6-8 weeks old, weighing 17-24g, were acclimatized for at least 6 days prior to trial registration. The mice had free access to food (ssniff MZ autoclavable Soest, Germany) and sterile water and were housed in a 12-hour light / dark cycle at 22°C ± 2°C and 55% ± 10% relative humidity. CT26 cells were obtained from ATCC® (catalog no. CRL-2638) and cultured in Roswell Park Memorial Institute medium (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) (Biochrom, catalog no. S0115) and GlutaMAX® (Life Technologies, catalog no. 61870-044) at 5% CO2 and 37°C. The aforementioned cells were collected using StemPro®Accutase® cell dissociation reagent (Life technologies, catalog number A1110501), resuspended in DPBS (Life technologies, catalog number 14190-169), and 0.5 × 10⁶ cells were collected per mouse. 6 Cells / 100μl were subcutaneously (SC) transplanted into the shorn right flank of female BALB / c Rj mice. Tumor volume was assessed every 2-3 days by calipas measurement, using formula: a 2 It was expressed as the product of the perpendicular diameters using ×b / 2. In ...

Claims

1. A bispecific antibody comprising a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, The first antigen-binding region is a. The HCDR1 sequence is the sequence shown in SEQ ID NO:9, the HCDR2 sequence is the sequence shown in SEQ ID NO:10, the HCDR3 sequence is the sequence shown in SEQ ID NO:11, the LCDR1 sequence is the sequence shown in SEQ ID NO:13, the LCDR2 sequence is the sequence shown as GAS, and the LCDR3 sequence is the sequence shown in SEQ ID NO:

14. A first heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a first light chain variable region (VL) comprising the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence; or b. The HCDR1 sequence is the sequence shown in SEQ ID NO:50, the HCDR2 sequence is the sequence shown in SEQ ID NO:51, the HCDR3 sequence is the sequence shown in SEQ ID NO:52, the LCDR1 sequence is the sequence shown in SEQ ID NO:54, the LCDR2 sequence is the sequence shown as SAS, and the LCDR3 sequence is the sequence shown in SEQ ID NO:

55. A first heavy chain variable region (VH) comprising the HCDR1 sequence, the HCDR2 sequence, and the HCDR3 sequence, and a first light chain variable region (VL) comprising the LCDR1 sequence, the LCDR2 sequence, and the LCDR3 sequence. Includes, The second antigen-binding region includes a second heavy chain variable region (VH) containing the HCDR1 sequence shown in SEQ ID NO:18, the HCDR2 sequence shown in SEQ ID NO:19, and the HCDR3 sequence shown in SEQ ID NO:20, and a second light chain variable region (VL) containing the LCDR1 sequence shown in SEQ ID NO:22, the LCDR2 sequence shown as DDN, and the LCDR3 sequence shown in SEQ ID NO:

23. Bispecific antibodies.

2. The bispecific antibody according to claim 1, wherein the second antigen-binding region that binds to human PD-L1 includes a second heavy chain variable region (VH) having at least 70% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:

17.

3. The bispecific antibody according to claim 1 or 2, wherein the second antigen-binding region that binds to human PD-L1 comprises a second heavy chain variable region (VH), and the VH comprises the sequence shown in SEQ ID NO:

17.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the second antigen-binding region that binds to human PD-L1 includes a second light chain variable region (VL) having at least 70% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:

21.

5. A bispecific antibody according to any one of claims 1 to 4, wherein the second antigen-binding region that binds to human PD-L1 includes a second light chain variable region (VL), and the VL includes the sequence shown in SEQ ID NO:

21.

6. A bispecific antibody according to any one of claims 1 to 5, wherein the second antigen-binding region that binds to human PD-L1 comprises a second heavy chain variable region (VH) and a second light chain variable region (VL), the VH comprising the sequence shown in SEQ ID NO:17, and the VL comprising the sequence shown in SEQ ID NO:

21.

7. The first antigen-binding region that binds to human CD137, a. A first heavy chain variable region (VH) containing a sequence having at least 70% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:15; or b. The first heavy chain variable region (VH) includes a sequence having at least 70% identity with the amino acid sequence of the VH sequence shown in SEQ ID NO:

49. A bispecific antibody according to any one of claims 1 to 6, comprising:

8. The first antigen-binding region that binds to human CD137, a. The first heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15; or b. The first heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49 A bispecific antibody according to any one of claims 1 to 7, comprising:

9. The first antigen-binding region that binds to human CD137, a. A first light chain variable region (VL) containing a sequence having at least 70% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:16; or b. A first light chain variable region (VL) containing a sequence having at least 70% identity with the amino acid sequence of the VL sequence shown in SEQ ID NO:

53. A bispecific antibody according to any one of claims 1 to 8, comprising:

10. The first antigen-binding region that binds to human CD137, a. The first light chain variable region (VL) containing the sequence shown in SEQ ID NO:16; or b. The first light chain variable region (VL) containing the sequence shown in SEQ ID NO:53 A bispecific antibody according to any one of claims 1 to 9, comprising:

11. The first antigen-binding region that binds to human CD137, a. The first heavy chain variable region (VH) contains the sequence shown in SEQ ID NO:15 and the first light chain variable region (VL) contains the sequence shown in SEQ ID NO:16, VH and VL; or b. The first heavy chain variable region (VH) contains the sequence shown in SEQ ID NO:49 and the first light chain variable region (VL) contains the sequence shown in SEQ ID NO:53, VH and VL A bispecific antibody according to any one of claims 1 to 10, comprising:

12. It comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, a. The first antigen-binding region is - The first heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15, or - The first heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49 Includes; and b. The second antigen-binding region includes a second heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17, A bispecific antibody according to any one of claims 1 to 11.

13. It comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, a. The first antigen-binding region is - A first heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:15, and a first light chain variable region (VL) containing the sequence shown in SEQ ID NO:16, or - A first heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:49, and a first light chain variable region (VL) containing the sequence shown in SEQ ID NO:

53. Includes; and b. The second antigen-binding region includes a second heavy chain variable region (VH) containing the sequence shown in SEQ ID NO:17, and a second light chain variable region (VL) containing the sequence shown in SEQ ID NO:

21. A bispecific antibody according to any one of claims 1 to 12.

14. A bispecific antibody according to any one of claims 1 to 13, which takes the form of a full-length antibody or an antibody fragment.

15. A bispecific antibody according to any one of claims 1 to 14, wherein each variable region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

16. The bispecific antibody according to claim 15, wherein the complementarity-determining region and the framework region are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

17. (i) a polypeptide comprising the first heavy chain variable region (VH) and further comprising a first heavy chain constant region (CH), and (ii) a polypeptide comprising the second heavy chain variable region (VH) and further comprising a second heavy chain constant region (CH), according to any one of claims 1 to 16.

18. (i) a polypeptide comprising the first light chain variable region (VL) and further comprising the first light chain constant region (CL), and (ii) a polypeptide comprising the second light chain variable region (VL) and further comprising the second light chain constant region (CL), according to claim 17.

19. The antibody comprises a first binding arm and a second binding arm. a. The first binding arm comprises (i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain steady region (CH), and (ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain steady region (CL); and b. The second binding arm comprises (iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain steady region (CH), and (iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain steady region (CL), The bispecific antibody according to claim 18.

20. The bispecific antibody according to any one of claims 17 to 19, wherein each of the first heavy chain constant region and the second heavy chain constant region (CH) comprises one or more constant region domain 1 region (CH1 region), hinge region, CH2 region, and CH3 region.

21. A bispecific antibody according to any one of claims 17 to 20, which is a bispecific antibody of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

22. A bispecific antibody according to any one of claims 17 to 21, which is a full-length IgG1 antibody.

23. A bispecific antibody according to any one of claims 17 to 22, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) includes a CH3 region, and the two CH3 regions include an asymmetric mutation.

24. A bispecific antibody according to any one of claims 17 to 23, wherein in the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering is substituted, and the first heavy chain constant region and the second heavy chain constant region are not substituted at the same position.

25. (i) The amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH), and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) The amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain constant region (CH), and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain constant region (CH), according to claim 24.

26. A bispecific antibody according to any one of claims 17 to 25, wherein the antibody induces effector function via Fc to a lower degree compared to another antibody comprising the same first antigen-binding region and second antigen-binding region and two heavy chain constant regions (CH) including the human IgG1 hinge, CH2 region, and CH3 region.

27. A bispecific antibody according to any one of claims 17 to 26, wherein in at least one of the first heavy chain constant region (CH) and the second heavy chain constant region (CH), one or more amino acids at positions corresponding to L234, L235, D265, N297, and P331 in the human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

28. The bispecific antibody according to claim 27, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E in the first heavy chain constant region (CH) and the second heavy chain constant region (CH), respectively.

29. The bispecific antibody according to claim 27, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A in the first heavy chain constant region (CH) and the second heavy chain constant region (CH), respectively.

30. The bispecific antibody according to claim 29, wherein the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is R and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is L.

31. The bispecific antibody according to claim 28, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to the EU numbering of both the first heavy chain constant region and the second heavy chain constant region are F and E, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to the EU numbering of the first heavy chain constant region is R and the position corresponding to F405 in the human IgG1 heavy chain according to the EU numbering of the second heavy chain constant region is L.

32. The first heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 24, and the second heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 25, or The bispecific antibody according to claim 27, wherein the first heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 25, and the second heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

24.

33. A bispecific antibody according to any one of claims 1 to 32, which induces and / or enhances the proliferation of T cells.

34. The aforementioned T cells are CD4 + T cells and / or CD8 + The bispecific antibody according to claim 33, wherein the antibody is a T cell.

35. The bispecific antibody according to any one of claims 1 to 34, wherein the bispecific antibody activates CD137 signaling only when the second antigen-binding domain is bound to PD-L1.

36. The bispecific antibody according to claim 33 or 34, wherein T cell proliferation is measured by co-culturing T cells expressing a specific T cell receptor (TCR) together with dendritic cells (DCs) that present a corresponding antigen recognized by the TCR on the major histocompatibility complex.

37. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, a. The first antigen-binding region comprises (i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and (ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), and b. The second antigen-binding region comprises (iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and (iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL), The first VH contains the amino acid sequence shown in SEQ ID NO:15, the first VL contains the amino acid sequence shown in SEQ ID NO:16, the second VH contains the amino acid sequence shown in SEQ ID NO:17, and the second VL contains the amino acid sequence shown in SEQ ID NO:

21. The first CH contains the amino acid sequence shown in SEQ ID NO:24, and the second CH contains the amino acid sequence shown in SEQ ID NO:25; or the first CH contains the amino acid sequence shown in SEQ ID NO:25, and the second CH contains the amino acid sequence shown in SEQ ID NO:

24. Bispecific antibodies.

38. A nucleic acid encoding a bispecific antibody or its polypeptide chain according to any one of claims 1 to 37.

39. An expression vector comprising the nucleic acid described in claim 38.

40. A cell comprising the nucleic acid according to claim 38 or the expression vector according to claim 39.

41. The cell according to claim 40, which is a mammalian cell or a Chinese hamster ovary cell.

42. A composition comprising a bispecific antibody according to any one of claims 1 to 37, a nucleic acid according to claim 38, an expression vector according to claim 39, or a cell according to claim 40 or 41.

43. The composition according to claim 42, which is a pharmaceutical composition.

44. The composition according to claim 43, further comprising a pharmaceutically acceptable carrier and / or excipient.

45. A bispecific antibody according to any one of claims 1 to 37, a nucleic acid according to claim 38, an expression vector according to claim 39, a cell according to claim 40 or 41, or a composition according to any one of claims 42 to 44, for use as a pharmaceutical.

46. A bispecific antibody, nucleic acid, expression vector, cell, or composition according to claim 45 for use in the treatment of cancer.

47. A composition according to any one of claims 42 to 44 for the treatment of a disease.

48. The composition according to claim 47, wherein the disease is cancer.

49. The bispecific antibody, nucleic acid, expression vector, cell, or composition for use according to claim 46 or the composition according to claim 48, wherein the cancer is characterized by the presence of a solid tumor, or is selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer.

50. A bispecific antibody, nucleic acid, expression vector, cell, or composition for use according to claim 49, or the composition according to claim 48, wherein the cancer is non-small cell lung cancer (NSCLC).

51. Use of a bispecific antibody according to any one of claims 1 to 37, a nucleic acid according to claim 38, an expression vector according to claim 39, a cell according to claim 40 or 41, or a composition according to any one of claims 42 to 44, for the manufacture of a pharmaceutical for treating cancer.

52. The use according to claim 51, wherein the cancer is a cancer characterized by the presence of a solid tumor, or a cancer selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer.

53. The use according to claim 52, wherein the lung cancer is non-small cell lung cancer (NSCLC).

54. The composition according to claim 47 or 48, comprising one or more additional therapeutic substances in combination.

55. The use according to claim 51, wherein the pharmaceutical product is used in combination with one or more further therapeutic substances.

56. The composition according to claim 54, wherein one or more further therapeutic substances are chemotherapeutic agents.

57. The use according to claim 55, wherein one or more further therapeutic substances are chemotherapeutic agents.

58. a. A step of culturing host cells that produce a first antibody comprising an antigen-binding region that binds to human CD137 as defined in any one of claims 1 and 7 to 12; b. A step of culturing host cells that produce a second antibody comprising an antigen-binding region that binds to human PD-L1 as defined in any one of claims 1 to 6; c. The step of incubating the first antibody together with the second antibody under conditions of sufficient reducing to allow cysteine ​​in the hinge region to undergo disulfide bond isomerization; and d. Steps to obtain CD137×PD-L1 bispecific antibodies A method for producing a bispecific antibody according to any one of claims 1 to 37, comprising:

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