Antibodies for use in therapy

Abstract

The present invention relates to a method for reducing or preventing tumor progression or treating cancer, comprising administering to a subject a binding agent comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1. The amount of binding agent administered in each treatment cycle is preferably about 0.3-5 mg / kg body weight, or about 25-400 mg in total.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods for reducing or preventing tumor progression or treating cancer by administering a binding agent comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1. [Background technology]

[0002] Background of the Invention CD137 (4-1BB, TNFRSF9) is a member of the tumor necrosis factor (TNF) receptor (TNFR) family. CD137 binds to CD8 + T cells and CD4 + It is a costimulatory molecule on the surface of T cells, regulatory T cells (Tregs), natural killer (NK) and NKT cells, B cells, and neutrophils. CD137 is not constitutively expressed on T cells but is induced upon T cell receptor (TCR) activation. Stimulation via its natural ligand, 4-1BBL, or agonist antibodies results in signaling using TNFR-associated factor (TRAF)-2 and TRAF-1 as adaptors. Initial signaling by CD137 involves a K-63 polyubiquitination reaction that ultimately activates the nuclear factor (NF)-κB and mitogen-activated protein (MAP)-kinase pathways. Signaling leads to increased T cell costimulation, proliferation, cytokine production, and maturation, and to CD8 +T cell survival is prolonged. Agonistic antibodies against CD137 have been shown to promote T cell-mediated anti-tumor control in various preclinical models (Murillo et al. 2008 Clin. Cancer Res. 14(21): 6895-6906 (Non-Patent Document 1)). Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including the human IgG4 antibody urelumab (WO2005035584 (Patent Document 1)) and the human IgG2 antibody utomilumab (Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733 (Non-Patent Document 2)).

[0003] Programmed cell death ligand 1 (PD-L1, PDL1, CD274, B7H1) is a 33 kDa single-pass 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-type domain and one Ig-like V-type domain. Freshly isolated T and B cells express only trace amounts of PD-L1, whereas CD14 + A small proportion of monocytes (approximately 16%) constitutively express PD-L1, however, interferon-γ (IFNγ) is known to upregulate PD-L1 on tumor cells.

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

[0005] PD-L1 blocking antibodies have shown clinical activity in several types of cancer known to overexpress PD-L1 (including melanoma and NSCLC). For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. Atezolizumab is currently undergoing clinical trials as an immunotherapy for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., 2017 Lancet 389:255-265), and has been approved for the treatment of non-small cell lung cancer and bladder cancer. The PD-L1 antibody avelumab (Kaufman et al., Lancet Oncol. 2016;17(10):1374-1385) has been approved by the FDA for the treatment of adult and pediatric patients aged 12 years or older with metastatic Merkel cell carcinoma and is undergoing clinical trials in several cancer indications, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, NSCLC, ovarian cancer, and kidney cancer. The PD-L1 antibody durvalumab has been approved for the treatment of locally advanced or metastatic urothelial cancer and is in clinical development in several solid tumors and hematological cancers (see, e.g., Massard et al., 2016 J Clin Oncol. 34(26):3119-25). Additional anti-PD-L1 antibodies are described, for example, in WO2004004771.

[0006] Horton et al. (J Immunother Cancer. 2015; 3(Suppl 2):O10) discloses the combination of an agonist 4-1BB antibody with a PD-L1 neutralizing antibody. WO2019 / 025545 (Patent Document 3) provides binding agents, such as a bispecific antibody that binds to human PD-L1 and human CD137.

[0007] However, despite these advances in the art, there is a great need for improved therapies that target PD-L1 and CD137. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2005035584 [Patent Document 2] WO2004004771 [Patent Document 3] WO2019 / 025545 [Non-patent literature]

[0009] [Non-Patent Document 1] Murillo et al. 2008 Clin. Cancer Res. 14(21): 6895-6906 [Non-patent document 2] Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733 [Non-patent document 3] Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6 [Non-patent document 4] Rittmeyer et al., 2017 Lancet 389:255-265 [Non-Patent Document 5] Kaufman et al Lancet Oncol. 2016;17(10):1374-1385 [Non-patent document 6] Massard et al., 2016 J Clin Oncol. 34(26):3119-25 [Non-Patent Document 7] Horton et al, J Immunother Cancer. 2015; 3(Suppl 2):O10 Summary of the Invention

[0010] It is an object of the present invention to provide a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject in at least one treatment cycle an appropriate amount of a binding agent comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1.

[0011] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) about 0.3 to 5 mg / kg body weight or about 25 to 400 mg in total; and / or b) Approximately 2.1×10 -9 ~3.4×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.7×10 -6 mol That's fine too.

[0012] A further object of the present invention is a composition comprising a binding agent comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1, wherein the amount of binding agent in the composition is about 25-400 mg or about 1.7 x 10 -7 ~2.7×10 -6 The object of the present invention is to provide a composition in which the [The present invention 1001] 1. A method for reducing or preventing tumor progression or treating cancer in a subject, comprising administering to the subject, in at least one treatment cycle, an appropriate amount of a binding agent comprising a first binding region that binds to human CD137, e.g., human CD137 having the sequence set forth in SEQ ID NO:24, and a second binding region that binds to human PD-L1, e.g., human PD-L1 having the sequence set forth in SEQ ID NO:26. [The present invention 1002] The method of claim 1001, wherein said amount of binding agent administered at each dose and / or treatment cycle results in proliferation, cytokine production, maturation, and long-term survival of T cells, and renders such T cells less susceptible to inhibition by PD-L1. [The present invention 1003] The amount of binding agent administered in each dose and / or treatment cycle is Within the range where more than 5%, preferably more than 10%, more preferably more than 15%, even more preferably more than 20%, even more preferably more than 25%, even more preferably more than 30%, even more preferably more than 35%, even more preferably more than 40%, even more preferably more than 45%, and most preferably more than 50% of the binding agent binds to both CD137 and PD-L1 Any of the methods of the present invention, wherein [The present invention 1004] The amount of binding agent administered at each dose and / or in each treatment cycle is a) about 0.3 to 5 mg / kg body weight or about 25 to 400 mg in total; and / or b) Approximately 2.1×10 -9 ~3.4×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.7×10 -6 mol Any of the methods of the present invention, [The present invention 1005] The amount of binding agent administered at each dose and / or in each treatment cycle is a) about 1.25 mg / kg body weight or about 100 mg in total; and / or b) Approximately 8.5×10 -9 mol / kg body weight or a total of approximately 6.8 × 10 -7 mol Any of the methods of the present invention, [The present invention 1006] Any of the aforementioned methods of the present invention, wherein the binding agent activates human CD137 upon binding to human CD137 and inhibits the binding of human PD-L1 to human PD-1 upon binding to PD-L1. [The present invention 1007] a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:1 and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:5; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:8, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:12; Any of the methods of the present invention. [The present invention 1008] a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3, and the CDR3 sequence shown in SEQ ID NO:4, and a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:6, the CDR2 sequence shown as GAS, and the CDR3 sequence shown in SEQ ID NO:7; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising 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 a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:13, the CDR2 sequence shown as DDN, and the CDR3 sequence shown in SEQ ID NO:14; Any of the methods of the present invention. [The present invention 1009] a) the first binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:5; and b) the second binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:8, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:12; Any of the methods of the present invention. [The present invention 1010] a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and b) the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:12; Any of the methods of the present invention. [The present invention 1011] Any of the aforementioned methods of the invention, wherein the binding agent is an antibody, a multispecific antibody, e.g., a bispecific antibody. [The present invention 1012] Any of the aforementioned methods of the invention, wherein the binding agent is in the form of a full-length antibody or an antibody fragment. [The present invention 1013] The method of any of claims 1007 to 1012, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4). [The present invention 1014] 1013. The method of claim 1013, wherein said complementarity determining regions and said framework regions are arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. [The present invention 1015] i) a polypeptide comprising, consisting of, or consisting essentially of a first heavy chain variable region (VH) and a first heavy chain constant region (CH); and ii) a polypeptide comprising, consisting of, or consisting essentially of a second heavy chain variable region (VH) and a second heavy chain constant region (CH). Any of the methods of the present invention, comprising: [The present invention 1016] i) a polypeptide comprising a first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) a polypeptide comprising a second light chain variable region (VL) and further comprising a second light chain constant region (CL). Any of the methods of the present invention, comprising: [The present invention 1017] the binding agent is an antibody comprising a first binding arm and a second binding arm, 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 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). Any of the methods of the present invention, comprising: [The present invention 1018] i) a first heavy chain and a first light chain comprising an antigen-binding region capable of binding to CD137; and ii) a second heavy chain and a second light chain comprising the antigen-binding region capable of binding to PD-L1; Any of the methods of the present invention, comprising: [The present invention 1019] The binding substance i) a first heavy chain and a first light chain comprising the antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a second light chain comprising the antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region. Any of the methods of the present invention, comprising: [The present invention 1020] Any of the methods of claims 1015 to 1019, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises one or more of a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region, preferably at least a hinge region, a CH2 region, and a CH3 region. [The present invention 1021] Any of the methods of claims 1015 to 1020, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) each comprise a CH3 region, and the two CH3 regions comprise asymmetric mutations. [The present invention 1022] Any of the methods of present inventions 1015 to 1021, wherein in the first heavy chain constant region (CH), at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain according to EU numbering, and in the second heavy chain constant region (CH), at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of the human IgG1 heavy chain according to EU numbering, and wherein the first heavy chain and the second heavy chain do not have substitutions at the same positions. [The present invention 1023] The method of invention 1022, wherein (i) the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L in said second heavy chain. [The present invention 1024] Any of the aforementioned methods of the invention, wherein the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions. [The present invention 1025] The method of claim 1024, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) have been modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising unmodified first and second heavy chain constant regions (CH). [The present invention 1026] 1025. The method of claim 1025, wherein each of said unmodified first heavy chain constant region (CH) and second heavy chain constant region (CH) comprises the amino acid sequence set forth in SEQ ID NO: 15. [The present invention 1027] The method of any of claims 1025 to 1026, wherein said Fc-mediated effector function is measured by binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. [The present invention 1028] 1027. The method of claim 1027, wherein said Fc-mediated effector function is measured by binding to C1q. [The present invention 1029] the first heavy chain constant region and the second heavy chain constant region are modified such that binding of the antibody to C1q is reduced 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%; C1q binding is preferably measured by ELISA. Any of the methods of 1024 to 1028 of the present invention. [The present invention 1030] Any of the aforementioned methods 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 positions L234, L235, D265, N297, and P331 of a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. [The present invention 1031] 1030. The method of claim 1030, wherein the positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering are F and E in said first and second heavy chains, respectively. [The present invention 1032] 1032. The method of claim 1030 or 1031, wherein the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A in said first heavy chain constant region and second heavy chain constant region (HC), respectively. [The present invention 1033] the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F and E, respectively; and (i) the first heavy chain constant region has L at a position corresponding to F405 of the human IgG1 heavy chain according to EU numbering, and R at a position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain; or (ii) the first heavy chain constant region has R at a position corresponding to K409 of the human IgG1 heavy chain according to EU numbering, and the second heavy chain has L at a position corresponding to F405 of the human IgG1 heavy chain according to EU numbering; Any of the methods 1030 to 1032 of the present invention. [The present invention 1034] the positions corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively; and (i) the first heavy chain constant region has L at a position corresponding to F405 of the human IgG1 heavy chain according to EU numbering, and R at a position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region; or (ii) the first heavy chain has R at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering, and the second heavy chain has L at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering; Any of the methods 1030 to 1033 of the present invention. [This invention 1035] the constant region of the first heavy chain and / or the second heavy chain comprises: a) the sequence shown in SEQ ID NO:15 or SEQ ID NO:30 [IgG1-FC]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution. 1035. The method of any of claims 1015 to 1034, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: [The present invention 1036] the constant region of the first heavy chain or the second heavy chain, e.g., the second heavy chain, comprises: a) the sequence shown in SEQ ID NO: 16 or SEQ ID NO: 31 [IgG1-F405L]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 9 substitutions compared to the amino acid sequence defined in a) or b), for example at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution. 1036. The method of any of claims 1015 to 1035, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: [This invention 1037] the constant region of the first heavy chain or the second heavy chain, e.g., the first heavy chain, comprises: a) the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 32 [IgG1-F409R]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. 1037. The method of any of claims 1015 to 1036, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: [The present invention 1038] the constant region of the first heavy chain and / or the second heavy chain comprises: a) the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 33 [IgG1-Fc_FEA]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 7 substitutions compared to the amino acid sequence defined in a) or b), for example at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. 1038. The method of any of claims 1015 to 1037, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: [This invention 1039] the constant region of the first heavy chain and / or the second heavy chain, e.g., the second heavy chain, comprises: a) the sequence shown in SEQ ID NO: 19 or SEQ ID NO: 34 [IgG1-Fc_FEAL]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions compared to the amino acid sequence defined in a) or b), for example at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. 1038. The method of any of claims 1015 to 1038, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: [The present invention 1040] the constant region of the first heavy chain and / or the second heavy chain, e.g., the first heavy chain, comprises: a) the sequence shown in SEQ ID NO:20 or SEQ ID NO:35 [IgG1-Fc_FEAR]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions compared to the amino acid sequence defined in a) or b), for example at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. 1039. The method of any of claims 1015 to 1039, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of: [The present invention 1041] Any of the aforementioned methods of the invention, wherein the binding agent comprises a kappa (κ) light chain constant region. [The present invention 1042] Any of the aforementioned methods of the present invention, wherein the binding agent comprises a lambda (λ) light chain constant region. [This invention 1043] Any of the aforementioned methods of the present invention, wherein said first light chain constant region is a kappa (κ) light chain constant region. [The present invention 1044] Any of the aforementioned methods of the present invention, wherein said second light chain constant region is a lambda (λ) light chain constant region. [This invention 1045] Any of the aforementioned methods of the present invention, wherein said first light chain constant region is a lambda (λ) light chain constant region. [The present invention 1046] Any of the aforementioned methods of the present invention, wherein said second light chain constant region is a kappa (κ) light chain constant region. [This invention 1047] the kappa (κ) light chain a) the sequence shown in SEQ ID NO:21; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. 1047. The method of any one of claims 1041 to 1046, comprising an amino acid sequence selected from the group consisting of: [This invention 1048] the lambda (λ) light chain a) the sequence shown in SEQ ID NO:22; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The method of any of claims 1042 to 1047, comprising an amino acid sequence selected from the group consisting of: [This invention 1049] Any of the aforementioned methods of the present invention, wherein the binding agent is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [The present invention 1050] Any of the aforementioned methods of the present invention, wherein the binding agent is a full-length IgG1 antibody. [This invention 1051] Any of the methods of the present invention, wherein the antibody is an antibody of the IgG1m(f) allotype. [This invention 1052] Any of the aforementioned methods of the present invention, wherein the subject is a human subject. [This invention 1053] Any of the aforementioned methods of the present invention, wherein the tumor or cancer is a solid tumor. [This invention 1054] Any of the aforementioned methods of the present invention, wherein the tumor or cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic cancer, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, ovarian cancer, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma. [This invention 1055] Any of the aforementioned methods of the present invention, wherein the tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial carcinoma (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx), and cervical cancer. [This invention 1056] Any of the aforementioned methods of the present invention, wherein the tumor or cancer is lung cancer. [This invention 1057] 1056. The method of claim 1056, wherein said lung cancer is non-small cell lung cancer (NSCLC), e.g., squamous or non-squamous NSCLC. [This invention 1058] 1057. The method of claim 1057, wherein said NSCLC does not have an epidermal growth factor (EGFR) sensitivity-increasing mutation and / or an anaplastic lymphoma (ALK) translocation / ROS1 rearrangement. [This invention 1059] Any of the methods of claims 1056 to 1058, wherein the subject has received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment. [The present invention 1060] The method of claim 1059, wherein said subject has undergone platinum-based chemotherapy. [This invention 1061] The method of claim 1059, wherein said subject is ineligible for platinum-based therapy and is undergoing treatment with another chemotherapy, for example, a gemcitabine-containing regimen. [This invention 1062] Any of the aforementioned methods of the present invention, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1063] Any of the aforementioned methods of the invention, wherein the subject has experienced disease progression during or after treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1064] Any of the aforementioned methods of the invention, wherein the subject has experienced disease progression during or after their last prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1065] The method of any of claims 1059 to 1064, wherein said subject has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment. [The present invention 1066] Any of the aforementioned methods of the present invention, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1067] Any of the aforementioned methods of the present invention, wherein the tumor or cancer is endometrial cancer. [The present invention 1068] 1068. The method of claim 1067, wherein said subject has epithelial endometrial tissue architecture, including endometrioid carcinoma, serous carcinoma, squamous cell carcinoma, clear cell carcinoma, or carcinosarcoma. [The present invention 1069] The method of any one of claims 1067 to 1068, wherein the subject has received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment. [The present invention 1070] Any of the methods of claims 1067 to 1069, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent targeting PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1071] Any of the methods of the preceding invention, wherein the tumor or cancer is urothelial cancer, including cancer of the bladder, ureter, urethra, or renal pelvis. [This invention 1072] The method of claim 1071, wherein the subject has received up to four systemic conditioning regimens for advanced / metastatic disease and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment. [This invention 1073] The method of any one of claims 1071 to 1072, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1074] 1073. The method of claim 1071 or 1072, wherein said subject has undergone platinum-based chemotherapy. [This invention 1075] The method of any of claims 1071 or 1072, wherein said subject is ineligible for platinum-based therapy and has been treated with another chemotherapy, for example, a gemcitabine-containing regimen. [This invention 1076] Any of the methods of the present invention, wherein the tumor or cancer is breast cancer, e.g., triple-negative breast cancer (TNBC). [This invention 1077] The method of claim 1076, wherein the TNBC is HER2-negative, e.g., HER2-negative as determined by measuring protein expression by fluorescent in situ hybridization (FISH) or immunohistochemistry, progesterone receptor-negative, and estrogen receptor-negative. [This invention 1078] The method of any one of claims 1076 to 1077, wherein the subject has received at least one systemic conditioning regimen for locally advanced / metastatic disease, for example, at least one systemic conditioning regimen including an anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimen. [This invention 1079] The method of claim 1078, wherein the subject has received up to four systemic conditioning regimens for locally advanced / metastatic disease, including at least one systemic conditioning regimen comprising an anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimen. [The present invention 1080] Any of the methods of claims 1076 to 1079, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1081] The method of claim 1080, wherein said subject has experienced disease progression, e.g., radiographically confirmed disease progression, during or after said pretreatment with a checkpoint inhibitor. [This invention 1082] Any of the methods of claims 1076 to 1079, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent targeting PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1083] Any of the methods of the present invention, wherein the tumor or cancer is head and neck cancer, e.g., squamous cell carcinoma of the head and neck (SCCHN). [This invention 1084] The method of claim 1083, wherein said tumor or cancer is recurrent or metastatic SCCHN. [This invention 1085] 108. The method of claim 1083 or 1084, wherein said tumor or cancer is a cancer of the oral cavity, pharynx, or larynx. [The present invention 1086] Any of the methods of claims 1083 to 1085, wherein the subject has received up to four systemic conditioning regimens for recurrent / metastatic disease and experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment. [This invention 1087] The method of claim 1086, wherein said subject has undergone platinum-based chemotherapy. [This invention 1088] The method of claim 1086, wherein said subject is ineligible for platinum-based therapy and is receiving another chemotherapy. [This invention 1089] Any of the methods of claims 1083 to 1088, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [The present invention 1090] The method of claim 1089, wherein said subject has experienced disease progression, e.g., radiographically confirmed disease progression, during or after said pretreatment with a checkpoint inhibitor. [This invention 1091] Any of the methods of claims 1083 to 1088, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent targeting PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1092] Any of the methods of the present invention, wherein the tumor or cancer is cervical cancer. [This invention 1093] 1092. The method of claim 1092, wherein said cervical cancer is squamous cell, adenocarcinoma, or adenosquamous histology cervical cancer. [This invention 1094] The method of claim 1092 or 1093, wherein the subject has received at least one systemic conditioning regimen for recurrent / metastatic disease, e.g., chemotherapy in combination with a treatment targeting vascular endothelial growth factor A, e.g., treatment with bevacizumab, and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment. [This invention 1095] The method of claim 1094, wherein the subject has received up to four systemic conditioning regimens for recurrent / metastatic disease, including chemotherapy in combination with a vascular endothelial growth factor A-targeted treatment, e.g., treatment with bevacizumab. [This invention 1096] The method of any one of claims 1092 to 1093, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. [This invention 1097] Any of the aforementioned methods of the present invention, wherein the binding agent is administered by systemic administration. [This invention 1098] Any of the aforementioned methods of the present invention, wherein the binding agent is administered by intravenous injection or infusion. [This invention 1099] Any of the aforementioned methods of the present invention, wherein each treatment cycle is 3 weeks (21 days). [The present invention 1100] Any of the aforementioned methods of the present invention, wherein one dose is administered every three weeks (1Q3W). [The present invention 1101] Any of the aforementioned methods of the present invention, wherein one dose is administered on day 1 of each treatment cycle. [The present invention 1102] Any of the methods of the invention described above, wherein each dose is infused over a minimum of 30 minutes, e.g., at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 240 minutes. [The present invention 1103] A composition comprising a binding substance comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1, wherein the amount of the binding substance in the composition is 25 to 400 mg or 1.7 × 10 -7 ~2.7×10 -6 mol. [The present invention 1104] 1103. The composition of claim 1103, comprising about 80 mg of said binding agent. [This invention 1105] The composition of any one of claims 1103 to 1104, wherein the binding substance is a binding substance defined in any one of claims 1001 to 1102. [The present invention 1106] The composition of any one of 1103 to 1105 of the present invention, which is for systemic administration. [This invention 1107] The composition of any one of 1103 to 1106 of the present invention, which is for injection or infusion, for example, for intravenous injection or infusion. [This invention 1108] 8. The composition of any of claims 1103 to 1107, wherein said binding substance is present in a volume of 50 to 500 mL, for example 100 to 250 mL, of an aqueous solution, for example 0.9% NaCl (saline). [This invention 1109] Any one of the compositions of 1103 to 1108 of the present invention, which is in unit dosage form. [Brief explanation of the drawings]

[0013] [Figure 1]Co-binding of GEN1046 to PD-L1-expressing K562 cells and CD137-expressing K562 cells induces doublet formation and a bell-shaped dose-response curve. Equal numbers of CellTrace™ Far Red-labeled K562 cells transfected with the CD137 gene (K562_h4-1BB) were co-incubated with CellTrace™ Violet-labeled K562 cells transfected with the PD-L1 gene (K562_hPD-L1) for 15 minutes in the presence of 0.001-100μg / mL of i) GEN1046 or ii) the control antibody combinations PD-L1-547-FEALxb12-FEAR and b12-FEALxCD137-009-HC7LC2-FEAR. Samples were analyzed by flow cytometry, and the percent CellTrace™ FarRed / CellTrace™ Violet double-positive doublets (A) were plotted as a function of GEN1046 concentration (B). Data shown are the mean ± standard deviation of n=3 technical replicates (symbols may need to be smaller to show SD). [Figure 2] Schematic diagram of the predicted mechanism of action of the CD137xPD-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 neutralizes T cell activation signals, ultimately resulting in T cell inhibition. (B) Addition of the CD137xPD-L1 bispecific antibody blocks the inhibitory PD-1:PD-L1 interaction via the PD-L1-specific arm. At the same time, the bispecific antibody mediates agonistic signaling to CD137 expressed on T cells through cell-cell interactions, resulting in potent T cell costimulation. [Figure 3]Relative luminescence units (RLU) as a function of antibody concentration in a luciferase-based CD137 activation reporter assay performed in the presence of PD-L1-expressing tumor cell lines. The human ovarian cancer cell line ES-2 (A) and breast cancer cell line MDA-MB-231 (B), which endogenously express PD-L1, were co-cultured with NFkB-Luc2P / 4-1BB Jurkat reporter cells in the presence of 0.00128–100 μg / mL of i) GEN1046 or ii) b12-FEAL control antibody for 6 hours. Induction of luciferase expression was determined by incubation with luciferase substrate and measurement of relative luminescence units. Data shown are the mean ± standard deviation of n=3 technical replicates. [Figure 4] Comparison of GEN1046 with control antibodies PD-L1-547-FEALxb12-FEAL or IgG1-b12-FEAL in a polyclonal T cell proliferation assay. CFSE-labeled PBMCs were incubated with a suboptimal concentration of anti-CD3 antibody (0.03 μg / mL) and cultured for 4 days in the presence of 0.0032–10 μg / mL of i) GEN1046, ii) PD-L1-547-FEALxb12-FEAL, or iii) b12-FEAL control antibody. T cell proliferation of total T cells (A) and CCR7+CD45RO+ central memory and CCR7-CD45RO+ effector memory T cell subsets among total T cells (B) was measured by flow cytometry. Data are presented as the mean expansion index of two replicates from one representative donor, calculated using FlowJo v10.4 software. Error bars (SD) indicate the variability of this experiment (two replicates using cells from one donor). [Figure 5]GEN1046 reversed PD-1 / PD-L1-mediated T cell inhibition and further costimulated CD8+ T cell proliferation in an antigen-specific T cell assay using an activated PD-1 / PD-L1 axis. CD8+ T cells were electroporated with 10 μg each of RNA encoding the α and β chains of the CLDN6-specific TCR, along with or without PD-1-encoding RNA (w / o PD-1), and were cocultured with CFSE-labeled immature DCs electroporated with 0.3 μg (A) or 1 μg (B) of CLDN6-encoding RNA. Electroporated CD8+ T cells and iDCs were cocultured for 4 days in the presence of GEN1046 (0.00015–1 μg / mL) or b12-FEAL (1 μg / mL). T cell proliferation was assessed by analyzing the CFSE dilution of CD8+ T cells using flow cytometry, and the T cell expansion index (e.g., the degree to which the total T cell population expanded by proliferation) was automatically calculated by FlowJo (version 10.3). Data shown are the mean ± SD of the expansion index of triplicate wells from one donor out of four donors included in two experiments. [Figure 6]The effect of GEN1046 on the secretion of inflammatory cytokines (IFNγ, TNFα, IL-13, and IL-8) in an antigen-specific T cell assay was investigated with or without PD-1 introduced into T cells by electroporation. CD8+ T cells were electroporated with RNA encoding the α and β chains of the CLDN6-specific TCR (10 μg each), together with or without PD-1-encoding RNA (2 μg), labeled with CFSE, and cocultured with immature DCs electroporated with 1 μg of CLDN6-encoding RNA. Electroporated CD8+ T cells and iDCs were cocultured in the presence of GEN1046 (0.00015–1 μg / mL) or b12-FEAL (1 μg / mL). Forty-eight hours after antibody addition, supernatant cytokine levels were determined by multiplex sandwich immunoassay using the MSD V-Plex Human Proinflammatory panel 1 (10-Plex) kit. Data shown are the mean ± SD of concentrations from six replicate wells from one representative donor of the two donors included in this experiment. [Figure 7] Ex vivo expansion of tumor-infiltrating lymphocytes (TILs) derived from resected human non-small cell lung cancer tissue by CD137-009-FEALxPD-L1-547-FEAR. Tumor fragments derived from the resected tissue were cultured with 10 U / mL IL-2 and the indicated concentrations of CD137-009-FEALxPD-L1-547-FEAR. After 10 days of culture, cells were harvested and analyzed by flow cytometry. (A) Number of TILs per 1,000 beads; (B) Number of CD3+CD8+ T cells per 1,000 beads; (C) Number of CD3+CD4+ T cells per 1,000 beads; (D) Number of CD3-CD56+ NK cells per 1,000 beads. Data are the mean cell counts ± SD of five individual wells, using two tumor fragments per well as starting material. *p<0.05 using ordinary one-way ANOVA with Dunnett's multiple comparison test. [Figure 8] Schematic diagram of clinical trial design. [Figure 9]Dose escalation; best percent change in tumor size from baseline, all patients. Data cutoff: September 29, 2020. Five patients did not have post-baseline scans. aMinimum duration of response (5 weeks) per RECIST v1.1 was not reached. bPR not confirmed on subsequent scan. NE, not evaluable; NSCLC, non-small cell lung cancer; PD, progressive; PD-(L)1, programmed cell death (ligand) 1; PR, partial response; SD, stable disease; SoD, sum of diameters; uPR, unconfirmed partial response. [Figure 10] Dose escalation; best change in tumor size from baseline, NSCLC patients. Data cutoff: September 29, 2020. aPR not confirmed on subsequent scan. bPD-L1 expression was assessed in archival tumor specimens. BOR, best overall response; CR, complete response; ICI, immune checkpoint inhibitor; NA, not available; PD, progressive; PD-(L)1, programmed cell death (ligand) 1; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumor; SD, stable disease; SoD, sum of diameters; TPS, tumor proportion score; uPR, unconfirmed partial response. [Figure 11]Expansion Cohort 1: A) Best change in tumor size from baseline, B) Change in target lesion SoD from baseline. Data cutoff: October 12, 2020. *Indicates patients with ongoing treatment. aPR was not confirmed on subsequent scans. bPD-L1 expression was assessed in tumor biopsies obtained before the start of GEN1046 treatment (22C3 pharmDx assay, HistoGeneX, Belgium). All patients who underwent at least one post-baseline tumor assessment (schedule every 6 weeks) and were therefore evaluable for clinical benefit were included. Six of 12 patients are still receiving treatment. Of the remaining 12 patients not shown, three patients had clinical progression before the first response assessment, and nine patients are still receiving treatment and never had their first response assessment. BOR and time point response were assessed using RECIST 1.1; NA: assessment after first PD. BOR, best overall response; ICI, immune checkpoint inhibitor; NA, not available; NE, not evaluable; NSCLC, non-small cell lung cancer; PD, progressive disease; PD-(L)1, programmed cell death (ligand) 1; PR, partial response; RECIST, Response Evaluation Criteria in Solid Tumors; SD, stable disease; SoD, sum of diameters; TPS, tumor proportion score; uPR, unconfirmed partial response. [Figure 12] Model-predicted maximum trimer formation and receptor occupancy for PD-L1 when administered at a 100 mg dose once every three weeks (1Q3W). [Figure 13]A-D: Pharmacodynamic evaluations, including changes in circulating levels of interferon-γ (IFN-γ) and interferon-γ-inducible protein 10 (IP-10) (A-B), proliferating effector memory CD8 T cells, and total CD8 T cells (C-D), were performed using blood samples from patients with advanced solid tumors enrolled in the dose-escalation phase of an open-label, multicenter safety study of GEN1046 (NCT03917381; data cutoff: January 19, 2021). A-B. Circulating levels of IFN-γ and IP-10 in serum samples were measured at baseline and at multiple time points after GEN1046 administration in Cycle 1 and Cycle 2 (Day 1 [2 hours and 4-6 hours post-dose], Day 2, Day 3, Day 8, and Day 15). IFN-γ and IP-10 levels in serum samples were determined by Meso Scale Discovery (MSD) multiplex immunoassay. Data shown are the maximum fold change from baseline measured during Cycle 1. Statistical analysis was performed using the Wilcoxon-Mann-Whitney test. C-D. Peripheral blood immunophenotyping was performed on whole blood collected at baseline and at multiple time points (days 2, 3, 8, and 15) after GEN1046 administration in cycles 1 and 2. The frequency of proliferating (Ki67+) total CD8 T cells and effector memory CD8 T cells (CD8+CD45RA-CCR7- T cells) in whole blood samples was assessed by flow cytometry. Data shown are the maximum fold change from baseline measured during cycle 1. Statistical analysis was performed using the Wilcoxon-Mann-Whitney test. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description of the Invention definition In the context of the present invention, the term "binding agent" refers to any agent capable of binding to a desired antigen. In certain embodiments of the present invention, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include synthetic, modified, or non-natural moieties, particularly non-peptide moieties. Such moieties may, for example, link the desired antigen-binding functionality or antigen-binding region, e.g., an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0015] 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 (L) chains and one pair of heavy (H) chains, with all four chains interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically comprises a heavy chain variable region (herein referred to as V H or VH) and a heavy chain constant region (herein referred to as C H The heavy chain constant region is typically composed 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 flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically contains a light chain variable region (herein referred to as V L or VL) and a light chain constant region (herein referred to as C Lor CL). The light chain constant region is typically composed of one domain, CL. The VH and VL regions may be further divided into hypervariable regions (or hypervariable regions that are hypervariable in sequence and / or can be in the form of structure-defined loops), also called complementarity-determining regions (CDRs), interspersed with conserved 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 carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196 , 901-917 (1987)). Unless otherwise specified or contradicted by context, CDR sequences herein are identified using DomainGapAlign according to 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); see also Internet http address www.imgt.org / . (See also ). Unless otherwise specified or contradicted by the context, the amino acid positions of the constant region in the present invention are described according to 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, SEQ ID NO:93 herein denotes amino acid positions 118 to 447 of the IgG1m(f) heavy chain constant region according to EU numbering.

[0016] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and should not be understood as limiting. Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups and a side chain (R group) that is unique to each amino acid. In the context of the present invention, amino acids may be classified based on their structure and chemical characteristics. Accordingly, amino acid classes may be reflected in one or both of the following tables:

[0017] Table 1. Major classifications based on the structure and general chemical characterization of the R group. TIFF0007809062000001.tif52128

[0018] Table 2. Alternative physical and functional classifications of amino acid residues TIFF0007809062000002.tif88128

[0019] Substitution of one amino acid for another may be classified as conservative or non-conservative substitution. In the context of the present invention, a "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical characteristics. Such substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above: for example, leucine may be substituted with isoleucine, since both leucine and isoleucine are aliphatic branched hydrophobic substances. Similarly, aspartic acid may be substituted with glutamic acid, since both aspartic acid and glutamic acid are small, negatively charged residues.

[0020] As used herein, the term "amino acid corresponding to position ..." refers to the amino acid position number of the human IgG1 heavy chain. 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 the like, typically using default settings, and that has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Techniques for aligning sequences or segments within a sequence, thereby determining positions within a sequence that correspond to amino acid positions according to the present invention, are considered well known in the art.

[0021] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with 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, 4 days, 5 days, 6 days, 7 days, or more, or any other relevant, functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody-antigen binding and / or a period sufficient for the antibody to enhance effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with an antigen. As used herein, the term "antigen-binding region" refers to the region that interacts with an antigen and includes both the VH and VL regions. The term "antibody" as used herein includes not only monospecific antibodies but also multispecific antibodies that contain multiple, e.g., two or more, e.g., 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 host tissues or host factors, including complement system components, such as C1q, the first component of the classical pathway of complement activation. As mentioned above, the term "antibody" as used herein includes antigen-binding fragments, i.e., antibody fragments that retain the ability to specifically bind to antigens, unless otherwise specified or clearly contradicted by the context. 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 encompassed within the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or the monovalent antibodies described in WO2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments in which two Fab fragments are linked by a disulfide bridge at the hinge region; (iii) Fd fragments, consisting essentially of the VH and CH1 domains; (iv) Fv fragments, consisting essentially of the VL and VH domains of a single arm of an antibody; and (v) domain antibodies, consisting essentially of a VH domain (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90), also known as dAb fragments (Ward et al., Nature 341 , 544-546(1989));(vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an 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)). See, e.g., Bird et al., Science 242 , 423-426(1988) and Huston et al., PNAS USA 85The fragments may be joined using recombinant techniques by synthetic linkers that allow them to be produced as a single protein chain forming a single chain (see, e.g., J. Immunol., 5879-5883 (1988)). Such single-chain antibodies are included within the term antibody unless otherwise specified or clearly indicated by context. While such fragments are generally included within the meaning of antibody, they are a unique feature of the present invention, collectively and individually exhibiting distinct biological properties and utilities. These and other useful antibody fragments and bispecific forms of such fragments in the context of the present invention are further discussed herein. The term antibody, unless otherwise specified, should also be understood 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 recombinant techniques. The antibodies produced may have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the sequence of the antibody is more similar to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibodies of the invention may be sequence variants of native IgG1 antibodies that include altered constant regions.

[0022] In the context of the present invention, the term "bispecific antibody" or "bs" refers to an antibody having two different antigen-binding regions defined by different antibody sequences. In some embodiments, the different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding regions bind to different target antigens. The bispecific antibody may be any format of bispecific antibody, including any of the bispecific antibody formats described herein below.

[0023] The term "full-length," when used in the context of an antibody, indicates that the antibody is not a fragment and contains all of the domains of a particular isotype that are normally found in nature for that isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains for an IgG1 antibody. In some embodiments, the term "full-length," when used in the context of an antibody, refers to an antibody (e.g., a parent antibody or a variant antibody) that contains one or two pairs of heavy and light chains normally found in a heavy-light chain pair in a wild-type antibody of that isotype, with each chain containing all of the heavy and light chain constant and variable domains. In full-length antibodies, the heavy and light chain constant and variable domains may contain amino acid substitutions that improve the functional properties of the antibody compared to the full-length parent antibody or wild-type antibody. These amino acid substitutions include substitutions aimed at reducing antibody effector function and substitutions that facilitate assembly of multispecific antibodies, e.g., bispecific antibodies. Full-length antibodies according to the invention can be produced by a method comprising (i) cloning the CDR sequences into a suitable vector containing the complete heavy and light chain sequences, and (ii) expressing the complete heavy and light chain sequences in a suitable expression system. It is within the knowledge of one skilled in the art to produce full-length antibodies when starting from either the CDR sequences or the complete variable region sequences.

[0024] As used herein, the term "human antibody" is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and a human immunoglobulin constant domain. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0025] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be accomplished by joining the six non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site with homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). To fully reproduce the binding affinity and specificity of the parent antibody, it may be necessary to substitute (backmutate) framework residues from the parent antibody (i.e., non-human antibody) into the human framework regions. Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications may be applied, although the modifications are not necessarily back mutations, to obtain humanized antibodies with favorable characteristics, such as affinity and biochemical properties.

[0026] As used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least a hinge region, a CH2 domain, and a CH3 domain.

[0027] As used herein, the term "Fc region" refers to the region of an antibody that, from the N-terminus to the C-terminus, comprises at least the hinge, CH2, and CH3 regions. The Fc region of an antibody may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0028] As used herein, the term "hinge region" refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to the EU numbering system set forth in Kabat (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region may also refer to any hinge region of the other subtypes described herein.

[0029] As used herein, the term "CH1 region" or "CH1 domain" refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the Eu numbering system set forth in Kabat (ibid.). However, the CH1 region may also be the CH1 region of any of the other subtypes described herein.

[0030] As used herein, the term "CH2 region" or "CH2 domain" refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the Eu numbering system set forth in Kabat (ibid.). However, the CH2 region may also be the CH2 region of any of the other subtypes described herein.

[0031] As used herein, the term "CH3 region" or "CH3 domain" refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the Eu numbering system set forth in Kabat (ibid.). However, the CH3 region may also be the CH3 region of any of the other subtypes described herein.

[0032] As used herein, the terms "binding" or "capable of binding" in the context of antibody binding to a given antigen or epitope typically refer to binding of an antibody to a given antigen or epitope with a binding affinity of about 10% or more when measured using biolayer interferometry (BLI) or, for example, when measured using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less K D The antibody binds with an affinity corresponding to a K for binding to a non-specific antigen other than the designated antigen or a closely related antigen (e.g., BSA, casein). D K that is at least 1 / 10, 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 The amount of increased affinity corresponds to the antibody's K D As a result, the K Dis very low (i.e., the antibody is highly specific), the affinity for the antigen may be at least 10,000-fold less than the affinity for a non-specific antigen.

[0033] As used herein, "k d " (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is k off Also called value.

[0034] As used herein, "K D The term "" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0035] The term "PD-L1," as used herein, refers to the programmed death-ligand 1 protein. PD-L1 is found in humans and other species, and therefore, the term "PD-L1" is not limited to human PD-L1 unless contradicted by context. The human PD-L1 sequence can be found by Genbank accession number NP_054862.1. The sequence of human PD-L1 is also set forth in SEQ ID NO:25, in which amino acids 1-18 are predicted to be a signal peptide. The mature polypeptide sequence is set forth in SEQ ID NO:26.

[0036] The term "PD-1," as used herein, refers to the human programmed cell death-1 protein, also known as CD279 (UniProtKB Q15116).

[0037] The term "programmed cell death-1 (PD-1) pathway" or "PD-1 pathway" refers to a molecular signaling pathway involving the cell surface receptor PD-1 and its ligands PD-L1 and PD-L2. Activation of this pathway induces immune tolerance, whereas inhibition can relieve T cell suppression, thereby leading to immune activation.

[0038] As used herein, the term "CD137" refers to the human cluster of differentiation 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. Human CD137 has the UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO:23, with amino acids 1-23 predicted to be a signal peptide. The mature sequence of human CD137 is shown in SEQ ID NO:24.

[0039] A "treatment cycle" is defined herein as the period of time during which the effects of separate doses of a binding agent add up due to their pharmacokinetics, or in other words, the period after which the subject's body is essentially cleared or eliminated from the administered binding agent. Within a short time window, e.g., within a short 2-24 hour period, e.g., within a 2-12 hour period, or multiple smaller doses on the same day may be equivalent to a larger single dose.

[0040] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions × 100). The percent identity between two nucleotide 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), incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)).

[0041] In the context of the present invention, the following notation is used to describe mutations, unless otherwise specified: i) A substitution of an amino acid at a certain position is written, for example, K409R, which means that lysine at position 409 of the protein is substituted with arginine. ii) For certain variants, a specific three-letter or one-letter notation is used, with the symbols Xaa and X indicating any amino acid residue. Thus, a substitution of lysine at position 409 with arginine is indicated by K409R, and a substitution of lysine at position 409 with any amino acid residue is indicated by K409X. In the case of a deletion of lysine at position 409, this is indicated by K409*.

[0042] In the context of the present invention, "inhibition of binding of PD-L1 to PD-1" refers to a detectably significant reduction in the binding of PD-L1 to PD-1 in the presence of an antibody capable of binding to PD-L1. Typically, inhibition refers to at least about a 10% reduction in the binding between PD-L1 and PD-1 caused by the presence of an anti-PD-L1 antibody, such as at least about a 15%, such as at least about a 20%, such as at least a 40% reduction. Inhibition of binding of PD-L1 to PD-1 can be determined by any suitable technique. In one embodiment, inhibition is determined as described in Example 6 of WO2019 / 025545.

[0043] The term "treatment" refers to the administration of an effective amount of a therapeutically active antibody of the invention with the intent to alleviate, ameliorate, arrest or eradicate (cure) the symptoms or disease state.

[0044] Resistance to treatment with the binding substance of the present invention, failure to respond to treatment with the binding substance of the present invention, and / or recurrence from treatment with the binding substance of the present invention can be assessed according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST criteria v1.1). The RECIST criteria are shown in the table below.

[0045] Table 3. Definition of response (RECIST v1.1) TIFF0007809062000003.tif159161

[0046] "Best overall response" is the best response recorded from the start of treatment until disease progression / relapse (the smallest measurement recorded since treatment began is used as the criterion for PD). Subjects with a CR or PR are considered to have an objective response. Subjects with a CR, PR, or SD are considered to have controlled disease. Subjects with NE are counted as non-responders. "Best overall response" is the best response recorded from the start of treatment until disease progression / relapse (the smallest measurement recorded since treatment began is used as the criterion for PD). Subjects with a CR, PR, or SD are considered to have controlled disease. Subjects with NE are counted as non-responders.

[0047] "Duration of response (DOR)" is defined as the time from the first documented objective tumor response (CR or PR) to the date of first PD or death from the underlying cancer, with the confirmed best overall response being CR or PR.

[0048] "Progression-free survival (PFS)" is defined as the number of days from Day 1 of Cycle 1 to first documented progression or death from any cause.

[0049] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 to death from any cause. If the subject is not known to have died, OS is adjusted to the last day the subject was known to be alive (on or before the cutoff date).

[0050] In the context of the present invention, the term "treatment regimen" refers to a systematic treatment plan designed to improve and maintain health.

[0051] In a first aspect, the present invention provides a method for reducing or preventing tumor progression or treating cancer in a subject, the method comprising administering to the subject, in at least one treatment cycle, an appropriate amount of a binding agent comprising a first binding region that binds to human CD137, e.g., human CD137 having the sequence set forth in SEQ ID NO:24, and a second binding region that binds to human PD-L1, e.g., human PD-L1 having the sequence set forth in SEQ ID NO:26.

[0052] Preferably, the amount of binding agent administered at each dose and / or treatment cycle results in the proliferation, cytokine production, maturation, and long-term survival of T cells, and renders such T cells less susceptible to inhibition by PD-L1.

[0053] The amount of binding agent administered at each dose and / or treatment cycle may in particular be within a range where more than 5%, preferably more than 10%, more preferably more than 15%, even more preferably more than 20%, even more preferably more than 25%, even more preferably more than 30%, even more preferably more than 35%, even more preferably more than 40%, even more preferably more than 45%, and most preferably more than 50% of the binding agent binds to both CD137 and PD-L1.

[0054] In a currently preferred embodiment, the amount of binding agent administered at each dose and / or in each treatment cycle is: a) about 0.3 to 5 mg / kg body weight or about 25 to 400 mg in total; and / or b) Approximately 2.1×10 -9 ~3.4×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.7×10 -6 mol is.

[0055] According to these embodiments, a dose defined in mg / kg can be converted to a fixed dose based on the median body weight of subjects to whom the binding agent is administered being 80 kg, and vice versa.

[0056] The amount of binding agent administered at each dose and / or in each treatment cycle may, inter alia, be about 0.3 to 4.0 mg / kg body weight or about 25 to 320 mg in total; and / or Approximately 2.1×10 -9 ~2.7×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.2×10 -6 mol; about 0.38 to 4.0 mg / kg body weight or about 30 to 320 mg in total; and / or Approximately 2.6×10 -9 ~2.7×10 -8 mol / kg body weight or a total of approximately 2.4 × 10 -7 ~2.2×10 -6 mol; about 0.5 to 3.3 mg / kg body weight or about 40 to 260 mg in total; and / or Approximately 3.4×10 -9 ~2.2×10 -8 mol / kg body weight or a total of approximately 2.7 × 10 -7 ~1.8×10 -6 mol; about 0.6 to 2.5 mg / kg body weight or about 50 to 200 mg in total; and / or Approximately 4.3×10 -9 ~1.7×10 -8 mol / kg body weight or a total of approximately 3.4 × 10 -7 ~1.4×10 -6 mol; about 0.8 to 1.8 mg / kg body weight or about 60 to 140 mg in total; and / or Approximately 5.1×10 -9 ~1.2×10 -8 mol / kg body weight or a total of approximately 4.1 × 10 -7 ~9.5×10 -7 mol; about 0.9 to 1.8 mg / kg body weight or about 70 to 140 mg in total; and / or Approximately 6.0×10 -9 ~1.2×10 -8 mol / kg body weight or a total of approximately 4.8 × 10 -7 ~9.5×10 -7 mol; about 1 to 1.5 mg / kg body weight or about 80 to 120 mg in total; and / or Approximately 6.8×10 -9 ~1.0×10 -8 mol / kg body weight or a total of approximately 5.5 × 10 -7 ~8.2×10 -7 mol; about 1.1 to 1.4 mg / kg body weight or about 90 to 110 mg in total; and / or Approximately 7.7×10 -9 ~9.4×10 -9 mol / kg body weight or a total of approximately 6.1 × 10 -7 ~7.5×10 -7 mol; about 1.2 to 1.3 mg / kg body weight or about 95 to 105 mg in total; and / or Approximately 6.8×10 -9 ~8.9×10 -9 mol / kg body weight or a total of approximately 6.5 × 10 -7 ~7.2×10 -7 mol, about 0.8-1.5 mg / kg body weight or about 65-120 mg in total; and / or Approximately 5.5×10 -9 ~1.0×10 -8 mol / kg body weight or a total of approximately 4.4 × 10 -7 ~8.2×10 -7 mol; about 0.9 to 1.3 mg / kg body weight or about 70 to 100 mg in total; and / or Approximately 6.0×10 -9 ~8.5×10 -9 mol / kg body weight or a total of approximately 4.8 × 10 -7 ~6.8×10 -7 mol, about 0.9 to 1.1 mg / kg body weight or about 75 to 90 mg in total; and / or Approximately 6.4×10 -9 ~7.7×10 -9 mol / kg body weight or a total of approximately 5.1 × 10 -7 ~6.1×10 -7 mol That's fine too.

[0057] Furthermore, the amount of binding agent administered at each dose and / or in each treatment cycle may be, inter alia: 0.3 to 4.0 mg / kg body weight or 25 to 320 mg in total; and / or 2.1×10 -9 ~2.7×10 -8 mol / kg body weight or a total of 1.7 × 10 -7 ~2.2×10 -6 mol; 0.38 to 4.0 mg / kg body weight or 30 to 320 mg in total; and / or 2.6×10 -9 ~2.7×10 -8 mol / kg body weight or a total of 2.4 × 10 -7 ~2.2×10 -6 mol; 0.5 to 3.3 mg / kg body weight or a total of 40 to 260 mg; and / or 3.4×10 -9 ~2.2×10 -8 mol / kg body weight or a total of 2.7 × 10 -7 ~1.8×10 -6 mol; 0.6 to 2.5 mg / kg body weight or 50 to 200 mg in total; and / or 4.3×10 -9 ~1.7×10 -8 mol / kg body weight or a total of 3.4 × 10 -7 ~1.4×10 -6 mol; 0.8 to 1.8 mg / kg body weight or a total of 60 to 140 mg; and / or 5.1×10 -9 ~1.2×10 -8 mol / kg body weight or a total of 4.1 x 10 -7 ~9.5×10 -7mol; 0.9 to 1.8 mg / kg body weight or 70 to 140 mg in total; and / or 6.0×10 -9 ~1.2×10 -8 mol / kg body weight or a total of 4.8 × 10 -7 ~9.5×10 -7 mol; 1 to 1.5 mg / kg body weight or a total of 80 to 120 mg; and / or 6.8×10 -9 ~1.0×10 -8 mol / kg body weight or a total of 5.5 × 10 -7 ~8.2×10 -7 mol; 1.1 to 1.4 mg / kg body weight or a total of 90 to 110 mg; and / or 7.7×10 -9 ~9.4×10 -9 mol / kg body weight or a total of 6.1 x 10 -7 ~7.5×10 -7 mol; 1.2 to 1.3 mg / kg body weight or a total of 95 to 105 mg; and / or 6.8×10 -9 ~8.9×10 -9 mol / kg body weight or a total of 6.5 × 10 -7 ~7.2×10 -7 mol, 0.8-1.5 mg / kg body weight or a total of 65-120 mg; and / or 5.5×10 -9 ~1.0×10 -8 mol / kg body weight or a total of 4.4 × 10 -7 ~8.2×10 -7 mol; 0.9 to 1.3 mg / kg body weight or 70 to 100 mg in total; and / or 6.0×10 -9 ~8.5×10 -9 mol / kg body weight or a total of 4.8 × 10 -7 ~6.8×10 -7 mol, 0.9–1.1 mg / kg body weight or 75–90 mg in total; and / or 6.4×10 -9 ~7.7×10 -9 mol / kg body weight or a total of 5.1 x 10-7 ~6.1×10 -7 mol That's fine too.

[0058] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) about 1.1 mg / kg body weight or about 80 mg in total; and / or b) Approximately 6.8×10 -9 mol / kg body weight or a total of approximately 5.5 × 10 -7 mol.

[0059] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) 1.1 mg / kg body weight or 80 mg in total; and / or b) 6.8 × 10 -9 mol / kg body weight or a total of 5.5 × 10 -7 mol.

[0060] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) about 1.0 mg / kg body weight or about 80 mg in total; and / or b) Approximately 6.8×10 -9 mol / kg body weight or a total of approximately 5.5 × 10 -7 mol.

[0061] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) 1.0 mg / kg body weight or 80 mg in total; and / or b) 6.8 × 10 -9 mol / kg body weight or a total of 5.5 × 10 -7 mol.

[0062] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) about 1.25 mg / kg body weight or about 100 mg in total; and / or b) Approximately 8.5×10 -9 mol / kg body weight or a total of approximately 6.8 × 10 -7 mol It is currently preferred that

[0063] The amount of binding agent administered at each dose and / or in each treatment cycle is: a) 1.25 mg / kg body weight or 100 mg in total; and / or b) 8.5 x 10 -9 mol / kg body weight or a total of 6.8 × 10 -7 mol It is equally preferable that

[0064] The binding agent may activate human CD137 upon binding to human CD137, and inhibit the binding of human PD-L1 to human PD-1 upon binding to PD-L1.

[0065] In the method according to the invention, the binding agent comprises: a) the first binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:1, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:5; and b) the second antigen-binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:8, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:12. It can also be something like this.

[0066] In the method according to the invention, the binding agent comprises: a) the first binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3, and the CDR3 sequence shown in SEQ ID NO:4, and a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:6, the CDR2 sequence shown as GAS, and the CDR3 sequence shown in SEQ ID NO:7; and b) the second antigen-binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO:13, the CDR2 sequence set forth as DDN, and the CDR3 sequence set forth in SEQ ID NO:14. It can also be something like this.

[0067] Furthermore, in the method according to the invention, the binding agent comprises: a) the first binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:5; and b) the second binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:8, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:12. It can also be something like this.

[0068] In the method according to the invention, the binding agent comprises: a) the first binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and b) the second binding region comprises, consists of, or consists essentially of a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:12; It is something.

[0069] The binding agent may in particular be an antibody, eg a multispecific antibody, or eg a bispecific antibody.

[0070] The binding agent may also be in the form of a full-length antibody or an antibody fragment.

[0071] More preferably, the antibody is a human or humanized antibody.

[0072] Each variable region may comprise three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0073] The complementarity determining regions and framework regions may be arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0074] The binding substance is i) a polypeptide comprising, consisting of, or consisting essentially of said first heavy chain variable region (VH) and first heavy chain constant region (CH); and ii) a polypeptide comprising, consisting of, or consisting essentially of said second heavy chain variable region (VH) and second heavy chain constant region (CH). may include:

[0075] In the method according to the invention, the binding agent 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). It may comprise, consist of, or consist essentially of.

[0076] The binding agent may be an antibody comprising a first binding arm and a second binding arm, the first binding arm comprising: 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 the second binding arm comprises, consists of, or consists essentially of 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). Comprising, consisting of, or consisting essentially of.

[0077] The binding substance is i) a first heavy chain and a first light chain comprising an antigen-binding region capable of binding to CD137; and ii) a second heavy chain and a second light chain comprising the antigen-binding region capable of binding to PD-L1; It may comprise, consist of, or consist essentially of.

[0078] The binding substance is i) a first heavy chain and a first light chain comprising said antigen-binding region capable of binding to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a second light chain comprising the antigen-binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region. It may comprise, consist of, or consist essentially of.

[0079] Each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) may comprise one or more of a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region, preferably at least the hinge region, the CH2 region, and the CH3 region.

[0080] The first heavy chain constant region (CH) and the second heavy chain constant region (CH) may each comprise a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0081] In the first heavy chain constant region (CH), at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain according to EU numbering may be substituted, and in the second heavy chain constant region (CH), at least one of the amino acids at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 of a human IgG1 heavy chain according to EU numbering may be substituted. In a specific embodiment, the first heavy chain and the second heavy chain are not substituted at the same positions.

[0082] The binding substance may be (i) in which the amino acid at the position corresponding to F405 of 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 of the human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) in which the amino acid at the position corresponding to K409 of 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 of the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0083] In the methods according to the invention, the binding agent may be one that induces Fc-mediated effector functions to a reduced extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions.

[0084] In particular, the methods may use binding agents in which the first and second heavy chain constant regions (CH) have been modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising unmodified first and second heavy chain constant regions (CH). In particular, each or both of the unmodified first and second heavy chain constant regions (CH) can comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO:15.

[0085] Fc-mediated effector function may be determined by measuring binding of a binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, Fc-mediated effector function may be determined by measuring binding of a binding agent to C1q.

[0086] The first heavy chain constant region and the second heavy chain constant region of the binding agent may be modified so that binding of the antibody to C1q is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and C1q binding is preferably measured by ELISA.

[0087] The binding agent used in the methods provided herein may be a binding agent in which, 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 positions L234, L235, D265, N297, and P331 of a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0088] In a binding agent for use according to the invention, the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain according to EU numbering may be F and E in said first and second heavy chains, respectively.

[0089] In particular, the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering may be F, E, and A in said first and second heavy chain constant regions (HC), respectively.

[0090] The binding substance used in the methods of the present invention may be a binding substance in which the positions corresponding to L234 and L235 of the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0091] The binding substance used in the methods of the present invention may be a binding substance in which the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0092] The binding agent used in the method according to the present invention may be one in which the constant region of the first heavy chain and / or the second heavy chain is a) the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 30 [IgG1-FC]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution. The binding agent may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0093] The binding agent used in the method according to the invention may be one in which the constant region of said first heavy chain or second heavy chain, e.g. the second heavy chain, is a) the sequence shown in SEQ ID NO: 16 or SEQ ID NO: 31 [IgG1-F405L]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 9 substitutions compared to the amino acid sequence defined in a) or b), for example at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution. The binding agent may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0094] The binding agent used in the method according to the invention may be one in which the constant region of said first heavy chain or second heavy chain, e.g. the first heavy chain, is a) the sequence shown in SEQ ID NO: 17 or 32 [IgG1-F409R] b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The binding agent may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0095] The binding agent used in the method according to the present invention may be one in which the constant region of the first heavy chain and / or the second heavy chain is a) the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 33 [IgG1-Fc_FEA]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 7 substitutions compared to the amino acid sequence defined in a) or b), for example at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The binding agent may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0096] The binding agent used in the method according to the invention may be one in which the constant region of said first heavy chain and / or second heavy chain, e.g. the second heavy chain, is a) the sequence shown in SEQ ID NO: 19 or SEQ ID NO: 34 [IgG1-Fc_FEAL]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions compared to the amino acid sequence defined in a) or b), for example at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The binding agent may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0097] The binding agent used in the method according to the invention may be one in which the constant region of said first heavy chain and / or second heavy chain, e.g. the first heavy chain, is a) the sequence shown in SEQ ID NO: 20 or SEQ ID NO: 35 [IgG1-Fc_FEAR]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions compared to the amino acid sequence defined in a) or b), for example at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The binding agent may comprise, consist essentially of, or consist of an amino acid sequence selected from the group consisting of:

[0098] The binding agent used in the methods according to the invention may comprise a kappa (κ) light chain constant region.

[0099] The binding agent used in the methods according to the invention may comprise a lambda (λ) light chain constant region.

[0100] The binding agent used in the method according to the present invention may be a binding agent wherein said first light chain constant region is a kappa (κ) light chain constant region.

[0101] The binding agent used in the method according to the present invention may be a binding agent wherein said second light chain constant region is a lambda (λ) light chain constant region.

[0102] The binding agent used in the method according to the present invention may be a binding agent wherein said first light chain constant region is a lambda (λ) light chain constant region.

[0103] The binding agent used in the method according to the invention may be a binding agent in which the second light chain constant region is a kappa (κ) light chain constant region.

[0104] The binding agent used in the method according to the invention is one in which the kappa (κ) light chain is a) the sequence shown in SEQ ID NO:21; b) a subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The binding agent may comprise an amino acid sequence selected from the group consisting of:

[0105] The binding agent used in the method according to the invention is one in which the lambda (λ) light chain is a) the sequence shown in SEQ ID NO:22; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The binding agent may comprise an amino acid sequence selected from the group consisting of:

[0106] The binding agent may be of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0107] In particular, the binding agent may be a full-length IgG1 antibody.

[0108] In a currently preferred embodiment, the antibody is of the IgG1m(f) allotype.

[0109] The subjects treated in accordance with the present invention are preferably human subjects.

[0110] The tumor or cancer is a solid tumor.

[0111] The tumor or cancer may be selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, ovarian cancer, endometrial or uterine carcinoma, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma.

[0112] In certain embodiments, the tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial carcinoma (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx), and cervical cancer.

[0113] The tumor or cancer may in particular be lung cancer.

[0114] The lung cancer may be non-small cell lung cancer (NSCLC), for example, squamous or non-squamous NSCLC.

[0115] Lung cancer is the most common malignant tumor and the leading cause of cancer death worldwide. Non-small cell lung cancer (NSCLC) accounts for 85–90% of all lung cancer cases (Jemal et al., 2011). The 5-year survival rate for NSCLC is approximately 18% (SEER, 2018). The major histological subtypes of NSCLC include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large cell carcinoma, carcinoid tumor, and other less common subtypes, with adenocarcinoma being the most common.

[0116] Standard treatment for patients with advanced or metastatic NSCLC who have progressed on or are no longer candidates for targeted therapy typically includes platinum-based chemotherapy. Platinum combinations have resulted in overall response rates (ORR) of approximately 25-35%, time to progression (TTP) of 4-6 months, and median survival of 8-10 months.

[0117] Tumor gene mutations / alterations have been identified and influence therapy selection. Identifying specific mutations or alterations in tumor genes, such as anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR), c-ROS oncogene 1 (ROS1), BRAF, KRAS, and programmed cell death ligand-1 (PD-L1), can help select potentially effective targeted therapies while avoiding the use of therapies unlikely to provide clinical benefit (NCCN, 2018c). Activating-sensitivity EGFR mutations predict response to EGFR tyrosine kinase inhibitors (TKIs) (e.g., gefitinib, erlotinib, afatinib, and osimertinib). Similarly, TKIs (e.g., alectinib, ceritinib, and crizotinib) are effective treatments for ALK and ROS1 mutations and are approved as first-line therapy for each mutation. Checkpoint inhibitor antibodies that block the interaction between PD1 and PD-L1 (e.g., pembrolizumab and nivolumab) have also been shown to be effective treatments, either alone or in combination with chemotherapy, for treating patients with advanced or metastatic NSCLC whose tumors express PD-L1.

[0118] Despite the availability of multiple treatment options, the prognosis for patients with stage IV NSCLC is ultimately poor, and lung cancer remains the number one cause of cancer death in both men and women. Cure rates decline with each line of therapy, either when patients succumb to their cancer or when they experience a decline in health that precludes further treatment.

[0119] The lung cancer may be NSCLC, and the NSCLC does not have an epidermal growth factor (EGFR)-sensitizing mutation and / or an anaplastic lymphoma (ALK) translocation / ROS1 rearrangement. An EGFR-sensitizing mutation refers to a mutation that confers sensitivity to an EGFR tyrosine kinase inhibitor (TKI), such as the approved tyrosine kinase inhibitors erlotinib, osimertinib, gefintinib, olmutinib, nazartinib, and avitinib.

[0120] The epidermal growth factor receptor (EGFR) amino acid sequence is provided herein as SEQ ID NO:27.

[0121] The amino acid sequence of the epidermal growth factor receptor (EGFR) sensitivity-increasing mutation is: i) an in-frame deletion, and optionally an insertion, of one or more amino acids at positions 746 to 751, e.g., any of the deletions and insertions defined in Table 4; ii) a single amino acid substitution at any one of positions 709, 715, 719, 720, 768, 858, and 861, e.g., any of the deletions and insertions defined in Table 5; and iii) an in-frame duplication and / or insertion selected from the duplications / insertions defined in Table 6 The amino acid numbers refer to the amino acid numbers of SEQ ID NO:27.

[0122] (Table 4) In-frame deletions within exon 19 of the human EGFR gene (modified from Shigematsu et al., Clinical and Biological Features Associated With Epidermal Growth Factor Receptor Gene Mutations in Lung Cancers, JNCI: Journal of the National Cancer Institute, Volume 97, Issue 5, 2 March 2005). del = deletion; ins = insertion. TIFF0007809062000004.tif91128

[0123] Table 5. Single-base substitutions and resulting amino acid changes within exon 21 of the human EGFR gene (adapted from Shigematsu et al., Clinical and Biological Features Associated With Epidermal Growth Factor Receptor Gene Mutations in Lung Cancers, JNCI: Journal of the National Cancer Institute, Volume 97, Issue 5, 2 March 2005) TIFF0007809062000005.tif76128

[0124] (Table 6) In-frame duplications and / or insertions within exon 20 of the human EGFR gene (adapted from Shigematsu et al., Clinical and Biological Features Associated With Epidermal Growth Factor Receptor Gene Mutations in Lung Cancers, JNCI: Journal of the National Cancer Institute, Volume 97, Issue 5, 2 March 2005). ins = insertion. TIFF0007809062000006.tif68128

[0125] The non-small cell lung cancer may be characterized by at least one mutation in the EGFR amino acid sequence selected from L747S, D761Y, T790M, C797S, T854A, e.g., T790M, C797S, D761Y, and double mutations T790M / D761Y and T790 / C797S, and / or the subject undergoing treatment may have at least one mutation in the EGFR amino acid sequence selected from L747S, D761Y, T790M, C797S, T854A, e.g., T790M, C797S, D761Y, and double mutations T790M / D761Y and T790 / C797S. The amino acid numbers refer to the amino acid numbers of SEQ ID NO: 27.

[0126] Non-small cell lung cancer is i. Wild-type human EGFR; for example, human EGFR comprising the sequence set forth in SEQ ID NO:27 or a mature polypeptide thereof; and ii. A human EGFR that is a variant of the EGFR of item i and does not have any sensitivity-increasing mutations when compared with the EGFR of item i. The antibody may be characterized by expression of an epidermal growth factor receptor (EGFR) selected from the group consisting of:

[0127] Non-small cell lung cancer is i) an in-frame deletion, and optionally an insertion, of one or more amino acids at positions 746 to 751, e.g., any of the deletions and insertions defined in Table 4; ii) a single amino acid substitution at any one of positions 709, 715, 719, 720, 768, 858, and 861, e.g., any of the deletions and insertions defined in Table 5; and iii) an in-frame duplication and / or insertion selected from the duplications / insertions defined in Table 6 The cancer may be not characterized by an epidermal growth factor receptor (EGFR) sensitivity-increasing mutation selected from the group consisting of: The amino acid numbers refer to the amino acid numbers of SEQ ID NO: 27. Similarly, a subject receiving treatment according to the present invention may be a subject who does not have such an EGFR sensitivity-increasing mutation.

[0128] The non-small cell lung cancer may be a cancer that is not characterized by a mutation in the EGFR amino acid sequence selected from L747S, D761Y, T790M, C797S, T854A, for example, T790M, C797S, D761Y, and double mutations T790M / D761Y and T790 / C797S. The amino acid numbers refer to the amino acid numbers of SEQ ID NO: 27. Similarly, the subject receiving treatment according to the present invention may not have any of the mutations.

[0129] Non-small cell lung cancers and / or subjects undergoing treatment according to the invention may be characterized as having a mutation in the gene encoding ALK tyrosine kinase (ALK) (UniProt Q9UM73) that causes rearrangement of the gene encoding ALK with a gene encoding a fusion partner to form a fusion oncogene.

[0130] Non-small cell lung cancer may be characterized by a mutation in the gene encoding ALK that causes a rearrangement between the gene encoding ALK and the gene encoding Echinoderm microtubule-associated protein-like 4 (EMAPL4) (EML4) (UniProt Q9HC35) (to form the EML4-ALK fusion oncogene), and / or a subject receiving treatment according to the invention may have a mutation in the gene encoding ALK that causes a rearrangement between the gene encoding ALK and the gene encoding Echinoderm microtubule-associated protein-like 4 (EMAPL4) (EML4) (UniProt Q9HC35) (to form the EML4-ALK fusion oncogene).

[0131] Non-small cell lung cancer is caused by a mutation in the gene encoding ALK tyrosine kinase (ALK) and i. KIF5B (UniProt P33176), which encodes kinesin-1 heavy chain (KINH); ii. KLC1 (UniProt Q07866), which encodes kinesin light chain 1 (KLC1); iii. TFG (UniProt Q92734), which encodes the protein TFG; iv. TPR (UniProt P12270), which encodes the nuclear protein TPR; v. HIP1 (UniProtKB-O00291), which encodes Huntington-interacting protein 1 (HIP-1); vi. STRN (UniProtKB-O43815), which encodes striatin; vii. DCTN1 (UniProt Q14203), which encodes dynactin subunit 1; viii. SQSTM1 (UniProtKB-Q13501), which encodes sequestosome-1; ix. NPM1 (UniProt P06748), which encodes nucleophosmin; BCL11A (UniProt Q9H165), encoding xB-cell lymphoma / leukemia 11A, and xi. BIRC6 (UniProt Q13490), encoding a baculoviral IAP repeat-containing protein and / or a subject undergoing treatment according to the present invention, characterized by a mutation in the gene encoding ALK that results in a rearrangement with a gene selected from the group consisting of KIF5B-ALK fusion oncogene, KLC1-ALK fusion oncogene, TFG-ALK fusion oncogene, TPR-ALK fusion oncogene, HIP1-ALK fusion oncogene, STRN-ALK fusion oncogene, DCTN1-ALK fusion oncogene, SQSTM1-ALK fusion oncogene, NPM1-ALK fusion oncogene, BCL11A-ALK fusion oncogene, and BIRC6-ALK fusion oncogene. may have a mutation in the gene encoding ALK tyrosine kinase (ALK) that causes a rearrangement of the gene encoding ALK with a gene selected from the group and the formation of a respective fusion oncogene selected from the group consisting of KIF5B-ALK fusion oncogene, KLC1-ALK fusion oncogene, TFG-ALK fusion oncogene, TPR-ALK fusion oncogene, HIP1-ALK fusion oncogene, STRN-ALK fusion oncogene, DCTN1-ALK fusion oncogene, SQSTM1-ALK fusion oncogene, NPM1-ALK fusion oncogene, BCL11A-ALK fusion oncogene, and BIRC6-ALK fusion oncogene.

[0132] The non-small cell lung cancer may be characterized by expression of wild-type human ALK tyrosine kinase, eg, human ALK tyrosine kinase comprising the sequence provided in UniProt Q9HC35, or the mature polypeptide thereof.

[0133] The non-small cell lung cancer may be characterized by not having a mutation in the gene encoding ALK tyrosine kinase (ALK) that causes rearrangement of ALK with a fusion partner to form a fusion oncogene, and / or the subject does not have such a mutation.

[0134] The non-small cell lung cancer may be characterized by not having a mutation in the gene encoding Echinoderm microtubule-associated protein-like 4 (EMAPL4) (EML4) (UniProt Q9HC35) that causes a rearrangement between the gene encoding ALK tyrosine kinase (ALK) (UniProt Q9HC35) and ALK to form the EML4-ALK fusion oncogene, and / or the subject may not have such a mutation.

[0135] The non-small cell lung cancer may be characterized by the absence of a mutation in any gene selected from the group consisting of the gene encoding ALK tyrosine kinase (ALK), the gene encoding Echinoderm microtubule-associated protein-like 4 (EMAPL4) (EML4) (UniProt Q9HC35).

[0136] Non-small cell lung cancer is - epidermal growth factor receptor (EGFR) mutations that increase susceptibility, - mutations in the gene encoding ALK tyrosine kinase (ALK), leading to rearrangements between EML4 and ALK and the formation of the EML4-ALK fusion oncogene; - a mutation in the EGFR amino acid sequence that induces or confers resistance to one or more EGFR tysrosine kinase inhibitors (EGFR-TKIs) in the subject; and the subject may have been treated with a programmed cell death-1 (PD-1) / programmed cell death-1 (PD-1) inhibitor (e.g., nivolumab, genolimuzumab, atezolizumab, durvalumab, or avelumab) or with chemotherapy (e.g., chemotherapy including platinum, taxane, pemetrexed, and / or gemcitabine), and may have failed such previous treatment.

[0137] Non-small cell lung cancer is - epidermal growth factor receptor (EGFR) mutations that increase susceptibility, - a mutation in the EGFR amino acid sequence that induces or confers resistance to one or more EGFR tyrosine kinase inhibitors (EGFR-TKIs) in the subject; - Mutations in the gene encoding ALK tyrosine kinase (ALK), leading to rearrangements between EML4 and ALK and the formation of the EML4-ALK fusion oncogene and the subject may have been treated with an EGFR inhibitor (e.g., erlotinib, osimertinib, gefintinib, olmutinib, nazartinib, and avitinib) or with a PD-1 / PD-L1 inhibitor (e.g., nivolumab, genolimu- zumab, atezolizumab, durvalumab, or avelumab), and may have failed such prior treatment.

[0138] The subject has received up to four systemic conditioning regimens for advanced / metastatic disease to treat lung cancer and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

[0139] Prior to receiving treatment according to the present invention, the subject has received platinum-based chemotherapy to treat lung cancer, or the subject may be ineligible for platinum-based therapy and have received treatment with another chemotherapy, for example, a gemcitabine-containing regimen.

[0140] The subject may have received prior treatment with a checkpoint inhibitor, e.g., an agent that targets programmed cell death-1 (PD-1) / programmed cell death-ligand 1 (PD-L1), e.g., a PD-1 / PD-L1 inhibitor, to treat lung cancer. Preferably, the subject should have received only one prior treatment with a PD-1 / PD-L1 inhibitor, alone or in combination.

[0141] In particular, the subject may have experienced disease progression during or after treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor. Further, the subject may have experienced disease progression during or after their last prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

[0142] Inhibitors of PD-1 and / or PD-L1 may include, inter alia, antibodies or antigen-binding fragments thereof capable of binding to PD-L1.

[0143] Known inhibitors of PD-1 and / or PD-L1 include pembrolizumab (Merck & Co), CBT-501 (genolimuzumab; Genor Bio / CBT Pharma), nivolumab (BMS), REGN2810 (cemiplimab; Regeneron), BGB-A317 (tislelizumab; BeiGene / Celgene), Amp-514 (MEDI0680) (Amplimmune), TSR-042 (dostallimab; Tesaro / AnaptysBio), JNJ-63723283 / JNJ-3283 (Johnson & Johnson), PF-06801591 (Pfizer), JS-001 (tripolibamab / toriparimab; Shanghai Junshi Bio), SHR-1210 / INCSHR-1210 (camrelizumab; Incyte corp), PDR001 (spartalizumab; Novartis), BCD-100 (BioCad), AGEN2034 (Agenus), IBI-308 (sintilimab; Innovent Biologics), RG7446 / MPDL-3280A (atezolizumab; Roche), MSB-0010718C (avelumab; Merck Serono / Pfizer), and MEDI-4736 (durvalumab; AstraZeneca), KN-035 (embafolimab; 3DMed / Alphamab Co.).

[0144] In particular, the subject may have experienced disease progression, eg, radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

[0145] Alternatively, a subject undergoing treatment according to the present invention may have not previously been treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor, e.g., any of the PD-1 / PD-L1 inhibitors listed above, to treat said lung cancer.

[0146] In other embodiments, the tumor or cancer is endometrial cancer. Endometrial cancer (EC) is the most common gynecological malignancy in the United States and other developed countries, and its incidence has been increasing worldwide. In the United States, an estimated 60,000 new cases and over 10,000 deaths were reported in 2016. In 2012, 527,600 women were diagnosed with uterine EC worldwide. The majority of EC cases are identified at an early stage and treated by surgery with or without radiation therapy or chemotherapy. However, the prognosis for patients with advanced disease is poor, with a 5-year survival rate of less than 50% for patients with lymph node metastasis and less than 20% for patients with peritoneal or distant metastasis.

[0147] Multiagent chemotherapy is the preferred treatment for metastatic, recurrent, or high-risk disease; however, there is no consensus regarding a standard regimen. Carboplatin and paclitaxel are increasingly being used in the first-line setting for advanced / metastatic or recurrent EC. Response rates to carboplatin and paclitaxel range from 40% to 62%, with overall survival (OS) ranging from approximately 13 to 29 months. Patients who progress on combination therapy or cannot tolerate multiagent chemotherapy may receive single-agent therapy, but chemotherapy options in this setting have only produced modest activity, particularly in the second-line and later settings. Response rates to single agents range from 21% to 36% in the first-line setting and from 4% to 27% in the second-line setting (NCCN, 2018d).

[0148] Most recently, pembrolizumab demonstrated antitumor activity in patients with locally advanced or metastatic PD-L1-positive EC who had experienced progression on or after standard therapy.

[0149] In particular, subjects or endometrial cancers treated according to the present invention may have epithelial endometrial histology, including endometrioid carcinoma, serous carcinoma, squamous cell carcinoma, clear cell carcinoma, or carcinosarcoma.

[0150] The subject may have received up to four systemic conditioning regimens for advanced / metastatic disease to treat said endometrial cancer and may have experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

[0151] The subject may have not been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor, to treat said endometrial cancer. For example, the PD-1 / PD-L1 inhibitor is selected from the list of PD-1 / PD-L1 inhibitors above.

[0152] According to other embodiments, the tumor or cancer is a urothelial cancer, including cancer of the bladder, ureter, urethra, or renal pelvis.

[0153] The subject may have received up to four systemic conditioning regimens for advanced / metastatic disease to treat said urothelial cancer and may have experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

[0154] The subject may have received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor; e.g., any one of the PD-1 / PD-L1 inhibitors listed above, to treat the urothelial cancer.

[0155] Furthermore, the subject may have previously undergone platinum-based chemotherapy, i.e., chemotherapy using agents that are coordination compounds of platinum, to treat the urothelial cancer. Examples of platinum-based chemotherapy include treatment with cisplatin, oxaliplatin, and carboplatin.

[0156] The subject may not be eligible for platinum-based therapy and may have been treated with another chemotherapy, for example, a gemcitabine-containing regimen.

[0157] In another embodiment according to the present invention, the tumor or cancer is breast cancer, for example, triple-negative breast cancer (TNBC).TNBC generally refers to breast cancer that lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2).TNBC may be particularly HER2-negative, for example, as determined by measuring protein expression by fluorescent in situ hybridization (FISH) or immunohistochemistry.

[0158] The subject may have received at least one systemic conditioning regimen for locally advanced / metastatic disease to treat said breast cancer, for example, at least one systemic conditioning regimen including an anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimen.

[0159] In a further embodiment, the subject may have received up to four systemic conditioning regimens for locally advanced / metastatic disease to treat said breast cancer, including at least one systemic conditioning regimen, such as an anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimen.

[0160] The subject may have received prior treatment with a checkpoint inhibitor to treat breast cancer, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor; e.g., any one of the PD- / PD-L1 inhibitors listed above.

[0161] The subject may have experienced disease progression, e.g., radiographically confirmed disease progression, during or after prior treatment with a checkpoint inhibitor to treat breast cancer.

[0162] In other embodiments, the subject may be a subject who has not been previously treated with a checkpoint inhibitor to treat breast cancer, e.g., a subject who has not been treated with an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor; e.g., a PD- / PD-L1 inhibitor listed above.

[0163] The tumor or cancer may be head and neck cancer, such as squamous cell carcinoma of the head and neck (SCCHN). SCCHN is a leading cause of death, with over 600,000 cases diagnosed annually worldwide. In 2018, approximately 64,690 people in the United States developed oral, pharyngeal, or laryngeal cancer, resulting in an estimated 13,740 deaths during the same period. Head and neck cancer can occur in the oral cavity, pharynx, larynx, nasal cavity, paranasal sinuses, thyroid, and salivary glands. Smoking and alcohol significantly increase the risk of developing head and neck cancer. Furthermore, human papillomavirus (HPV) infection is causally associated with squamous cell carcinoma of the oropharynx (particularly the tonsils and base of the tongue), and recent evidence suggests that HPV may also be associated with a higher risk of laryngeal squamous cell carcinoma. Patients with localized HPV-positive head and neck cancer have improved outcomes in terms of response to treatment, PFS, and OS compared with HPV-negative tumors.

[0164] Treatment of head and neck cancer is complex and requires a multidisciplinary approach. The prognosis for patients with recurrent or metastatic SCCHN is generally poor, with median survival ranging from approximately 6 to 12 months, depending on the patient's performance status and disease-related factors. For appropriate patients, first-line therapy includes cetuximab and cisplatin or carboplatin plus 5-fluorouracil (5-FU). The addition of cetuximab has been associated with improved survival (10.1 vs. 7.4 months) and mPFS (3.3 vs. 5.6 months) compared with platinum and 5-FU alone. Single-agent chemotherapy is recommended for patients with poor performance status. To date, the most commonly used single agents have included platinum compounds, taxanes, nab-paclitaxel, methotrexate, fluorouracil, and cetuximab.

[0165] In the United States and some other countries, pembrolizumab and nivolumab are approved for patients with progressive disease (PD) after platinum-containing chemotherapy. Data from trials exploring single-agent activity of PD-1 targets have appeared promising, but response rates remain low.

[0166] In particular, the tumor or cancer may be a recurrence of metastatic SCCHN.

[0167] In particular embodiments relating to SCCHN, the tumor or cancer is a cancer of the oral cavity, pharynx, or larynx.

[0168] The subject may have received up to four systemic conditioning regimens for recurrent / metastatic disease to treat SCCHN and may have experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

[0169] The subject may have undergone treatment with platinum-based chemotherapy to treat SCCHN, for example, treatment with cisplatin, oxaliplatin, and carboplatin.

[0170] Alternatively, the subject may not be eligible for platinum-based therapy and may have received another chemotherapy regimen to treat SCCHN.

[0171] The subject may have previously been treated with a checkpoint inhibitor to treat SCCHN, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor; e.g., a PD- / PD-L1 inhibitor listed above.

[0172] The subject may have experienced disease progression, eg, radiographically confirmed disease progression, during or after prior treatment with a checkpoint inhibitor.

[0173] In other embodiments, the subject may be a subject who has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor, e.g., a subject who has not received treatment with any of the PD- / PD-L1 inhibitors listed above.

[0174] In a further embodiment, the tumor or cancer is cervical cancer. Cervical cancer poses a significant medical problem worldwide, with an estimated incidence of over 500,000 new cases. In the United States, it is estimated that approximately 12,800 new cases and 4,210 deaths will occur in 2017. The median age at diagnosis of cervical cancer in the United States is 49 years old, and is younger in developing countries. In the United States, the five-year survival rate for patients diagnosed with localized disease is 91%, but the prognosis for patients with advanced disease remains poor. The five-year survival rate for advanced / metastatic disease is less than 35%.

[0175] First-line treatment for recurrent or metastatic cervical cancer consists of bevacizumab in combination with paclitaxel and a platinum (cisplatin or carboplatin) or paclitaxel and topotecan. Despite a 48% ORR and a median OS of approximately 18 months, nearly all patients relapse after this first-line treatment. Regarding second-line therapy, pembrolizumab is approved in the United States for the treatment of patients with recurrent or metastatic cervical cancer whose disease has progressed during or after chemotherapy and whose tumors express PD-L1, as confirmed by an FDA-approved test. No other approved therapies are available. However, patients are often treated with single-agent modalities, including, but not limited to, pemetrexed, topotecan, docetaxel, nab-paclitaxel, vinorelbine, and, in some cases, bevacizumab. Response rates with single-agent treatment are very low (range: 0-15%), and for this reason cervical cancer remains a population with a very high unmet medical need.

[0176] The cervical cancer may be, in particular, a cervical cancer of squamous cell, adenocarcinoma, or adenosquamous histology.

[0177] A subject to be treated according to the present invention may have received at least one systemic conditioning regimen for recurrent / metastatic disease to treat said cervical cancer, e.g., chemotherapy in combination with a treatment targeting vascular endothelial growth factor A, e.g., treatment with bevacizumab, and have experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

[0178] Subjects treated according to the present invention may have received up to four systemic conditioning regimens for recurrent / metastatic disease, including chemotherapy in combination with a vascular endothelial growth factor A-targeted treatment, e.g., treatment with bevacizumab.

[0179] In some embodiments, the subject treated in accordance with the present invention has not been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor, e.g., has not been treated with any of the PD- / PD-L1 inhibitors listed above.

[0180] Preferably, the subject is female.

[0181] The binding agents used according to the present invention may in particular be administered by systemic administration.

[0182] Preferably, the binding agent is administered to the subject by intravenous injection or infusion.

[0183] Each treatment cycle may be 2 weeks (14 days), 3 weeks (21 days), or 4 weeks (28 days).

[0184] In certain embodiments of the invention, each dose is administered or infused every two weeks (1Q2W), every three weeks (1Q3W), or every four weeks (1Q4W).

[0185] In some embodiments, the or each dose is administered or infused on day 1 of each treatment cycle.

[0186] Each dose may be administered or infused over a minimum of 30 minutes, for example, over a minimum of 60 minutes, a minimum of 90 minutes, a minimum of 120 minutes, or a minimum of 240 minutes.

[0187] A further aspect of the invention provides a composition, e.g., a pharmaceutical composition, comprising a binding agent comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1, wherein the amount of the binding agent in the composition is about 25 to 400 mg or about 1.7 x 10 -7 ~2.7×10 -6 mol, e.g., 25-400 mg or 1.7 × 10 -7 ~2.7×10-6 mol.

[0188] The amount of binding agent administered in the composition is, in particular, about 25 to 320 mg or about 1.7 x 10 -7 ~2.2×10 -6 mol, e.g., 25-320 mg or 1.7 × 10 -7 ~2.2×10 -6 mol; approximately 30 to 320 mg or approximately 2.4 × 10 -7 ~2.2×10 -6 mol; e.g., 30-320 mg or 2.4 × 10 -7 ~2.2×10 -6 mol approx. 40-260 mg or approx. 2.7 × 10 -7 ~1.8×10 -6 mol, e.g., 40-260 mg or 2.7 × 10 -7 ~1.8×10 -6 mol; approximately 50 to 200 mg or approximately 3.4 × 10 -7 ~1.4×10 -6 mol, e.g., 50-200 mg or 3.4 x 10 -7 ~1.4×10 -6 mol; approximately 60 to 140 mg or approximately 4.1 × 10 -7 ~9.5×10 -7 mol, e.g., 60-140 mg or 4.1 x 10 -7 ~9.5×10 -7 mol; approximately 70 to 140 mg or approximately 4.8 × 10 -7 ~9.5×10 -7 mol, e.g., 70-140 mg or 4.8 x 10 -7 ~9.5×10 -7 mol; approximately 80 to 120 mg or approximately 5.5 × 10 -7 ~8.2×10 -7 mol, e.g., 80-120 mg or 5.5 × 10 -7 ~8.2×10 -7 mol; approximately 90-110 mg or approximately 6.1 × 10 -7 ~7.5×10 -7 mol, e.g., 90-110 mg or 6.1 x 10 -7 ~7.5×10 -7mol; approximately 95 to 105 mg or approximately 6.5 × 10 -7 ~7.2×10 -7 mol, e.g., 95-105 mg or 6.5 x 10 -7 ~7.2×10 -7 mol; approximately 65 to 120 mg or approximately 4.4 × 10 -7 ~8.2×10 -7 mol, e.g., 65-120 mg or 4.4 × 10 -7 ~8.2×10 -7 mol; approximately 70 to 100 mg or approximately 4.8 × 10 -7 ~6.8×10 -7 mol, e.g., 70-100 mg or 4.8 x 10 -7 ~6.8×10 -7 mol; or approximately 75-90 mg or approximately 5.1 × 10 -7 ~6.1×10 -7 mol, e.g., 75-90 mg or 5.1 x 10 -7 ~6.1×10 -7 Mol is also acceptable.

[0189] The composition or pharmaceutical composition can be prepared by conventional techniques, e.g., by the method described in Remington: The Science and Practice of Pharmacy, 1999. th The pharmaceutical compositions may be formulated using carriers, excipients, and / or diluents, as well as any other ingredients suitable for pharmaceutical compositions, including known adjuvants, according to the techniques disclosed in "The Antibody and Antibody Conjugates of the Invention," Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutically acceptable carriers or diluents, as well as any known adjuvants and excipients, must be suitable for the antibody or antibody conjugate of the invention and the selected method of administration. The suitability of carriers and other ingredients for pharmaceutical compositions is determined based on the absence of a significant adverse effect on the desired biological properties of the selected compound or pharmaceutical composition of the invention (e.g., less than a significant effect on antigen binding [e.g., 10% or less relative inhibition, 5% or less relative inhibition, etc.]).

[0190] Pharmaceutical compositions of the present invention may include diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0191] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with the compounds of the present invention.

[0192] 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 such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffer solutions. Other carriers are well known in the pharmaceutical field.

[0193] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions.The use of such media and agents for pharmaceutically active substances is well known in the art.Except where conventional media or agents are incompatible with active compounds, they are intended to be used in the pharmaceutical compositions of the present invention.

[0194] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, e.g., ascorbic acid palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0195] The pharmaceutical compositions of the present invention may also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0196] The pharmaceutical compositions of the present invention may also contain one or more auxiliary agents suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, antiseptics, or buffers, which can enhance the shelf life or effectiveness of the compositions.The combination of compounds of the present invention may also be prepared using carriers that protect the compounds from rapid release, such as sustained-release formulations, including implants, transdermal patches, and microencapsulated delivery systems.Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with 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.See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0197] In one embodiment, the binding substance used according to the present invention can be formulated to be suitable for dispersal in vivo.Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions.The use of such media and agents for pharmaceutically active substances is known in the art.Unless conventional media or agents are incompatible with the active compound, their use in the compositions of the present invention is contemplated.Other active compounds or therapeutic compounds can also be incorporated into the composition.

[0198] Injectable pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. It is often preferable to include isotonic agents, such as sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride, in the compositions. Prolonged absorption of injectable compositions can be achieved by including absorption-delaying agents, such as monostearate salts and gelatin, in the compositions. Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one of the components or a combination of components, for example, as listed above, as needed, and then carrying out sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other components that are required, for example, other components that are required from those listed above.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which produces the powder of active ingredient and any other desired components from the solution of active ingredient and any other desired components that has been previously sterilized by filtration.

[0199] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or a combination of ingredients listed above as needed, and then carrying out sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other ingredients required from those listed above.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which produces the powder of active ingredient+any other desired ingredient from the solution of active ingredient+any other desired ingredient that has been previously sterilized by filtration.

[0200] In one embodiment, the composition according to the present invention comprises about 5.5 x 10 -7 mol or approximately 80 mg, e.g., 5.5 x 10 -7 mol or 80 mg of said binding agent.

[0201] In a currently preferred embodiment, the composition according to the present invention comprises about 6.8 x 10 -7 mol or about 100 mg of the binding agent, e.g., 6.8×10 -7 mol or 100 mg of said binding agent.

[0202] In the composition according to the invention, the binding agent may be as defined above, for example, the binding agent may comprise any of the variable and constant regions defined above.

[0203] The present invention further includes unit dosage forms of the binding agents or compositions disclosed above.

[0204] Preferably, the unit dosage form is a unit dosage form for systemic administration. In certain embodiments, the unit dosage form is for injection or infusion into a subject, for example, intravenous injection or infusion.

[0205] In the composition or unit dosage form, the binding substance is preferably present in an aqueous solution, such as 0.9% NaCl (saline). The volume of the unit dosage form may be 50 to 500 mL, for example, 50 to 250 mL, 50 to 500 mL, 100 to 500 mL, or 100 to 250 mL.

[0206] In yet a further aspect, the present application provides a binding agent for use in treating cancer, comprising a first binding region that binds to human CD137 and a second binding region that binds to human PD-L1.

[0207] The binding agent can be administered in any suitable amount. In particular, the amount of binding agent administered in each dose and / or in each treatment cycle can be: a) about 0.3 to 5 mg / kg body weight or about 25 to 400 mg in total; and / or b) Approximately 2.1×10 -9 ~3.4×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~2.7×10 -6 mol That's fine too.

[0208] Preferably, the amount of binding agent administered at each dose and / or in each treatment cycle is: a) about 1.25 mg / kg body weight or about 100 mg in total, e.g., 1.25 mg / kg body weight or 100 mg in total; and / or b) Approximately 8.5×10 -9 mol / kg body weight or a total of approximately 6.8 × 10 -7 mol, e.g., 8.5 x 10 -9 mol / kg body weight or a total of 6.8 × 10 -7 mol is.

[0209] Further items of this disclosure include: 1. A method for reducing or preventing tumor progression or treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a binding agent, wherein the binding agent comprises a first antigen-binding region that binds to human CD137 and a second antigen-binding region that binds to human PD-L1, and wherein the binding agent is administered in at least one treatment cycle, and wherein the therapeutically effective amount of the binding agent is (i) about 25 mg to about 400 mg in total; or (ii) A total of approximately 1.7 × 10 -7 mol~about 2.7×10 -6 mol That's the method. 2. A therapeutically effective amount of the binding agent is (i) about 80 mg to about 240 mg in total; or (ii) A total of approximately 5.5 × 10 -7 mol~about 1.6×10 -6 mol The method of item 1. 3. A therapeutically effective amount of the binding agent is (i) about 80 mg in total; or (ii) A total of approximately 5.5 × 10 -7 mol The method of item 1. 4. A therapeutically effective amount of the binding agent is (i) about 100 mg in total; or (ii) A total of approximately 6.8 × 10 -7 mol The method of item 1. 5. a) the first antigen-binding region comprises a heavy chain variable region (VH) comprising CDR1 (HCDR1), CDR2 (HCDR2), and CDR3 (HCDR3), and a light chain variable region (VL) comprising CDR1 (LCDR1), CDR2 (LCDR2), and CDR3 (LCDR3); HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:2; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:3; HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:4; LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6; LCDR2 comprises the amino acid sequence GAS, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:7; b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising CDR1 (HCDR1), CDR2 (HCDR2), and CDR3 (HCDR3), and a light chain variable region (VL) comprising CDR1 (LCDR1), CDR2 (LCDR2), and CDR3 (LCDR3); HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:10; HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:11; LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; LCDR2 comprises the amino acid sequence DDN, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14; Any of the methods listed in items 1 to 4. 6. The first binding region comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1; (ii) a light chain variable region (VL) containing amino acids that are at least 95% identical to the amino acid sequence set forth in SEQ ID NO:5; and the second binding region comprises (iii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:8; (iv) a light chain variable region (VL) containing at least 95% of the amino acid sequence of SEQ ID NO: 12; Any of the methods described in items 1 to 4, including: 7. The first binding region comprises: (i) a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1; (ii) a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and the second binding region comprises (iii) a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8; (iv) a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 12; Any of the methods described in items 1 to 6, including: 8. The binding substance is (1)(a) a first heavy chain comprising a first heavy chain variable region (VH1) and a first heavy chain constant region (CH1); and (b) a first light chain comprising a first light chain variable region (VL1) and a first light chain constant region (CL1); a first polypeptide comprising: (2)(c) a second heavy chain comprising a second heavy chain variable region (VH2) and a second heavy chain constant region (CH2); and (d) a second light chain comprising a second light chain variable region (VL2) and a second light chain constant region (CL2); a second polypeptide comprising Including, VH1 comprises the amino acid sequence set forth in SEQ ID NO:1; VL1 comprises the amino acid sequence set forth in SEQ ID NO:5, VH2 comprises the amino acid sequence set forth in SEQ ID NO:8; and VL2 comprises the amino acid sequence shown in SEQ ID NO: 12; Any of the methods listed in items 1 to 7. 9. The method of item 8, wherein CH1 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in the amino acid sequence of SEQ ID NO: 19 or 34, with 1 to 10 consecutive amino acids deleted, and CH2 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in the amino acid sequence of SEQ ID NO: 20 or 35, with 1 to 10 consecutive amino acids deleted. 10. The method of any of items 1 to 9, wherein the binding agent is an antibody or a fragment thereof. 11. Any of the methods of items 1 to 10, wherein the tumor or cancer in the subject is non-small cell lung cancer (NSCLC). 12. NSCLC subjects: (i) had received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; (ii) have a histological or cytological diagnosis of non-squamous NSCLC without epidermal growth factor (EGFR) sensitivity-increasing mutations and / or anaplastic lymphoma (ALK) translocations / ROS1 rearrangements; (iii) have received platinum-based therapy or other chemotherapy due to platinum ineligibility; and (iv) had disease progression after prior treatment with a PD-1 / PD-L1 inhibitor; Item 11 method. 13. NSCLC subjects: (i) had received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; (ii) have a histological or cytological diagnosis of non-squamous NSCLC without epidermal growth factor (EGFR) sensitivity-increasing mutations and / or anaplastic lymphoma (ALK) translocations / ROS1 rearrangements; (iii) have received platinum-based therapy or other chemotherapy due to platinum ineligibility; and (iv) no prior treatment with a PD-1 / PD-L1 inhibitor; Item 11 method. 14. Any of the methods of items 1 to 10, wherein the tumor or cancer in the subject is urothelial carcinoma (UC). 15. UC target is (i) had received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; (ii) had disease progression after prior treatment with a PD-1 / PD-L1 inhibitor; and (iii) had received platinum-based chemotherapy or were not eligible for platinum-based or cisplatin-containing chemotherapy; Item 14 method. 16. Any of the methods of items 1 to 10, wherein the tumor or cancer in the subject is endometrial cancer (EC). 17. EC subject, (i) had received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; (ii) have epithelial endometrial histology, including endometrioid carcinoma, serous carcinoma, squamous cell carcinoma, clear cell carcinoma, or carcinosarcoma; and (iii) no prior treatment with a PD-1 / PD-L1 inhibitor; Item 16 method. 18. Any of the methods of items 1 to 10, wherein the tumor or cancer in the subject is triple-negative breast cancer (TNBC). 19. TNBC subjects: (i) had TNBC defined as HER2-negative; (ii) had received 1 to 4 prior systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; Item 18 method. 20. The method of item 18, wherein the TNBC subject has had disease progression after prior treatment with a PD-1 / PD-L1 inhibitor. 21. The method of item 18, wherein the TNBC subject has not received prior treatment with a PD-1 / PD-L1 inhibitor. 22. Any of the methods of items 1 to 10, wherein the tumor or cancer in the subject is squamous cell carcinoma of the head and neck (SCCHN). 23. SCCHN subject: (i) have received up to four systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; (ii) had prior platinum-based chemotherapy and shown disease progression, or, if not eligible for platinum-based chemotherapy, had prior treatment with another combination and shown disease progression; Item 22 method. 24. The method of any of items 1 to 10, wherein the tumor or cancer in the subject is cervical cancer. 25. The target of cervical cancer is (i) had received 1 to 4 systemic conditioning regimens for advanced / metastatic disease and experienced disease progression during or after the last systemic conditioning regimen; (ii) have cervical cancer of squamous cell, adenocarcinoma, or adenosquamous histology; and (iii) no prior treatment with a PD-1 / PD-L1 inhibitor; Item 24 method. 26. The method of any of the preceding items, wherein the therapeutically effective amount of the binding agent is administered in a fixed dose regardless of body weight. 27. The method of any of the preceding items, wherein the binding agent is administered by systemic administration. 28. The method of any of the preceding items, wherein the binding agent is administered by intravenous injection or infusion. 29. The method of any of the preceding items, wherein each treatment cycle is 3 weeks (21 days). 30. The method of any of the preceding items, wherein one dose is administered every three weeks (1Q3W). 31. The method of any of the preceding items, wherein one dose is administered on day 1 of each treatment cycle.

[0210] array (Table 7) TIFF0007809062000007.tif201158TIFF0007809062000008.tif243158TIFF0007809062000009.tif244158 TIFF0007809062000010.tif243158TIFF0007809062000011.tif244158TIFF0007809062000012.tif151158

[0211] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0212] Example 1: Generation of CD137 antibody Antibodies CD137-005 and CD137-009 were produced as described in Example 1 of WO2016 / 110584. Briefly, rabbits were immunized with a protein mixture containing a human CD137-Fc fusion protein. Single B cells were sorted from the blood and screened for the production of CD137-specific antibodies by ELISA and flow cytometry. RNA was extracted from the B cells that screened positively and sequenced. The heavy and light chain variable regions were gene synthesized and cloned into a human IgG1κ or IgG1λ expression vector containing a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A, and F405L(FEAL) or F405L(FEAL). The numbers for the amino acid positions are according to EU numbering (corresponding to SEQ ID NO: 20). The variable region sequences of the chimeric CD137 antibody (CD137-009) are shown in the sequence listing herein, SEQ ID NO:28 and SEQ ID NO:29.

[0213] 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 generated using germline humanization (CDR grafting) technology. Humanized V-region genes were designed based on human germline sequences with the closest homology to the rabbit antibody VH and Vκ amino acid sequences. A series of seven VH and three Vκ (VL) germline humanized V-region genes were designed. Structural models of the non-human parent antibody V-regions were generated using the Swiss PDB and analyzed to identify amino acids in the V-region framework that may be important for the binding properties of the antibody. These amino acids were targeted for incorporation into antibodies grafted with one or more variant CDRs. The germline sequences used as the basis for the humanization design are listed in Table 8.

[0214] Table 8. Best-matched human germline V and J segment sequences TIFF0007809062000013.tif41164

[0215] Variant sequences with the lowest incidence of potential T cell epitopes were then selected using iTope™ and TCED™ (T Cell Epitope Database), in silico technologies 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; 20 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.

[0216] The variable region sequences of the humanized CD137 antibody (CD137-009-HC7LC2) are shown in the sequence listing herein, SEQ ID NO: 1 and SEQ ID NO: 5.

[0217] Example 3: Generation of PD-L1 antibodies Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). cDNA encoding amino acids 19-238 of human PD-L1 was cloned into an expression plasmid under Aldevron's intellectual property rights. Antibody PD-L1-547 was generated by immunizing OmniRat animals (transgenic rats expressing a diverse antibody repertoire with fully human idiotypes; Ligand Pharmaceuticals Inc., San Diego, USA) with intradermal application of human PD-L1 cDNA-coated gold particles using a handheld particle bombardment device ("gene gun"). Serum samples were collected after each series of immunizations and tested by flow cytometry against HEK cells transiently transfected with the above expression plasmid to express human PD-L1. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. RNA from hybridomas producing PD-L1-specific antibodies was extracted and sequenced. The heavy and light chain variable regions (SEQ ID NOs: 8 and 12) were gene synthesized and cloned into a human IgG1λ expression vector containing a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A, and K409R (FEAR). Amino acid position numbers are according to EU numbering (corresponding to SEQ ID NO: 19).

[0218] Example 4: Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific IgG1 antibodies were generated by Fab arm exchange under controlled reducing conditions. The basis of this method is the use of complementary CH3 domains to promote heterodimer formation under specific assay conditions, as described in WO2011 / 131746. To create an antibody pair with complementary CH3 domains, F405L and K409R (EU numbering) mutations were introduced into the relevant antibodies.

[0219] To generate bispecific antibodies, two parental complementary antibodies, each at a final concentration of 0.5 mg / mL, were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL of PBS for 5 h at 31°C. 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.

[0220] Bispecific antibodies were generated by combining the following antibodies from Examples 1 and 4: - CD137-009-FEAL antibody in combination with PD-L1-547-FEAR antibody - PD-L1-547-FEAL antibody in combination with CD137-009-FEAR antibody - GEN1046 (PD-L1-547-FEAL antibody in combination with CD137-009-HC7LC2-FEAR antibody) - b12-FEAL antibody in combination with PD-L1-547-FEAR antibody, CD137-009-FEAR, or 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. - PD-L1-547-FEAL or CD137-009-FEAL with the b12-FEAR antibody.

[0221] Example 5: Simultaneous binding of GEN1046 to PD-L1-expressing cells and CD137-expressing cells To measure the dose-response of GEN1046 binding to human PD-L1- and CD137-expressing cells simultaneously, transgenic K562 cells were differentially labeled with fluorescent dyes and doublet formation was analyzed by flow cytometry.

[0222] Human PD-L1 gene-transfected K562 cells (K562_hPD-L1; 6 × 10 6 6 × 10 cells) were fluorescently labeled using the CellTrace™ Violet Cell Proliferation Kit (Cat. No. C34557, Thermo Fisher Scientific GmbH, Dreieich, Germany) in 2 mL of 2.5 μM staining solution at 37°C for 10 minutes. In parallel, human CD137 gene-transfected K562 cells (K562_h4-1BB; 6 × 10 cells) were transfected with the human CD137 gene. 6 1.25 × 10 cells) were fluorescently labeled using the CellTrace™ Far Red Cell Proliferation Kit (Cat. No. C34564, Thermo Fisher Scientific GmbH, Dreieich, Germany) in 2 mL of 0.5 μM staining solution for 10 min at 37°C. Staining was stopped by adding 4 mL of fetal bovine serum (FBS; Cat. No. S0115, Biochrom GmbH, Berlin, Germany). After washing once with RPMI 1640 supplemented with 10% FBS (Cat. No. 11875093, Thermo Fisher Scientific GmbH, Dreieich, Germany), the stained K562_hPD-L1 cells and K562_h4-1BB cells were combined at a 1:1 ratio and plated at 1.25 × 10 cells in RPMI 1640, 10% FBS. 6 The combined K562_hPD-L1 and K562_h4-1BB cells were transferred to a polystyrene 5 mL round-bottom tube (catalog no. 10579511, Fisher Scientific, Schwerte, Germany) and adjusted to 1 × 10 cells / mL. 6Cells were incubated with serial dilutions of antibody (ranging from 0.001 to 100 μg / mL in 10-fold dilutions) in RPMI 1640 with 10% FBS for 15 minutes at 37°C. Samples were immediately analyzed using a FACS Canto™ II flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany) without premixing to preserve any doublets that formed. The K562_hPD-L1 / K562_h4-1BB doublet was identified as a CellTrace™ Violet / CellTrace™ FarRed double-positive population using FlowJo 10.4 software. The percent double-positive cells were plotted as a function of antibody concentration using GraphPad Prism version 8.01 (GraphPad Software, Inc.).

[0223] Figure 1A shows that the addition of GEN1046 induced the formation of CellTrace™ Violet / CellTrace™ FarRed doublets. While K562_hPD-L1 / K562_h4-1BB cocultures incubated with an intermediate concentration of 0.1 μg / mL of GEN1046 exhibited the most significant doublet formation, only moderate doublet formation was observed at GEN1046 concentrations as low as 0.001 μg / mL, and minimal to no doublet formation was detectable at GEN1046 concentrations as high as 100 μg / mL. This observation is consistent with the bell-shaped dose-response curve shown in Figure 1B, which covers the range of antibody concentrations tested, from 0.001 μg / mL to 100 μg / mL. In contrast to GEN1046, the combination of monovalent PD-L1 and CD137 control antibodies PD-L1-547-FEALxb12-FEAR and b12-FEALxCD137-009-HC7LC2-FEAR did not form doublets at all concentrations tested.

[0224] Example 6: Effect of GEN1046 in CD137 reporter assay A schematic diagram of the predicted mechanism of action of the PD-L1xCD137 bispecific antibody is shown in Figure 2.

[0225] To measure the dose-response of GEN1046, which mediates PD-L1 binding-dependent CD137 agonist activity, a luciferase-based CD137 activation reporter assay was performed using adherent, growing human tumor cell lines as a PD-L1 source.

[0226] Human ES-2 (ovarian clear cell carcinoma; ATCC® CRL-1978™) and MDA-MB-231 (breast adenocarcinoma; ATCC® HTB-26™) cells, which endogenously express PD-L1, were cultured at 3 × 10 cells / well in DMEM (catalog no. 10566016, Thermo Fisher Scientific GmbH, Dreieich, Germany) in white flat-bottom 96-well plates (catalog no. 136101, Thermo Fisher Scientific GmbH, Dreieich, Germany). 4Cells were seeded at a density of 1000 cells / well and incubated overnight at 37°C. Cryopreserved Thaw-and-use GloResponse™ NFkB-Luc2P / 4-1BB Jurkat reporter cells (catalog no. CS196003, Promega GmbH, Walldorf, Germany) were thawed the next day, and the contents of one vial were transferred to a 15 mL tube containing 9.5 mL of prewarmed RPMI-1640 supplemented with 1% FBS. The culture medium of adherent ES-2 and MDA-MB-231 cells was discarded, and coculture was initiated by seeding 50 μL of the NFkB-Luc2P / 4-1BB Jurkat cell suspension on top of the ES-2 or MDA-MB-231 cell monolayer. Cells were incubated for 6 hours at 37°C with serial dilutions of antibody (assay concentrations ranging from 0.00128 to 100 μg / mL in 5-fold dilutions) dissolved in RPMI 1640 with 10% FBS. The assay plate was then removed from the incubator and allowed to equilibrate to room temperature (RT) for 10 minutes. Bio-Glo™ Luciferase Reagent (Cat. No. G7941, Promega GmbH, Walldorf, Germany) was reconstituted and pre-warmed to RT. 75 μL of luciferase reagent was added per well and incubated in the dark at RT for 10 minutes. Induced luminescence was measured using an Infinite F200 Pro plate reader (Tecan Deutschland GmbH, Crailsheim, Germany).

[0227] Addition of GEN1046 to ES-2:Jurkat (Figure 3A) and MDA-MB-231:Jurkat reporter cell cocultures (Figure 3B) effectively induced luciferase expression, a readout of CD137 agonist activation, in a concentration-dependent manner following a bell-shaped dose-response curve. A mid-dose level of GEN1046, approximately 0.1 μg / mL, produced the most prominent luminescence signal, whereas lower and higher dose levels were less effective at inducing luciferase expression. Importantly, luciferase expression was undetectable at very low (0.00128 μg / mL GEN1046) and very high (100 μg / mL GEN1046) GEN1046 concentrations. For both cocultures analyzed, incubation with the b12-FEAL control antibody did not result in luciferase expression.

[0228] Example 7: Polyclonal T cell proliferation assay to measure the effect of a bispecific antibody that binds PD-L1 and CD137 To measure T cell proliferation induction in polyclonal activated T cells, PBMCs were incubated with a suboptimal concentration of anti-CD3 antibody (clone UCHT1) combined with the bispecific antibody GEN1046 or a control antibody for T cell activation. Within the PBMC population, cells expressing PD-L1 can bind to the PD-L1-specific arm of the bispecific antibody. In contrast, activated T cells within the population can bind to the CD137-specific arm. In this assay, T cell transactivation via the CD137-specific arm, induced by bispecific antibody-mediated cross-linking of PD-L1-expressing cells and blockade of the PD-L1:PD-1 interaction, is measured as T cell proliferation.

[0229] PBMCs were obtained from buffy coats of healthy donors (Sanquin, Amsterdam, The Netherlands) using a Ficoll gradient (Lonza, lymphocyte separation medium, catalog no. 17-829E) and labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, catalog no. C34554) in PBS according to the manufacturer's instructions. 75,000 CFSE-labeled PBMCs were seeded per well in a 96-well round-bottom plate (Greiner bio-one, catalog no. 650180) and incubated for 4 days at 37°C, 5% CO2 with a suboptimal concentration of anti-CD3 antibody (Stemcell, clone UCHT1, catalog no. 60011; 0.03 μg / mL final concentration), previously determined to induce suboptimal T cell proliferation, and bispecific or control antibodies (0.0032–10 μg / mL) in 200 μL of IMDM GlutaMAX supplemented with 5% human AB serum and 1% penecilin / streptomycin.

[0230] The proliferation of different T cell subsets was analyzed by flow cytometry. Cells were washed with PBS and stained with Fixable Viability Stain 510 (50 μL / well; BD Biosciences, Cat. No. 564406) for 20 min at 4°C to exclude dead cells. After one additional wash with FACS buffer, the cells were stained with PE-CF594-conjugated CD56-specific antibody (BD BioSciences, catalog no. 564849), Pacific Blue-conjugated CD4-specific antibody (BioLegend, catalog no. 300521), AF700-conjugated CD8-specific antibody (BioLegend, catalog no. 301028), BV711-conjugated CD197-specific antibody (CCR7; BioLegend, catalog no. 353228), PE-Cy7-conjugated CD45RO-specific antibody (BioLegend, catalog no. 304230), APC-conjugated CD274-specific antibody (PD-L1; BioLegend catalog no. 329708), and BV605-conjugated CD137-specific antibody (BioLegend, catalog no. 309822) in FACS buffer for 30 minutes at 4°C to distinguish various cell subsets. The cells were washed three times with FACS buffer, then dissolved in 80 μL FACS buffer and measured by FACS Fortessa (BD Biosciences). CFSE dilution was measured in total T cells as well as in different T cell subsets (e.g., CCR7 + CD45RO + Central memory T cells and CCR7 - CD45RO + The expansion index was measured in effector memory T cells (EFCs). Detailed analysis of T cell proliferation based on CFSE peaks, which indicate cell division, was performed using FlowJo 10.4 software. Exported expansion index values ​​were used to plot dose-response curves in GraphPad Prism version 6.04 (GraphPad Software, Inc). The expansion index determines the fold expansion of the entire culture. An expansion index of 2.0 represents a doubling of cell number, whereas an expansion index of 1.0 represents no change in total cell number.

[0231] Figure 4A shows that the bispecific antibody GEN1046 induced T cell expansion. This T cell expansion was increased compared to CD3 prestimulation alone, the isotype control antibody b12-FEAL, and the monovalent PD-L1-control antibody PD-L1-547-FEALxb12-FEAR, which has one irrelevant arm and one arm corresponding to the parent bivalent antibody PD-L1-547-FEAR. GEN1046-induced T cell proliferation was optimal at 0.4 μg / mL, while T cell expansion was less pronounced at lower and higher concentrations. CCR7 + CD45RO + Central memory T cells and CCR7 - CD45RO + When effector memory T cells were analyzed separately (Figure 4B), a similar pattern emerged in which GEN1046 enhanced T cell proliferation, with T cell proliferation being optimal at 0.4 μg / mL.

[0232] Example 8: Antigen-specific CD8 to measure the effect of a bispecific antibody that binds PD-L1 and CD137 + T cell proliferation assay 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-transcribed RNA (IVT-RNA) to express the claudin-6 antigen. T cells were transfected with PD-1 IVT-RNA and a claudin-6-specific HLA-A2-restricted T cell receptor (TCR). This TCR can recognize a claudin-6-derived epitope presented on HLA-A2 on DCs. GEN1046, a PD-L1xCD137 bispecific antibody, crosslinks PD-L1 endogenously expressed on monocyte-derived dendritic cells or tumor cells with CD137 on T cells, blocking the inhibitory PD-1 / PD-L1 interaction and simultaneously clustering CD137, resulting in T cell proliferation. Clustering of the CD137 receptor expressed on T cells activates the CD137 receptor, thereby delivering costimulatory signals to T cells.

[0233] HLA-A2 + Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (Transfusionszentrale, University Hospital, Mainz, Germany). Monocytes were isolated from PBMCs by magnetically activated cell sorting (MACS) using anti-CD14 microbeads (Miltenyi; catalog number 130-050-201) according to the manufacturer's instructions. Peripheral blood lymphocytes (PBLs, CD14-negative fraction) were frozen for future T cell isolation. 1 × 10 cells were used for differentiation into immature DCs (iDCs). 6Monocytes / ml were cultured for 5 days in RPMI GlutaMAX (Life technologies GmbH, catalog no. 61870-044) containing 5% human AB serum (Sigma-Aldrich Chemie GmbH, catalog no. H4522-100ML), sodium pyruvate (Life technologies GmbH, catalog no. 11360-039), non-essential amino acids (Life technologies GmbH, catalog no. 11140-035), 100 IU / ml penicillin-streptomycin (Life technologies GmbH, catalog no. 15140-122), 1000 IU / ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi, catalog no. 130-093-868), and 1000 IU / ml interleukin-4 (IL-4; Miltenyi, catalog no. 130-093-924). Half of the medium was replaced with fresh medium once during these 5 days. iDCs were harvested by collecting nonadherent cells, and adherent cells were detached by incubating with PBS containing 2 mM EDTA for 10 minutes at 37°C. After washing, iDCs were frozen in RPMI GlutaMAX containing 10% v / v DMSO (AppliChem GmbH, Cat. No. A3672,0050) + 50% v / v human AB serum for future antigen-specific T cell assays.

[0234] Antigen specific CD8 + Frozen PBLs and iDCs derived from the same donor were thawed 1 day before starting the T cell proliferation assay. +T cells were isolated from PBLs by MACS technique using anti-CD8 microbeads (Miltenyi, catalog no. 130-045-201) according to the manufacturer's instructions. Approximately 10–15 × 10 T cells were isolated in a 4 mm electroporation cuvette (VWR International GmbH, catalog no. 732-0023) containing 250 μL X-Vivo15 (Biozym Scientific GmbH, catalog no. 881026) using a BTX ECM® 830 Electroporation System (BTX; 500 V, 1 × 3 ms pulse). 6 CD8 + T cells were electroporated with 10 μg of in vitro translated (IVT)-RNA encoding the α chain and 10 μg of IVT-RNA encoding the β chain of claudin-6-specific mouse TCR (HLA-A2-restricted; described in WO2015150327A1) and 0.4–10 μg of IVT-RNA encoding PD-1. Immediately after electroporation, cells were transferred to fresh IMDM medium (Life Technologies GmbH, catalog no. 12440-061) supplemented with 5% human AB serum and allowed to rest for at least 1 h at 37°C and 5% CO2. T cells were labeled with 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Invitrogen, catalog no. C34564) dissolved in PBS according to the manufacturer's instructions and incubated overnight in IMDM medium supplemented with 5% human AB serum.

[0235] 5 × 10 cells were electroporated in 250 μL of X-Vivo15 medium using the electroporation system described above (300 V, 1 × 12 ms pulse). 6 Up to 100 thawed iDCs were electroporated with 0.3–1 µg of full-length claudin-6-encoding IVT-RNA and incubated O / N in IMDM medium supplemented with 5% human AB serum.

[0236] The next day, cells were harvested. The cell surface expression of claudin-6 and PD-L1 on DCs and TCR and PD-1 on T cells was examined by flow cytometry. DCs were stained with an Alexa647-conjugated CLDN6-specific antibody (not commercially available; generated in-house) and an anti-human CD274 antibody (PD-L1, eBioscienes, catalog no. 12-5983), while T cells were stained with an anti-mouse TCR β chain antibody (Becton Dickinson GmbH, catalog no. 553174) and an anti-human CD279 antibody (PD-1, eBioscienes, catalog no. 17-2799). Five thousand electroporated DCs were incubated with 50,000 electroporated CFSE-labeled T cells in the presence of bispecific or control antibodies in a 96-well round-bottom plate containing IMDM GlutaMAX supplemented with 5% human AB serum. Five days later, T cell proliferation was measured by flow cytometry. Detailed analysis of T cell proliferation based on CFSE peaks, indicating cell division, was performed using FlowJo 10.4 software. Exported expansion index values ​​were used to plot dose-response curves in GraphPad Prism version 6.04 (GraphPad Software, Inc.). The expansion index determines the fold expansion of the entire culture. An expansion index of 2.0 represents a doubling of cell numbers, whereas an expansion index of 1.0 represents no change in total cell numbers.

[0237] Figure 5 shows that GEN1046 dose-dependently enhanced T cell proliferation compared to the isotype control antibody b12-FEAL, as reflected by an increased proliferation index at concentrations ≥0.004 μg / mL. GEN1046-induced T cell proliferation was optimal at 0.03–0.11 μg / mL and decreased slightly at the highest concentration tested, demonstrating a bell-shaped dose-response curve.

[0238] Example 9: Antigen-specific CD8 to measure cytokine release induced by a bispecific antibody that binds PD-L1 and CD137 + T cell proliferation assay Induction of cytokine release by the bispecific antibody GEN1046, which targets PD-L1 and CD137, was measured in antigen-specific assays performed essentially as described in Example 8.

[0239] T cells were electroporated with 10 μg of TCR α-chain-encoding RNA and 10 μg of β-chain-encoding RNA, with or without 2 μg of PD-1-encoding IVT RNA. Electroporated T cells were not CFSE-labeled (as described above), but were transferred immediately after electroporation to fresh IMDM medium (Life Technologies GmbH, catalog no. 12440-061) supplemented with 5% human AB serum. iDCs were electroporated with 5 μg of claudin-6 (CLDN6)-encoding RNA as described above. After O / N incubation, DCs were stained with Alexa647-conjugated CLDN6-specific antibodies, and T cells were stained with anti-mouse TCR β-chain and anti-human CD279 antibodies, as described above.

[0240] Five thousand electroporated DCs were incubated with 50,000 electroporated T cells in the presence of various concentrations of the bispecific antibody GEN1046 or the control antibody b12-FEAL in a 96-well round-bottom plate containing IMDM GlutaMAX supplemented with 5% human AB serum. After the 48-hour incubation period, the plate was centrifuged at 500 × g for 5 minutes, and the supernatant was carefully transferred from each well to a fresh 96-well round-bottom plate and stored at -80°C until cytokine analysis on the MSD® platform. Supernatants collected from the antigen-specific proliferation assay were analyzed for cytokine levels of 10 different cytokines using the MSD V-Plex Human Proinflammatory panel 1 (10-Plex) kit (Meso Scale Diagnostics, LLC., catalog number K15049D-2) on a MESO QuickPlex SQ 120 instrument (Meso Scale Diagnostics, LLC., catalog number R31QQ-3) according to the manufacturer's instructions.

[0241] The addition of GEN1046 resulted in a concentration-dependent increase in the secretion of primarily IFN-γ, TNF-α, IL-13, and IL-8 (Figure 6). This was optimal at concentrations between 0.04 and 0.33 μg / mL. Lower dose levels, as well as a dose as high as 1 μg / mL, were less effective in inducing these cytokines, demonstrating a bell-shaped dose-response curve. When comparing T cell:DC cocultures in which T cells were not electroporated with PD-1 RNA to T cell:DC cocultures in which T cells were electroporated with 2 μg of PD-1 RNA, slightly higher cytokine levels were detectable in the cocultures without PD-1 RNA electroporation. This was observed for both the GEN1046 dose-response curve and the b12-FEAL control antibody values.

[0242] Example 10: Ex vivo TIL expansion assay to evaluate the effect of CD137xPD-L1 bispecific antibodies on tumor-infiltrating lymphocytes To evaluate the effect of CD137-009-FEALxPD-L1-547-FEAR on tumor-infiltrating lymphocytes (TILs), ex vivo culture of human tumor tissue was performed as follows. Freshly excised human tumor tissue specimens were washed three times by transferring the isolated tumor mass from one well of a 6-well plate (Fisher Scientific Catalog No. 10110151) to the next well using a spatula or serological pipette. The wash 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, tumors were dissected with a surgical blade (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, 10% human serum albumin (HSA, CSL Behring, catalog number PZN-6446518), 1% Pen / Strep, 1% Fungizone, and 10 U / mL IL-2 (Proleukin® S, Novartis Pharma, catalog number 02238131)). CD137-009-FEALxPD-L1-547-FEAR was added at the indicated final concentrations. The culture plate was incubated at 37°C and 5% CO2. After 72 hours, 1 mL of fresh TIL medium containing the indicated concentrations of bispecific antibodies was added to each well. The wells were monitored microscopically every other day for the development of TIL clusters. When more than 25 TIL microclusters were detected in each well, the wells were transferred individually. To split the TIL cultures, 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. An additional 2 mL of TIL medium was then added to each well.

[0243] After a total culture period of 10–14 days, TILs were collected and analyzed by flow cytometry. Cells were stained with the following reagents: 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) diluted 1:50. To quantitatively compare the cells obtained between different treatment groups, the cell pellet was resuspended after a final washing step in FACS buffer supplemented with BD™ CompBeads (BD biosciences, catalog number 51-90-9001291). Flow cytometry analysis was performed using a BD FACSCanto™ II flow cytometer (Becton Dickinson), and the resulting data was analyzed using FlowJo 7.6.5 software. By normalizing the obtained 7AAD-negative cell fraction to the obtained bead number, the relative live TIL count per 1,000 beads, CD3 + CD8 + T cell count, CD3 + CD4 + T cell count and CD3 - CD56 + The number of NK cells was calculated.

[0244] Figure 7 shows the analysis of TIL expansion from human non-small cell lung cancer tissue specimens. Here, the following concentrations of CD137-009-FEALxPD-L1-547-FEAR were added: 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL. Tissue specimens from the same patients without antibody served as negative controls. After 10 days of culture, TILs were collected and analyzed by flow cytometry. Five samples (from the five initial wells) were measured for each antibody concentration from different wells of a 24-well plate. In all samples cultured with the bispecific antibody, the number of live TILs increased compared to control samples without antibody. Overall, a significant increase (up to 10-fold) in live TILs was observed when 0.1 μg / mL CD137-009-FEALxPD-L1-547-FEAR was added to the cultures (Figure 7A). When analyzed separately, CD3 + CD8 + A strong effect on T cell expansion was observed, which was significant at 0.1 μg / mL CD137-009-FEALxPD-L1-547-FEAR (Figure 7B; 7.4-fold expansion compared to control). + CD4 + T cells expanded only slightly, and these expansions were not significant compared to cultures without antibody (Fig. 7C). - CD56 + The most significant TIL expansion was observed for NK cells (Figure 7D; up to 64-fold increase compared to control), which was significant at 0.1 μg / mL CD137-009-FEALxPD-L1-547-FEAR.

[0245] Example 11: Pharmacodynamic evaluation of GEN1046 in peripheral blood in patients with advanced solid tumors To investigate the biological activity of various dose levels of GEN1046 in patients with advanced tumors, blood and serum samples were collected at baseline and at multiple time points during treatment. Based on the mechanism of action of GEN1046, biologically active dose levels were predicted to modulate circulating levels of interferon-γ (IFN-γ) and interferon-γ-inducible protein 10 (IP-10) and induce proliferation of peripheral CD8 T cells.

[0246] Serum samples were collected from patients at baseline and at multiple time points after GEN1046 administration in Cycles 1 and 2 (Day 1 [2 and 4-6 hours post-dose], Day 2, Day 3, Day 8, and Day 15) to determine serum levels of IFN-γ and IP-10. Serum levels of IFN-γ and IP-10 were measured by Meso Scale Discovery (MSD) multiplex immunoassay (catalog number K15209G) according to the manufacturer's instructions.

[0247] To measure peripheral modulation of immune cell subsets, peripheral blood immunophenotyping was performed on whole blood collected in EDTA tubes at baseline and multiple time points (days 2, 3, 8, and 15) after GEN1046 administration in cycles 1 and 2. 100 μL of whole blood was added to fluorochrome-conjugated monoclonal antibodies that specifically bind to cell surface antigens: CD45RA-FITC (clone LEU-18, BD Biosciences, catalog number 335039), CCR7-BV510 (clone 3D12, BD Biosciences, catalog number 563449), and CD8-PerCP-Cy5.5 (clone RPA-T8, BD Biosciences, catalog number 560662). After incubation on ice, the stained samples were treated with FACS Lysing Solution (BD Biosciences, catalog number 349202) to lyse red blood cells. Excess antibody and cell debris were removed by washing with Stain Buffer (BD Biosciences, Cat. No. 554656). After lysis / washing, cells were fixed and permeabilized by incubation with Permeabilizing Solution 2 buffer (BD Biosciences, Cat. No. 340973). Cells were then washed, resuspended in Stain Buffer, and incubated on ice with an antibody against Ki67 (BV421 B56, BD Biosciences, Cat. No. 562899) to detect proliferating cells. After incubation, excess antibody was removed by washing with Stain Buffer. Cells were resuspended in Stain Buffer and stained and acquired within 1 hour on a BD FACSCanto™ II flow cytometer (Becton Dickinson).

[0248] Administration of GEN1046 to cancer patients modulated circulating levels of IFN-γ and IP-10 as well as proliferating effector memory CD8 T cells (Table 9 and Figure 13). In a preliminary data set shown in Table 9, IFN-γ levels increased more than two-fold in the first treatment cycle across all dose levels tested. The greatest increases were detected at the 50 mg and 80 mg dose levels, with most patients (75%) in the 80 mg cohort experiencing a more than two-fold increase (Table 9). GEN1046 inhibited Ki67 + CD8 + CD45RA - CCR7 - It also induced the proliferation of effector memory CD8+ T cells as measured by an increase in the frequency of T cells. The increase in the frequency of proliferating CD8+ T cells was comparable to the changes observed with modulation of circulating levels of IFNγ. + The greatest and more consistent modulation of effector memory T cells was observed in patients in the 80 mg cohort. Notably, the magnitude of changes in circulating levels of both IFN-γ and proliferating effector memory CD8 T cells was smaller in the 400 mg cohort compared to the 25-200 mg cohort. These results demonstrate that GEN1046 induces an immune response characterized by modulation of immune effector cells and soluble factors critical for the generation of antitumor immune responses, with the 80 mg dose level producing a large magnitude of response.

[0249] In the data set shown in Figure 13, increases in IFN-γ and IP-10 were observed at dose levels ≤ 200 mg in the first treatment cycle (Figures 13A-B). Increases in IFN-γ and IP-10 were also observed at dose levels ≥ 400 mg, although the maximum fold change from baseline during the first treatment cycle was significantly less compared to lower dose levels. GEN1046 also inhibited Ki67 + CD8 + T cells and Ki67 + CD8 + CD45RA - CCR7 - Total CD8 as measured by increased frequency of T cells +It also induced proliferation of T cells and effector memory CD8+ T cells (Figure 13C-D). The changes in proliferating CD8+ T cells were comparable to those observed with modulation of circulating levels of IFNγ and IP-10. + The greatest and more consistent modulation of effector memory T cells was observed in patients treated at dose levels ≤200 mg. In the ≥400 mg cohort, the magnitude of changes in proliferating effector memory CD8 T cells and total CD8 T cells was significantly less than in the 25-200 mg cohort. These results demonstrate that GEN1046 induces an immune response characterized by modulation of immune effector cells and soluble factors critical for the generation of antitumor immune responses, with responses of greater magnitude occurring at dose levels ≤200 mg.

[0250] Table 9. GEN1046 Modulation of Peripheral Pharmacodynamic Endpoints in Cancer Patients: Peak Fold Change from Baseline by Dose Level During Cycle 1 a TIFF0007809062000014.tif89162Preliminary data as of January 27, 2020. n: number of patients per dose cohort; Min: smallest measured value; Q1: 25th percentile; Q3: 75th percentile; Max: largest measured value. a Pharmacodynamic evaluations, including changes in circulating levels of interferon-γ and effector memory T cells, were performed using blood samples from patients with advanced solid tumors enrolled in the dose-escalation phase of an open-label, multicenter safety study of GEN1046 (NCT03917381). b Circulating levels of interferon-γ in serum samples were measured at baseline and at multiple time points after GEN1046 administration (Day 1 [2 hours and 4-6 hours post-dose], Day 2, Day 3, Day 8, and Day 15) in Cycles 1 and 2. Interferon-γ levels in serum samples were determined by Meso Scale Discovery (MSD) multiplex immunoassay. cPeripheral blood immunophenotyping was performed on whole blood collected at baseline and at multiple time points (days 2, 3, 8, and 15) after GEN1046 administration in cycles 1 and 2. Proliferation (Ki67 + ) effector memory CD8 T cells (CD8 + CD45RA - CCR7 - The frequency of erythrocytes (e.g., erythrocytes ... and thymocytes) was assessed in whole blood samples by flow cytometry.

[0251] Example 12: Preliminary data from clinical trials: Study design The clinical trial for GCT1046-01 (ClinicalTrials.gov Identifier: NCT03917381) was designed as a two-part study, including an ongoing dose-escalation part and a planned expansion part.

[0252] This study was designed as an open-label, multicenter, Phase I / IIa safety study of GEN1046 (DuoBody® - PD-L1x4-1BB). The study consisted of two parts: first-in-human (FIH) dose escalation (Phase I) and expansion (Phase IIa). Figure 8 shows a schematic diagram of the clinical trial design.

[0253] Dose escalation Dose escalation was designed to evaluate GEN1046 in subjects with solid malignancies with the goal of determining the maximum tolerated dose (MTD) or maximum administered dose (MAD) and / or recommended phase 2 dose (RP2D).

[0254] For dose escalation, subjects were required to be male or female, aged 18 years or older, and to have measurable disease per RECIST 1.1.

[0255] Subjects were required to have metastatic or unresectable histologically or cytologically confirmed non-CNS solid tumors, for which standard therapies likely to provide clinical benefit are unavailable or who are not candidates for such available therapies, and who, in the opinion of the investigator, may benefit from experimental therapy with GEN1046.

[0256] In dose escalation, subjects received an infusion of GEN1046 once every three weeks (1Q3W) until protocol-defined criteria for treatment discontinuation were met, e.g., radiographic disease progression or clinical progression. GEN1046 was administered using an iv infusion over a minimum of 60 minutes on day 1 of each 3-week treatment cycle (21 days). The concept of the study design is shown in Figure 8.

[0257] The 1Q3W dose escalation was designed to potentially (depending on data collected during the study) evaluate GEN1046 at seven main dose levels: a constant 25 mg, 80 mg, 200 mg, 400 mg, 800 mg, 1200 mg, and 1600 mg, and six optional intermediate dose levels: a constant 50 mg, 140 mg, 300 mg, 600 mg, 1000 mg, and 1400 mg.

[0258] The recommended phase 2 dose (RP2D) was based on a review of available safety and dosing information and could be less than the maximum tolerated dose (MTD).

[0259] Increase The purpose of the expansion is to provide further data on the safety, tolerability, MoA, PK, and antitumor activity of the selected dose / schedule.

[0260] The expansion is designed to begin recruitment in up to six tumor types (seven parallel cohorts): NSCLC, EC, UC, TNBC, SCCHN, and cervical cancer. Further expansion cohorts for additional tumor types may be established based on preliminary efficacy signals generated in dose escalation. The sponsor will determine the priority for establishing disease-specific expansion cohorts based on data generated in dose escalation.

[0261] NSCLC expansion cohort The NSCLC expansion cohort must include subjects with squamous histology as well as subjects with non-squamous histology.

[0262] Because response rates and other disease-related outcomes may differ in PD-1 / PD-L1-naive versus PD-1 / L1-pretreated populations, NSCLC patients were divided into different cohorts to ensure sufficient evidence of preliminary efficacy. Cohort 2 aims to explore preliminary efficacy in PD-1 / L1-naive NSCLC patients for whom SOC with PD-1 / L1 inhibitors is limited or unavailable. If preliminary clinical evidence, as confirmed by DMC review of the totality of the data, suggests substantial improvement over available therapies in populations with high unmet medical need (e.g., PD-L1-poor or -negative), sponsors may request that Cohort 2 be established in regions where access to PD-1 / L1 inhibitors is not limited.

[0263] UC expansion cohort The UC cohort was designed to include both platinum-based chemotherapy eligible and ineligible subjects.

[0264] SCCHN and TNBC growth cohort The SCCHN and TNBC cohorts may include subjects who have received prior treatment with a PD-1 / PD-L1 inhibitor, as well as subjects who have not received prior treatment with a PD-1 / L1 inhibitor.

[0265] Inclusion criteria Subjects are eligible for inclusion in the study only if all of the following criteria are met:

[0266] Subjects must be male or female, aged 18 years or older, and must have measurable disease according to RECIST 1.1.

[0267] Subjects must have a histologically or cytologically confirmed diagnosis of relapsed or refractory, advanced and / or metastatic NSCLC, EC, UC, TNBC, SCCHN, or cervical cancer that is no longer a candidate for or has refused standard therapy (if the subject was available and eligible for each treatment) and has failed anti-cancer therapy as follows:

[0268] Expansion cohort 1 (NSCLC): pretreated with PD-1 / L1 NSCLC subjects who have received up to four systemic conditioning regimens (adjuvant and maintenance therapy are considered part of one line of treatment) for advanced / metastatic disease and have radiographic disease progression during or after the last conditioning therapy.

[0269] Subjects must have a histologic or cytologic diagnosis of non-squamous NSCLC without epidermal growth factor receptor (EGFR) sensitizing mutations and / or anaplastic lymphoma (ALK) translocations / ROS1 rearrangements. EGFR sensitizing mutations are those amenable to treatment with approved tyrosine kinase inhibitors (TKIs). Documentation of EGFR and ALK status must be available per local assessment. If documentation of EGFR and ALK status is not available, sponsor medical monitor approval is required prior to enrollment.

[0270] Subjects must have received platinum-based therapy (or another chemotherapy regimen due to platinum ineligibility, eg, a gemcitabine-containing regimen).

[0271] Subjects must have received prior treatment with a PD-1 / L1 inhibitor, alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with SD or BOR of PD while on a CPI-containing regimen with treatment duration up to 16 weeks.

[0272] Expansion Cohort 2 (NSCLC) - PD-1 / L1 naive NSCLC subjects who have received up to four systemic conditioning regimens (adjuvant and maintenance therapy are considered part of one line of treatment) for advanced / metastatic disease and have radiographic disease progression during or after the last conditioning therapy.

[0273] Subjects must have a histologic or cytologic diagnosis of non-squamous NSCLC without epidermal growth factor receptor (EGFR) sensitizing mutations and / or anaplastic lymphoma kinase (ALK) translocations / ROS1 rearrangements. EGFR sensitizing mutations are those amenable to treatment with approved tyrosine kinase inhibitors (TKIs). Documentation of EGFR and ALK status must be available per local assessment. If documentation of EGFR and ALK status is not available, sponsor medical monitor approval is required prior to enrollment.

[0274] Subjects must have received platinum-based therapy (or another chemotherapy regimen due to platinum ineligibility, eg, a gemcitabine-containing regimen).

[0275] Subjects must have received prior treatment with a PD-1 / L1 inhibitor. It should not happen .

[0276] Expansion Cohort 3 (UC): UC subjects who have received up to four systemic conditioning regimens (adjuvant and maintenance therapy are considered part of one line of treatment) for locally advanced / metastatic disease and have radiographic disease progression (of the bladder, ureter, urethra, or renal pelvis) during or after the last conditioning regimen.

[0277] Subjects must have received prior treatment with a PD-1 / L1 inhibitor, alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with SD or BOR of PD while on a CPI-containing regimen with treatment duration up to 16 weeks.

[0278] Regional results from the most recent PD-L1 trial (if available) must be provided prior to enrollment.

[0279] Cohort 3a: For subjects eligible to receive platinum-based therapy: Subjects must have received platinum-based chemotherapy.

[0280] Cohort 3b: For subjects not eligible for platinum-based therapy: Subjects must not be eligible for platinum-based or cisplatin-containing chemotherapy.

[0281] Expansion Cohort 4 (EC): EC subjects who have received up to four systemic conditioning regimens (adjuvant and maintenance therapy are considered part of one line of treatment) for advanced / metastatic disease and have radiographic disease progression during or after their last conditioning regimen.

[0282] Subjects must have epithelial endometrial histology, including endometrioid carcinoma, serous carcinoma, squamous cell carcinoma, clear cell carcinoma, or carcinosarcoma. Sarcomas and mesenchymal ECs are excluded.

[0283] Subjects must have received prior treatment with a PD-1 / L1 inhibitor. It should not happen(Established local labels / access must be respected).

[0284] Expansion Cohort 5 (TNBC): TNBC determined to be HER2-negative [HER2 negative by FISH] assay (HER2 to CEP17 non-amplified ratio <2.0, single-probe mean HER2 gene copy number <4 signals / cell); or Alternatively, HER2 protein expression by IHC result is 1+ negative or IHC 0- negative according to regional evaluation and ER and PgR negative status (defined as <1% hormone receptor expressing cells by IHC analysis).Subjects who have received at least one but no more than four systemic conditioning regimens (adjuvant and maintenance therapy are considered part of one line of treatment) for locally advanced / metastatic disease, including but not limited to anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimens, and have radiographic disease progression during or after the last conditioning therapy.

[0285] Subjects with a prior history of phenotypically distinct breast cancer must have confirmed TNBC in a biopsy obtained after the subject's last prior systemic therapy.

[0286] Cohort 5a - Subjects who have received prior PD-1 / L1 inhibitor therapy: Subjects must have received prior treatment with a PD-1 / L1 inhibitor, alone or in combination, and must have radiographic disease progression at the time of treatment.

[0287] Cohort 5b - PD-1 / L1 inhibitor-naive subjects: Subjects must have received prior treatment with a PD-1 / L1 inhibitor. It should not happen .

[0288] Expansion Cohort 6 (SCCHN): Subjects with recurrent or metastatic SCCHN (oral cavity, pharynx, larynx) who have received up to four systemic conditioning regimens (adjuvant and maintenance therapy are considered part of one line of treatment) for recurrent / metastatic disease and have radiographic PD during or after the last conditioning regimen.

[0289] Subjects must have disease progression during or after prior treatment with platinum-based chemotherapy (alternative concomitant chemotherapy is permitted if the subject has documented platinum-ineligible status).

[0290] Cohort 6a - Subjects who have received prior PD-1 / L1 inhibitor therapy: Subjects must have received prior treatment with a PD-1 / L1 inhibitor, alone or in combination, and must have radiographic disease progression on treatment. Sponsor approval is required for subjects with SD or BOR of PD while on a CPI-containing regimen with treatment duration up to 16 weeks.

[0291] Cohort 6b - PD-1 / L1 inhibitor-naive subjects: Subjects must have received prior treatment with a PD-1 / L1 inhibitor. It should not happen .

[0292] Expansion Cohort 7 (Cervical Cancer): Cervical cancer subjects who have received at least one, but not more than four, systemic conditioning regimens for recurrent / metastatic disease, including a combination of chemotherapy and bevacizumab (according to applicable labeling), unless the subject is bevacizumab-ineligible according to local criteria, and have radiographic disease progression during or after the last conditioning regimen (chemotherapy administered in the adjuvant or neoadjuvant setting or in combination with radiation therapy must not have been included in the previous line of therapy).

[0293] Subjects must have cervical cancer of squamous cell, adenocarcinoma, or adenosquamous histology.

[0294] Subjects must have received prior treatment with a PD-1 / L1 inhibitor. It should not happen (Established local labels / access must be respected).

[0295] result Dose escalation The following preliminary results were obtained during dose escalation: Table 10 shows the best overall response (RECIST v1.1) by dose level at enrollment and dosing for a total of 30 patients (data extraction date: February 3, 2020).

[0296] Tables 11 and 12 show the objective response rate and confirmed objective response rate, respectively, by dose level at enrollment and dosing for a total of 61 patients (RECIST v1.1) (data cutoff: October 12, 2020).

[0297] The best percent change in tumor size from baseline in all patients is shown in Figure 9. Disease control occurred in 40 / 61 (65.6%) patients in the dose-escalation phase. Partial responses (PRs) were observed in four patients with triple-negative breast cancer, ovarian cancer, or non-small cell lung cancer (NSCLC). Thirty-six patients maintained stable disease.

[0298] The clinical activity (best change in tumor size from baseline) observed in NSCLC patients is shown in Figure 10 (data cutoff: October 12, 2020). Of six NSCLC patients, all of whom had previously received checkpoint immunotherapy, two had an unconfirmed PR, two maintained stable disease, and two progressed.

[0299] Table 10. Best overall response by dose level (RECIST v1.1) TIFF0007809062000015.tif20680 1 uPR

[0300] Table 11. Objective Response Rate - Dose Escalation TIFF0007809062000016.tif23674

[0301] Table 12. Confirmed Response Rate - Dose Escalation TIFF0007809062000017.tif23984

[0302] Increase: Expansion Cohort 1: As of October 12, 2020, 24 patients have been enrolled in Expansion Cohort 1, which includes patients with NSCLC (pretreated with PD-1 / L1). Twelve patients with confirmed progression on or after checkpoint inhibitor therapy were evaluable after baseline (Figure 11).

[0303] Conclusion: GEN1046 is a first-in-class, next-generation PD-L1x4-1BB bispecific antibody with an acceptable safety profile and promising early clinical activity, unlike existing 4-1BB agonists.

[0304] In the dose-escalation phase of this Phase I / IIa study, GEN1046 demonstrated a manageable safety profile and preliminary clinical activity in a heavily pretreated advanced solid tumor population.

[0305] Most adverse events were mild to moderate. Treatment-related grade 3 transaminase elevations resolved with corticosteroids. No treatment-related bilirubin or grade 4 transaminase elevations were observed. Six patients experienced dose-limiting toxicities (DLTs). The maximum tolerated dose (MTD) was not reached.

[0306] Clinical benefit was observed across different dose levels in patients, including those who were resistant to previous immunotherapy and those with tumors that were generally insensitive to immune checkpoint inhibitors (ICIs).

[0307] Disease control was achieved in 65.6% of patients, including partial responses in triple-negative breast cancer (1), ovarian cancer (1), and ICI-pretreated NSCLC (2).

[0308] Modulation of pharmacodynamic endpoints was observed across a wide range of dose levels, demonstrating biological activity.

[0309] Example 13: Pharmacokinetic / Pharmacodynamic Model We developed an integrated semi-mechanistic PK / PD (pharmacokinetic / pharmacodynamic) model that assumes distribution of GEN1046 to the central and peripheral PK compartments, as well as distribution to the tumor and lymphatic compartments. The model utilizes PK and pharmacodynamic data from the literature and physiological parameters to parameterize PD-L1 and 4-1BB expression and T cell trafficking to these compartments. The model compartments consist of well-mixed 2D and 3D spaces and free drug movement between all compartments. Furthermore, the model incorporates the dynamic binding of GEN1046 to PD-L1 and 4-1BB to predict trimer formation (crosslinking with PD-L1 and 4-1BB) and receptor occupancy (RO) for PD-L1 and 4-1BB in the tumor. Simulations showed that trimer formation was optimal at an 80 mg dose. The model-predicted RO for PD-L1 and 4-1BB in the tumor appeared to be sufficient at doses of 80–140 mg. Increasing the dose to ≥200 mg reduced trimer formation. Furthermore, based on available clinical pharmacodynamic data, dose levels ≤200 mg demonstrated greater magnitude and consistent modulation of peripheral pharmacodynamic endpoints (IFNγ and expanded Ki67+ effector memory CD8+ T cells). Considering PK / pharmacodynamic modeling predictions and available clinical data, the optimal dose of GEN1046 was predicted to be in the range of 80–140 mg. A 100 mg dose administered 1 Q3W maintained maximal trimer formation and mean RO(%) for PD-L1 at reasonable levels throughout the entire dosing interval.

[0310] The model-predicted maximum trimer formation and receptor occupancy for 100 mg 1Q3W PDL1 is shown in FIG.

Claims

1. 1. A medicament for use in a method for reducing or preventing tumor progression or treating cancer in a subject, comprising a binding agent, the binding agent comprises a first binding region that binds to human CD137, e.g., human CD137 having the sequence set forth in SEQ ID NO:24, and a second binding region that binds to human PD-L1, e.g., human PD-L1 having the sequence set forth in SEQ ID NO:26; a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3, and the CDR3 sequence shown in SEQ ID NO:4, and a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:6, the CDR2 sequence shown as GAS, and the CDR3 sequence shown in SEQ ID NO:7; and b) the second binding region comprises a heavy chain variable region (VH) comprising 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 a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:13, the CDR2 sequence shown as DDN, and the CDR3 sequence shown in SEQ ID NO:14; the binding agent is an antibody or a multispecific antibody, and The method comprises administering the binding agent in at least one treatment cycle at each dose and / or each treatment cycle: a) about 0.3 to 2.5 mg / kg body weight or about 25 to 200 mg in total; and / or b) Approximately 2.1×10 -9 ~1.7×10 -8 mol / kg body weight or a total of approximately 1.7 × 10 -7 ~1.4×10 -6 mol The pharmaceutical composition comprising a step of administering to the subject an amount of

2. 2. The method of claim 1, wherein said amount of binding agent results in proliferation, cytokine production, maturation, and long-term survival of T cells, and renders such T cells less susceptible to inhibition by PD-L1.

3. said amount of binding agent being a) about 1.25 mg / kg body weight or about 100 mg in total; and / or b) Approximately 8.5×10 -9 mol / kg body weight or a total of approximately 6.8 × 10 -7 mol The pharmaceutical composition according to claim 1 or 2,

4. The pharmaceutical agent according to any one of claims 1 to 3, wherein the binding substance activates human CD137 when bound to human CD137, and inhibits the binding of human PD-L1 to human PD-1 when bound to PD-L1.

5. a) the first binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:1, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:5; and b) the second binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:8, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO:12; The pharmaceutical composition according to any one of claims 1 to 4.

6. a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:5; and b) the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:8 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:12; The pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical according to any one of claims 1 to 6, wherein the binding substance is a bispecific antibody.

8. The pharmaceutical of any one of claims 1 to 7, wherein the binding substance is in the form of a full-length antibody or an antibody fragment.

9. The pharmaceutical according to any one of claims 1 to 8, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

10. The pharmaceutical according to claim 9, wherein the complementarity determining regions and the framework regions are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

11. i) a polypeptide comprising, consisting of, or consisting essentially of a first heavy chain variable region (VH) and a first heavy chain constant region (CH); and ii) a polypeptide comprising, consisting of, or consisting essentially of a second heavy chain variable region (VH) and a second heavy chain constant region (CH). The pharmaceutical composition according to any one of claims 1 to 10, comprising:

12. i) a polypeptide comprising a first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) a polypeptide comprising a second light chain variable region (VL) and further comprising a second light chain constant region (CL). The pharmaceutical composition according to any one of claims 1 to 11, comprising:

13. the binding agent is an antibody comprising a first binding arm and a second binding arm, 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 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). The pharmaceutical composition according to any one of claims 1 to 12, comprising:

14. i) a first heavy chain and a first light chain comprising said first binding region capable of binding to CD137; and ii) a second heavy chain and a second light chain comprising said second binding region capable of binding to PD-L1; The pharmaceutical composition according to any one of claims 1 to 13, comprising:

15. The binding substance i) a first heavy chain and a first light chain comprising the first binding region capable of binding to CD137, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a second light chain comprising the second binding region capable of binding to PD-L1, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region. The pharmaceutical composition according to any one of claims 1 to 14, comprising:

16. The pharmaceutical agent according to any one of claims 11 to 15, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises one or more of a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region, preferably at least a hinge region, a CH2 region, and a CH3 region.

17. The pharmaceutical agent according to any one of claims 11 to 16, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

18. The medicament of any one of claims 11 to 17, 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 of a human IgG1 heavy chain according to EU numbering has been 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 of a human IgG1 heavy chain according to EU numbering has been substituted, and the first heavy chain and the second heavy chain do not have substitutions at the same positions.

19. The pharmaceutical agent of claim 18, wherein (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.

20. The medicament of any one of claims 1 to 19, wherein the binding substance induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2, and CH3 regions.

21. The pharmaceutical agent of claim 20, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) have been modified so as to induce Fc-mediated effector function to a lesser extent than an identical antibody except that the antibody comprises unmodified first heavy chain constant region (CH) and second heavy chain constant region (CH).

22. The pharmaceutical agent of claim 21, wherein each of the unmodified first heavy chain constant region (CH) and second heavy chain constant region (CH) comprises the amino acid sequence shown in SEQ ID NO:

15.

23. The pharmaceutical agent of any one of claims 21 to 22, wherein the Fc-mediated effector function is measured by binding to an Fcγ receptor, binding to C1q, or induction of Fc-mediated cross-linking of an Fcγ receptor.

24. The pharmaceutical agent of claim 23, wherein the Fc-mediated effector function is measured by binding to C1q.

25. the first heavy chain constant region and the second heavy chain constant region are modified such that binding of the antibody to C1q is reduced 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%; C1q binding is preferably measured by ELISA. The pharmaceutical composition according to any one of claims 20 to 24.

26. The medicament of any one of claims 11 to 25, 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 positions L234, L235, D265, N297, and P331 of a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

27. The pharmaceutical agent of claim 26, wherein the positions corresponding to positions L234 and L235 of the human IgG1 heavy chain according to EU numbering are F and E in the first heavy chain and the second heavy chain, respectively.

28. The pharmaceutical agent of claim 26 or 27, wherein the positions corresponding to positions L234, L235, and D265 of the human IgG1 heavy chain according to EU numbering are F, E, and A in the first heavy chain constant region and the second heavy chain constant region (HC), respectively.

29. the positions corresponding to positions L234 and L235 of a human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F and E, respectively; and (i) the first heavy chain constant region has L at a position corresponding to F405 of the human IgG1 heavy chain according to EU numbering, and R at a position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain; or (ii) the first heavy chain constant region has R at a position corresponding to K409 of the human IgG1 heavy chain according to EU numbering, and the second heavy chain has L at a position corresponding to F405 of the human IgG1 heavy chain according to EU numbering; The pharmaceutical composition according to any one of claims 26 to 28.

30. the positions corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F, E, and A, respectively; and (i) the first heavy chain constant region has L at a position corresponding to F405 of the human IgG1 heavy chain according to EU numbering, and R at a position corresponding to K409 of the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region; or (ii) the first heavy chain has R at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering, and the second heavy chain has L at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering; The pharmaceutical composition according to any one of claims 26 to 29.

31. the constant region of the first heavy chain and / or the second heavy chain comprises: a) the sequence shown in SEQ ID NO:15 or SEQ ID NO:30 [IgG1-FC]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution.

31. The medicament of any one of claims 11 to 30, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

32. the constant region of the first heavy chain or the second heavy chain, e.g., the second heavy chain, comprises: a) the sequence shown in SEQ ID NO: 16 or SEQ ID NO: 31 [IgG1-F405L]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 9 substitutions compared to the amino acid sequence defined in a) or b), for example at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 substitution.

32. The medicament of any one of claims 11 to 31, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

33. the constant region of the first heavy chain or the second heavy chain, e.g., the first heavy chain, comprises: a) the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 32 [IgG1-F409R]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution.

33. The medicament of any one of claims 11 to 32, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

34. the constant region of the first heavy chain and / or the second heavy chain comprises: a) the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 33 [IgG1-Fc_FEA]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 7 substitutions compared to the amino acid sequence defined in a) or b), for example at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution.

34. The medicament of any one of claims 11 to 33, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

35. the constant region of the first heavy chain and / or the second heavy chain, e.g., the second heavy chain, comprises: a) the sequence shown in SEQ ID NO: 19 or SEQ ID NO: 34 [IgG1-Fc_FEAL]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions compared to the amino acid sequence defined in a) or b), for example at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution.

35. The medicament of any one of claims 11 to 34, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

36. the constant region of the first heavy chain and / or the second heavy chain, e.g., the first heavy chain, comprises: a) the sequence shown in SEQ ID NO:20 or SEQ ID NO:35 [IgG1-Fc_FEAR]; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions compared to the amino acid sequence defined in a) or b), for example at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution.

36. The medicament of any one of claims 11 to 35, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of:

37. The pharmaceutical of any one of claims 1 to 36, wherein the binding substance comprises a kappa (κ) light chain constant region.

38. The medicament of any one of claims 1 to 37, wherein the binding substance comprises a lambda (λ) light chain constant region.

39. The pharmaceutical agent of any one of claims 1 to 38, wherein the first light chain constant region is a kappa (κ) light chain constant region.

40. The pharmaceutical agent of any one of claims 1 to 39, wherein the second light chain constant region is a lambda (λ) light chain constant region.

41. The pharmaceutical agent of any one of claims 1 to 38 and 40, wherein the first light chain constant region is a lambda (λ) light chain constant region.

42. The pharmaceutical agent of any one of claims 1 to 39 and 41, wherein the second light chain constant region is a kappa (κ) light chain constant region.

43. the kappa (κ) light chain a) the sequence shown in SEQ ID NO:21; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The pharmaceutical composition of any one of claims 37 to 42, comprising an amino acid sequence selected from the group consisting of:

44. the lambda (λ) light chain a) the sequence shown in SEQ ID NO:22; b) a subsequence of the sequence in a), e.g., a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions compared to the amino acid sequence defined in a) or b), for example at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions, or at most 1 substitution. The pharmaceutical composition of any one of claims 38 to 43, comprising an amino acid sequence selected from the group consisting of:

45. The pharmaceutical according to any one of claims 1 to 44, wherein the binding substance is a binding substance of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

46. The pharmaceutical according to any one of claims 1 to 45, wherein the binding substance is a full-length IgG1 antibody.

47. The pharmaceutical composition of any one of claims 1 to 46, wherein the antibody is an IgG1m(f) allotype antibody.

48. The pharmaceutical of any one of claims 1 to 47, wherein the subject is a human subject.

49. The pharmaceutical of any one of claims 1 to 48, wherein the tumor or cancer is a solid tumor.

50. 50. The medicament of any one of claims 1 to 49, wherein the tumor or cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic cancer, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, ovarian cancer, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma.

51. The pharmaceutical agent of any one of claims 1 to 50, wherein the tumor or cancer is selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC), urothelial cancer (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g., triple-negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g., cancer of the oral cavity, pharynx, or larynx), and cervical cancer.

52. The pharmaceutical agent of any one of claims 1 to 51, wherein the tumor or cancer is lung cancer.

53. 53. The pharmaceutical of claim 52, wherein the lung cancer is non-small cell lung cancer (NSCLC), such as squamous or non-squamous NSCLC.

54. The pharmaceutical agent of claim 53, wherein the NSCLC does not have an epidermal growth factor (EGFR) sensitivity-increasing mutation and / or an anaplastic lymphoma (ALK) translocation / ROS1 rearrangement.

55. The pharmaceutical agent of any one of claims 52 to 54, wherein the subject has received up to four systemic conditioning regimens for advanced / metastatic disease and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

56. 56. The method of claim 55, wherein the subject has undergone platinum-based chemotherapy.

57. 56. The method of claim 55, wherein the subject is ineligible for platinum-based therapy and is undergoing treatment with another chemotherapy, for example, a gemcitabine-containing regimen.

58. The pharmaceutical agent of any one of claims 1 to 57, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

59. The medicament of any one of claims 1 to 58, wherein the subject has experienced disease progression during or after treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

60. The medicament of any one of claims 1 to 59, wherein the subject has experienced disease progression during or after the last prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

61. The method of any one of claims 55 to 60, wherein the subject has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

62. The medicament of any one of claims 1 to 57, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

63. The pharmaceutical agent of any one of claims 1 to 51, wherein the tumor or cancer is endometrial cancer.

64. 64. The method of claim 63, wherein the subject has epithelial endometrial histology, including endometrioid carcinoma, serous carcinoma, squamous cell carcinoma, clear cell carcinoma, or carcinosarcoma.

65. The pharmaceutical agent of claim 63 or 64, wherein the subject has received up to four systemic conditioning regimens for advanced / metastatic disease and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

66. The medicament of any one of claims 63 to 65, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

67. The pharmaceutical agent of any one of claims 1 to 51, wherein the tumor or cancer is urothelial cancer, including cancer of the bladder, ureter, urethra, or renal pelvis.

68. 68. The pharmaceutical agent of claim 67, wherein the subject has received up to four systemic conditioning regimens for advanced / metastatic disease and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

69. The pharmaceutical agent of claim 67 or 68, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

70. 69. The method of claim 67 or 68, wherein the subject has undergone platinum-based chemotherapy.

71. 69. The medicament of any one of claims 67 or 68, wherein the subject is ineligible for platinum-based therapy and has been treated with another chemotherapy, for example, a gemcitabine-containing regimen.

72. The pharmaceutical of any one of claims 1 to 51, wherein the tumor or cancer is breast cancer, for example, triple-negative breast cancer (TNBC).

73. The pharmaceutical agent of claim 72, wherein the TNBC is HER2-negative, for example, HER2-negative as determined by measuring protein expression by fluorescent in situ hybridization (FISH) or immunohistochemistry, progesterone receptor-negative, and estrogen receptor-negative.

74. The pharmaceutical agent of claim 72 or 73, wherein the subject has received at least one systemic conditioning regimen for locally advanced / metastatic disease, such as at least one systemic conditioning regimen including an anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimen.

75. The pharmaceutical agent of claim 74, wherein the subject has received up to four systemic conditioning regimens for locally advanced / metastatic disease, including at least one systemic conditioning regimen comprising an anthracycline-containing, taxane-containing, antimetabolite-containing, or microtubule inhibitor-containing regimen.

76. The pharmaceutical agent of any one of claims 72 to 75, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

77. The method of claim 76, wherein the subject has experienced disease progression, e.g., radiographically confirmed disease progression, during or after said pretreatment with a checkpoint inhibitor.

78. The medicament of any one of claims 72 to 75, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

79. The pharmaceutical agent of any one of claims 1 to 51, wherein the tumor or cancer is head and neck cancer, for example, squamous cell carcinoma of the head and neck (SCCHN).

80. The pharmaceutical agent of claim 79, wherein the tumor or cancer is recurrent or metastatic SCCHN.

81. 81. The pharmaceutical of claim 79 or 80, wherein the tumor or cancer is cancer of the oral cavity, pharynx, or larynx.

82. The medicament of any one of claims 79 to 81, wherein the subject has received up to four systemic conditioning regimens for recurrent / metastatic disease and has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

83. 83. The method of claim 82, wherein the subject has undergone platinum-based chemotherapy.

84. 83. The method of claim 82, wherein the subject is ineligible for platinum-based therapy and is receiving another chemotherapy.

85. The pharmaceutical agent of any one of claims 79 to 84, wherein the subject has been previously treated with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

86. The method of claim 85, wherein the subject has experienced disease progression, e.g., radiographically confirmed disease progression, during or after the pretreatment with a checkpoint inhibitor.

87. The medicament of any one of claims 79 to 84, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

88. The pharmaceutical agent of any one of claims 1 to 51, wherein the tumor or cancer is cervical cancer.

89. 89. The method of claim 88, wherein the cervical cancer is squamous cell, adenocarcinoma, or adenosquamous histology cervical cancer.

90. The pharmaceutical agent of claim 88 or 89, wherein the subject has received at least one systemic conditioning regimen for recurrent / metastatic disease, such as chemotherapy in combination with a treatment targeting vascular endothelial growth factor A, such as treatment with bevacizumab, and has experienced disease progression, such as radiographically confirmed disease progression, during or after the last systemic conditioning treatment.

91. The pharmaceutical agent of claim 90, wherein the subject has received up to four systemic conditioning regimens for recurrent / metastatic disease, including chemotherapy in combination with a treatment targeting vascular endothelial growth factor A, e.g., treatment with bevacizumab.

92. The pharmaceutical agent of claim 88 or 89, wherein the subject has not received prior treatment with a checkpoint inhibitor, e.g., an agent that targets PD-1 / PD-L, e.g., a PD-1 / PD-L1 inhibitor.

93. The method of any one of claims 1 to 92, wherein the binding substance is administered by systemic administration.

94. The method of any one of claims 1 to 93, wherein the binding agent is administered by intravenous injection or infusion.

95. 95. The method of any one of claims 1 to 94, wherein each treatment cycle is 3 weeks (21 days).

96. 96. The method of any one of claims 1 to 95, wherein one dose is administered every three weeks (1Q3W).

97. 97. The medicament of any one of claims 1 to 96, wherein one dose is administered on day 1 of each treatment cycle.

98. 98. The medicament of any one of claims 1 to 97, wherein each dose is infused over a minimum of 30 minutes, such as at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 240 minutes.

99. A composition comprising a binding agent comprising a first binding domain that binds to human CD137 and a second binding domain that binds to human PD-L1, a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3, and the CDR3 sequence shown in SEQ ID NO:4, and a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:6, the CDR2 sequence shown as GAS, and the CDR3 sequence shown in SEQ ID NO:7; and b) the second binding region comprises a heavy chain variable region (VH) comprising 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 a light chain variable region (VL) comprising the CDR1 sequence shown in SEQ ID NO:13, the CDR2 sequence shown as DDN, and the CDR3 sequence shown in SEQ ID NO:14; the binding agent is an antibody or a multispecific antibody, and The amount of binding substance in the composition is 25 to 200 mg or 1.7×10 -7 ~1.4×10 -6 mol, The composition.

100. 100. The composition of claim 99, comprising about 100 mg of said binding agent.

101. The composition of any one of claims 99 to 100, wherein the binding agent is a binding agent as defined in any one of claims 1 to 98.

102. 102. The composition of any one of claims 99 to 101, for systemic administration.

103. 103. The composition of any one of claims 99 to 102, which is for injection or infusion, for example for intravenous injection or infusion.

104. 104. The composition of any one of claims 99 to 103, wherein the binding agent is present in a volume of 50 to 500 mL, such as 100 to 250 mL, of an aqueous solution, such as 0.9% NaCl (saline).

105. The composition of any one of claims 99-104, in unit dosage form.

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