CD137 binding molecules and uses thereof

Abstract

To provide CD137 binding molecules, in particular CD137×TA binding molecules capable of binding to both an epitope of CD137 and an epitope of a tumor antigen.SOLUTION: The present invention is directed to binding molecules that possess one or more epitope-binding sites specific for an epitope of CD137, including antibodies, and molecules comprising epitope-binding fragments thereof. The invention is further directed to multispecific binding molecules comprising one or more epitope-binding sites specific for an epitope of CD137 and one or more epitope-binding sites specific for an epitope of a tumor antigen ("TA") (e.g., a "CD137×TA Binding Molecule"). In one embodiment, such CD137×TA binding molecules are bispecific molecules, and in particular are bispecific tetravalent diabodies having two epitope-binding sites each specific for an epitope of CD137 and two epitope-binding sites each specific for an epitope of TA.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Patent Application No. 62 / 980,000 (filed February 21, 2020; pending), U.S. Patent Application No. 63 / 104,685 (filed October 23, 2020; pending), and U.S. Patent Application No. 63 / 147,565 (filed February 9, 2021; pending), each of which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy created on February 12, 2021, is titled MAC-0111-PC_SL.txt, is 224,061 bytes in size, and is incorporated by reference in its entirety.

[0003] The present technology is directed to CD137-binding molecules, such as monospecific antibodies and molecules comprising epitope-binding fragments thereof, capable of binding to an epitope on CD137. The technology is further directed to multispecific CD137-binding molecules (e.g., bispecific antibodies, bispecific diabodies, BiTEs, trivalent binding molecules, etc.) capable of binding to both an epitope on CD137 and an epitope on a second antigen, particularly a tumor antigen ("TA") (e.g., "CD137xTA binding molecules"). The technology also provides novel PD-L1-binding molecules, such as monospecific antibodies and molecules comprising epitope-binding fragments thereof, capable of binding to an epitope on PD-L1, as well as derivatives thereof and uses thereof. The technology is also directed to pharmaceutical compositions comprising the above molecules. The technology also includes the use of the above molecules in the treatment of diseases, particularly cancer, or diseases or conditions associated with or characterized by the presence of a suppressed immune system. [Background technology]

[0004] CD137 (also known as 4-1BB and "TNF receptor superfamily member 9" (TNFRSF9)) is a costimulatory receptor member of the tumor necrosis factor receptor superfamily that mediates CD28-dependent and -independent T cell costimulation (NPL 1, 2). CD137 is inducibly expressed by T cells, natural killer (NK) cells, dendritic cells (DCs), B cells, and other cells of the immune system. Ligation of CD137 by its ligand CD137L (4-1BBL; TNFSF9) or by agonistic antibodies elicits various T cell responses, including cell proliferation, increased cytokine secretion, and prevention of activation-induced cell death. Thus, antibodies that stimulate CD137 can enhance antitumor immune responses by inducing T cell survival and proliferation. This recognition has led to the proposal that activating the immune system with immunospecific antibodies against CD137 could provide a therapy for cancer (NPL 3, 4). The anti-CD137 antibodies utomilumab and urelumab have been described, but their clinical development has been hampered by poor efficacy (utomilumab) or severe hepatotoxicity (urelumab). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Vinay, DS and Kwon, BS (1998) “Role of 4-1BB in immune responses,” SeminImmunol. 10:481-489 [Non-patent document 2] Croft, M. (2009) “The Role Of TNF Superfamily Members In T-Cell Function And Diseases,” Nat. Rev. Immunol. 9:271-285 [Non-patent document 3] Li, SY et al. (2013) “Immunotherapy Of Melanoma With The ImmunecostimulatoryMonoclonal Antibodies Targeting CD137,” Clin. Pharmacol. 5:47-53 [Non-patent document 4] Bartkowiak, T. et al. (2015) “4-1BB Agonists: Multi-Potent Potentiators Of Tumor Immunity,” Frontiers Oncol. 5:117 Summary of the Invention [Problem to be solved by the invention]

[0006] Improved compositions are provided that can more potently stimulate and direct the body's immune system to attack cancer cells while avoiding the toxicity associated with antibodies that exhibit high activity in the absence of cross-linking. While the adaptive immune system can be a powerful defense mechanism against cancer and disease, it is often thwarted by immunosuppressive / evasive mechanisms in the tumor microenvironment mediated by reduced / absent costimulatory activity of CD137. Furthermore, co-inhibitory molecules expressed by tumor cells, immune cells, and stromal cells within the tumor environment can significantly attenuate T cell responses against cancer cells. [Means for solving the problem]

[0007] CD137-binding molecules, particularly CD137xTA-binding molecules, are provided that can bind to both an epitope on CD137 and an epitope on a tumor antigen. Such bispecific molecules can bind to tumor antigens expressed on the surface of tumor cells and colocalize CD137-expressing immune cells to such tumor cells. Such colocalization upregulates immune cells, promoting activation or continued activation of the immune system (e.g., stimulating a cytotoxic T cell response against tumor cells). These attributes may make the bispecific molecules useful for stimulating the immune system and, in particular, for the treatment of cancer. The present technology addresses these and other goals.

[0008] Thus, in certain embodiments, provided are CD137-binding molecules, such as monospecific antibodies and molecules comprising epitope-binding fragments thereof, capable of binding to an epitope on CD137. The present invention is further directed to multispecific CD137-binding molecules (e.g., bispecific antibodies, bispecific diabodies, BiTEs, trivalent binding molecules, etc.) capable of binding to both an epitope on CD137 and an epitope on a second antigen, particularly a tumor antigen ("TA") (e.g., "CD137xTA binding molecules"). The present invention also provides novel PD-L1-binding molecules, such as monospecific antibodies and molecules comprising epitope-binding fragments thereof, capable of binding to an epitope on PD-L1, as well as derivatives thereof, and uses thereof. The present invention is also directed to pharmaceutical compositions comprising the above-described molecules. The present invention also includes the use of the above-described molecules in the treatment of disease, particularly cancer, or diseases or conditions associated with or characterized by the presence of a suppressed immune system.

[0009] The present invention provides novel CD137-binding molecules that exhibit desirable properties, particularly when incorporated into multispecific molecules. The present invention also relates to multispecific CD137×TA-binding molecules composed of polypeptide chains that, upon association with each other, form two binding sites, each specific for an epitope on CD137, and two binding sites, each specific for an epitope on TA. Such CD137×TA-binding molecules of the present invention are referred to as "bispecific tetravalent." The present invention also relates to CD137×TA-binding molecules composed of polypeptide chains that, upon association with each other, form two binding sites, each specific for an epitope on CD137, and one binding site, specific for an epitope on TA. Such CD137×TA-binding molecules of the present invention are referred to as "bispecific trivalent." A binding molecule (e.g., a CD137-binding molecule) of the present invention optionally comprises a first binding site and does not comprise a second binding site that immunospecifically binds to an antigen different from the antigen bound by the first binding site. Thus, a binding molecule of the present invention optionally comprises a first binding site and does not comprise a second binding site that immunospecifically binds to an antigen different from the antigen bound by the first binding site. A binding molecule that comprises only a first binding site and a first light chain variable domain and a first heavy chain variable domain, but does not comprise a second binding site, a second light chain variable domain, or a second heavy chain variable domain that binds to a different antigen from the first binding site; non-limiting examples of such binding molecules include scFv, antibodies, and Fab binding molecules.

[0010] The present invention provides CD137xTA-binding molecules comprising four polypeptide chains ("first," "second," "third," and "fourth" polypeptide chains), wherein the first and second polypeptide chains are covalently linked to each other, the third and fourth polypeptide chains are covalently linked to each other, and the first and third polypeptide chains are covalently linked to each other. Also provided are CD137xTA-binding molecules comprising five polypeptide chains ("first," "second," "third," "fourth," and "fifth" polypeptide chains), wherein the first and second polypeptide chains are covalently linked to each other, the third and fourth polypeptide chains are covalently linked to each other, the third and fifth polypeptide chains are covalently linked to each other, and the first and third polypeptide chains are covalently linked to each other.

[0011] In particular, the present invention provides CD137 binding molecules comprising a first binding site that immunospecifically binds to an epitope of CD137, wherein said first binding site comprises a CDR L 1. CD R L 2 and CDR L a first light chain variable domain comprising CDRs H 1. CDR H 2 and CDR H a first heavy chain variable domain comprising: (A) CDRs of the first light chain variable domain L 1. CDR L 2 and CDR L 3 is CD1 37 light chain CDRs of MAB-6 VL1 (SEQ ID NO: 50); (B) CDRs of the first heavy chain variable domain H 1. CDR H 2 and CDR H 3 is CD1 37 are the heavy chain CDRs of MAB-6 VH1 (SEQ ID NO: 46).

[0012] The present invention further relates to the embodiment of the CD137 binding molecule described above, wherein the first heavy chain variable domain comprises the amino acid sequence of hCD137 MAB-6 VH1 (SEQ ID NO: 46).

[0013] The present invention further provides a method for producing a light chain variable domain comprising the steps of: (A) hCD137 MAB-6 VLx (SEQ ID NO: 54); (B) hCD137 MAB-6 VL1 (SEQ ID NO: 50); (B) hCD137 MAB-6 VL2 (SEQ ID NO: 55); or (C) hCD137 MAB-6 VL3 (SEQ ID NO: 56) The present invention relates to an embodiment of the above CD137 binding molecule, comprising the amino acid sequence:

[0014] The present invention further comprises: (A) the first heavy chain variable domain comprises the amino acid sequence of: hCD137 MAB-6 VH1 (SEQ ID NO: 46); (B) The embodiment of the CD137 binding molecule, wherein the first light chain variable domain comprises the amino acid sequence of: hCD137 MAB-6 VL3 (SEQ ID NO: 56).

[0015] The present invention further provides a bispecific molecule, wherein the molecule comprises a second binding site that immunospecifically binds to a TA, and the second binding site comprises a CDR L 1. CDR L 2 and CDR L Including 3 a second light chain variable domain comprising: H 1. CDR H 2 and CDR H 3, a second heavy chain containing and a variant domain.

[0016] The present invention further relates to embodiments of the CD137-binding molecules described above, wherein the TA is selected from the tumor antigens presented in Tables 1-2.

[0017] The present invention further provides an antibody against a PD-L1-associated leukemia virus (LAV) comprising: (A) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3, hPD - L1 is the light chain CDRs of MAB-2 VLx (SEQ ID NO: 63); (B) CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, hPD -L1 relates to an embodiment of the CD137 binding molecule described above, which is the heavy chain CDRs of MAB-2 VHx (SEQ ID NO: 59).

[0018] The present invention further comprises: (A)(1) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of hPD-L1 MAB-2 VL1 (SEQ ID NO: 58); or (2) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of PD-L1 MAB-2 VL2 (SEQ ID NO: 72); (B)(1) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is, the heavy chain CDRs of hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 67); (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH3 (SEQ ID NO: 68); (4) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 69); (5) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 70); or (6) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h

[0033] In one embodiment of the CD137 binding molecule described above, the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 71) are

[0019] The present invention further relates to a method for producing a heavy chain variable domain comprising the steps of: (A) hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (B) hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (C) hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (D) hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (E) hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (F) hPD-L1 MAB-2 VH6 (SEQ ID NO: 71) The present invention relates to an embodiment of the above CD137 binding molecule, comprising the amino acid sequence:

[0020] The present invention further provides a method for producing a light chain variable domain comprising the steps of: (A) hPD-L1 MAB-2 VL1 (SEQ ID NO: 58); or (B) hPD-L1 MAB-2 VL2 (SEQ ID NO: 72) The present invention relates to an embodiment of the above CD137 binding molecule, comprising the amino acid sequence:

[0021] The present invention further relates to a method for treating a leukemia, wherein the TA is 5T4: (A)(1) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of 5T4 MAB-1 VL (SEQ ID NO: 93); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, 5 the heavy chain CDRs of T4 MAB-1 VH (SEQ ID NO: 92); or (B)(1) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of 5T4 MAB-2 VL (SEQ ID NO: 95); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, 5 The embodiment of the CD137 binding molecule described above is the heavy chain CDRs of T4 MAB-2 VH (SEQ ID NO: 96).

[0022] The present invention further relates to the embodiment of the above-described CD137 binding molecule, wherein the second heavy chain variable domain comprises the amino acid sequence of 5T4 MAB-1 VH (SEQ ID NO: 92).

[0023] The present invention further relates to the embodiment of the CD137 binding molecule described above, wherein the second light chain variable domain comprises the amino acid sequence of 5T4 MAB-1 VL (SEQ ID NO: 93).

[0024] The present invention further relates to a method for treating a HER2-associated ...TA comprising: (A) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3, but hHE R2-MAB-1 VLx (SEQ ID NO: 79) light chain CDRs; (B) CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, but hHE R2-MAB-1 VHx (SEQ ID NO: 78) heavy chain CDRs of the CD137 binding molecule.

[0025] The present invention further comprises: (A)(1) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of hHER2-MAB-1 VL1 (SEQ ID NO: 83); (2) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of HER2-MAB-1 VL2 (SEQ ID NO: 84); or (3) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of HER2-MAB-1 VL3 (SEQ ID NO: 85); (B)(1) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is, the heavy chain CDRs of hHER2-MAB-1 VH1 (SEQ ID NO: 80); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of HER2-MAB-1 VH2 (SEQ ID NO: 81); or (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h The embodiments of the CD137 binding molecules described above are directed to the heavy chain CDRs of HER2-MAB-1 VH3 (SEQ ID NO: 82).

[0026] The present invention further relates to a method for producing a heavy chain variable domain comprising the steps of: (A) hHER2-MAB-1 VHx (SEQ ID NO: 78); (B) hHER2-MAB-1 VH1 (SEQ ID NO: 80); (C) hHER2-MAB-1 VH2 (SEQ ID NO: 81); or (D) hHER2-MAB-1 VH3 (SEQ ID NO: 82) The present invention relates to an embodiment of the above CD137 binding molecule, comprising the amino acid sequence:

[0027] The present invention further provides a method for producing a light chain variable domain comprising the steps of: (A) hHER2-MAB-1 VLx (SEQ ID NO: 79); (B) hHER2-MAB-1 VL1 (SEQ ID NO: 83); (C) hHER2-MAB-1 VL2 (SEQ ID NO: 84); or (D) hHER2-MAB-1 VL3 (SEQ ID NO: 85) The present invention relates to an embodiment of the above CD137 binding molecule, comprising the amino acid sequence:

[0028] The present invention further relates to all of the above-mentioned embodiments of the CD137 binding molecule, wherein the molecule is an antibody, a bispecific tetravalent Fc-bearing diabody, or a bispecific trivalent molecule.

[0029] The present invention further relates to embodiments of the above-described CD137 binding molecules, wherein the molecules are bispecific and tetravalent and comprise a first, second, third, fourth, and optionally a fifth polypeptide chain, wherein the polypeptide chains form a covalent complex.

[0030] The present invention further relates to embodiments of the CD137 binding molecules described above, wherein the molecules are bispecific and trivalent and comprise first, second, third, and fourth polypeptide chains, wherein the polypeptide chains form a covalent complex.

[0031] The present invention further relates to the embodiment of all of the above CD137 binding molecules, wherein said molecule comprises an Fc region of the IgG1, IgG2, IgG3, or IgG4 isotype, and optionally said molecule further comprises a hinge domain.

[0032] The present invention further provides a variant Fc region, wherein the Fc region is a variant Fc region comprising one or more amino acid modifications that reduce the affinity of the variant Fc region for FcγR and / or increase the serum half-life of the variant Fc region, more particularly, the modifications are: (A) L234A; L235A; (B) L234A and L235A; (C) M252Y; M252Y and S254T; (D) M252Y and T256E; (E) M252Y, S254T, and T256E; or (F) K288D and H435K; wherein the numbering is that of the EU index as set forth in Kabat.

[0033] The present invention further relates to an antibody against PD-L1, wherein the TA is PD-L1: (A) the first polypeptide chain and the third polypeptide chain comprise the amino acid sequences of SEQ ID NO:116, SEQ ID NO:118, and SEQ ID NO:120; (B) The second polypeptide chain and the fourth polypeptide chain comprise the amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 139.

[0034] The present invention further relates to a method for producing a molecule comprising: (A) SEQ ID NO: 116 and SEQ ID NO: 117; (B) SEQ ID NO: 118 and SEQ ID NO: 119; (C) SEQ ID NO: 120 and SEQ ID NO: 119; (D) SEQ ID NO: 118 and SEQ ID NO: 121; (E) SEQ ID NO: 120 and SEQ ID NO: 121; (F) SEQ ID NO: 120 and SEQ ID NO: 122; (G) SEQ ID NO: 120 and SEQ ID NO: 123; (H) SEQ ID NO: 120 and SEQ ID NO: 124; (I) SEQ ID NO: 120 and SEQ ID NO: 125; (J) SEQ ID NO: 120 and SEQ ID NO: 126; or (K) SEQ ID NO: 120 and SEQ ID NO: 139 The present invention relates to an embodiment of the above CD137 binding molecule, comprising:

[0035] The present invention further relates to an antibody against PD-L1, wherein the TA is PD-L1: (A) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127, SEQ ID NO: 133, or SEQ ID NO: 135; (B) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: 134, or SEQ ID NO: 136; (C) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, or SEQ ID NO: 131; (D) The embodiment of the CD137 binding molecule, wherein the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 132.

[0036] The present invention further relates to a method for producing a molecule comprising: (A) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130; (B) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 131, and SEQ ID NO: 132; (C) SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 131, and SEQ ID NO: 132; or (D) SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 131, and SEQ ID NO: 132. The present invention relates to an embodiment of the above CD137 binding molecule, comprising:

[0037] The present invention further relates to pharmaceutical compositions comprising any of the CD137 binding molecules described above and a physiologically acceptable carrier.

[0038] The present invention further relates to the use of said CD137 binding molecule or said pharmaceutical composition in the treatment of cancer characterized by expression of TA.

[0039] The present invention further provides a method for producing a CDR L 1. CDR L 2 and CDR L a light chain variable domain comprising CD R H 1. CDR H 2 and CDR H 3, and a heavy chain variable domain comprising: (A) CDRs of the light chain variable domain L 1. CDR L 2 and CDR L 3, hPD-L 1 the light chain CDRs of MAB-2 VL2 (SEQ ID NO: 72); (B)(1) CDRs of the heavy chain variable domain H 1. CDR H 2 and CDR H 3, hPD -L1 are the heavy chain CDRs of MAB-2 VH2 (SEQ ID NO: 67); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH3 (SEQ ID NO: 68); (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH4 (SEQ ID NO: 69); (4) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of PD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (5) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h PD-L1 binding molecule, which is the heavy chain CDRs of PD-L1 MAB-2 VH6 (SEQ ID NO: 71).

[0040] The present invention further provides a method for the production of a heavy chain variable domain comprising: (A) hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (B) hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (C) hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (D) hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (E) hPD-L1 MAB-2 VH6 (SEQ ID NO: 71)

[0033] In another embodiment of the present invention, the PD-L1-binding molecule comprises the amino acid sequence:

[0041] The invention further relates to the embodiment of the above-mentioned PD-L1 binding molecules, wherein the light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VL2 (SEQ ID NO: 72).

[0042] The invention further relates to the embodiment of the above-mentioned PD-L1 binding molecules, wherein the molecule is an antibody or antigen-binding fragment thereof.

[0043] The present invention further relates to pharmaceutical compositions comprising any of the PD-L1 binding molecules described above and a physiologically acceptable carrier.

[0044] The present invention further provides a method for treating diseases or conditions associated with a suppressed immune system or characterized by expression of PD-L1. The present invention relates to the use of said PD-L1 binding molecule or said pharmaceutical composition in the treatment of a condition.

[0045] The present invention further relates to the above-mentioned use, wherein the condition associated with a suppressed immune system or characterized by expression of PD-L1 is cancer.

[0046] The present invention further relates to any of the above use embodiments, wherein the cancer is selected from the group consisting of: bladder cancer, bone cancer, cerebrospinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), stomach cancer, testicular cancer, thymic cancer, and uterine cancer.

[0047] The present invention further relates to a method of enhancing the activity of a tumor-targeting agent, comprising administering the tumor-targeting agent in combination with any of the CD137-binding molecules described above, any of the PD-L1-binding molecules described above, or any of the pharmaceutical compositions described above.

[0048] The present invention further relates to methods for treating a disease or condition associated with a suppressed immune system or characterized by the development of TA, comprising administering to a subject in need thereof any of the CD137-binding molecules described above, any of the PD-L1-binding molecules described above, or any of the pharmaceutical compositions described above.

[0049] The present invention further relates to the above method, further comprising the step of administering a tumor targeting agent.

[0050] The present invention further relates to the above method, wherein the condition associated with a suppressed immune system or characterized by the expression of tumor TA is cancer.

[0051] The present invention further relates to all of the above-mentioned embodiments of the method, wherein the tumor targeting agent is an antibody, an epitope-binding fragment of an antibody, or an agent that mediates T cell targeted killing of a target cell.

[0052] The present invention further relates to embodiments of the above methods, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, cerebrospinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), stomach cancer, testicular cancer, thymic cancer, and uterine cancer.

[0053] The invention further relates to nucleic acids encoding the CD137-binding molecules of any of the above-mentioned embodiments, or the PD-L1-binding molecules of any of the above-mentioned embodiments.

[0054] The present invention further relates to an expression vector comprising the nucleic acid.

[0055] The present invention further relates to a cell comprising a nucleic acid according to any of the above embodiments, or an expression vector of any of the above embodiments.

[0056] The present invention further relates to the above-described cell, wherein the cell is a mammalian cell. [Brief explanation of the drawings]

[0057] [Figure 1]Figures 1A-1D provide schematic diagrams illustrating representative covalent diabodies containing an Fc Region. Figures 1A-1D show schematic diagrams of representative tetravalent diabodies with four epitope-binding sites composed of two pairs of polypeptide chains (i.e., four total polypeptide chains). One polypeptide in each pair has a CH2 and CH3 domain, such that the associated chains form all or part of an Fc Region. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. The polypeptide chains of the two pairs can be identical. In embodiments in which the VL and VH domains recognize different epitopes (as shown in Figures 1A-1B), the resulting molecule has four epitope-binding sites and is bispecific and bivalent for each epitope it binds. In embodiments in which the VL and VH domains recognize the same epitope (e.g., the same VL domain CDRs and the same VH domain CDRs are used for both chains), the resulting molecule has four epitope-binding sites and is monospecific and tetravalent for a single epitope. Alternatively, the two pairs of polypeptides can be different. In embodiments in which the VL and VH domains of each pair of polypeptides recognize different epitopes (as shown in Figure 1C), the resulting molecule has four epitope-binding sites and is tetraspecific and monovalent for each epitope it binds. Figure 1A shows an Fc diabody containing peptide Heterodimer-Promoting Domains that contain cysteine ​​residues. Figure 1B shows an Fc diabody composed of two pairs of polypeptide chains (i.e., four polypeptide chains total), each containing an E-coil or K-coil Heterodimer-Promoting Domain. The wavy lines (WWW) in this figure, and in all figures providing schematic diagrams of the domains of a binding molecule, represent any one or more Heterodimer-Promoting Domains that are present. As shown, cysteine ​​residues may be present within the linker (main diagram) and / or within the Heterodimer-Promoting Domain (boxed). One polypeptide chain of each pair has a cysteine-containing linker (which may include all or part of the hinge region) and CH2 and CH3 Domains, such that the associated chains form all or part of an Fc region.Figure 1C shows an Fc Region-containing diabody containing antibody CH1 and CL Domains. Figure 1D shows a schematic diagram of a representative covalently linked diabody molecule with two epitope-binding sites composed of three polypeptide chains. Two of the polypeptide chains contain CH2 and CH3 Domains, such that the associated chains form all or part of an Fc Region. The polypeptide chains containing the VL and VH Domains further contain a Heterodimer-Promoting Domain, shown here as containing a cysteine ​​residue. [Figure 2] Figure 2 provides a schematic diagram of a representative covalent binding molecule with four epitope-binding sites composed of five polypeptide chains. Two of the polypeptide chains have a cysteine-containing linker (which may include all or part of a hinge region) and CH2 and CH3 domains, such that the associated chains form an Fc region containing all or part of the Fc region. The polypeptide chain containing the linked VL and VH domains further comprises a linker and a Heterodimer-Promoting Domain (further described in Figure 1B). VL and VH domains that recognize the same epitope are shown using the same shading or fill pattern. The variable domains can be selected to result in a CD137xTA binding molecule with two non-diabody-type binding domains specific for TA and two diabody-type binding domains specific for CD137. Alternatively, the variable domains can be selected to yield a resulting CD137xTA binding molecule with two non-diabody-type binding domains specific for CD137 and two diabody-type binding domains specific for a TA. Such molecules are bispecific, having two binding sites for CD137 (which may bind to the same or different CD137 epitopes) and two binding sites for a TA (which may bind to the same or different TA epitopes). [Figure 3]Figures 3A-3C provide schematic diagrams of representative Fc Region-containing trivalent binding molecules having three epitope-binding sites. Figure 3A shows a schematic diagram of the domains of a trivalent binding molecule comprising two diabody-type binding domains (further described in Figure 1B) covalently linked via a linker / heterodimer-promoting domain and a Fab-type binding domain in which the binding domain is N-terminal to the Fc Region. The molecule in Figure 3A comprises four chains. Figures 3B-3C show schematic diagrams of the domains of trivalent binding molecules comprising two diabody-type binding domains and a Fab-type binding domain in which the light and heavy chains are linked via a polypeptide spacer, or comprising an scFv-type binding domain, respectively. The trivalent binding molecules in Figures 3B-3C comprise three chains. VL and VH domains that recognize the same epitope are indicated using the same shading or fill pattern. [Figure 4] FIG. 4 shows the ability of the CD137×TA binding molecules DART-A, TRIDENT-A, the comparator molecule TRIDENT-2, and the negative control hIgG1 to bind to CD137 expressed on the surface of modified CHO cells. [Figure 5] Figures 5A-5B show the ability of the CD137xTA binding molecules DART-A, TRIDENT-A, hPD-L1 MAB-2(1.1), and the negative control hIgG1 to bind to PD-L1 expressed on the surface of engineered CHO cells (Figure 5A) and to block the PD-L1 / PD-1 interaction in a PD-L1 reporter assay (Figure 5B). [Figure 6] Figure 6 shows the ability of the CD137xTA binding molecules DART-A, TRIDENT-A, comparator molecules: DART-2, and TRIDENT-2, DART-3, r-urelumab, and negative controls: DART-1 and hIgG1, to mediate target-dependent signaling in a CD137 reporter assay. [Figure 7]Figures 7A-7B show the ability of the CD137xTA binding molecules DART-A, TRIDENT-A, comparator molecules: DART-2, and TRIDENT-2, DART-3, r-urelumab, and negative controls: DART-1 and hIgG1, to mediate target-dependent release of the cytokines INF-γ (Figure 7A) and IL-2 (Figure 7B) in a primary T cell cytokine release assay. [Figure 8] Figures 8A-8C show serum levels of the CD137 x TA binding molecule TRIDENT-A and induction of immune cell proliferation by the CD137 x TA binding molecule TRIDENT-A. Pharmacokinetics (serum clearance) (Figure 8A), CD8+ T cell proliferation (Figure 8A), and NK cell proliferation (Figure 8A) are plotted from days 20 to 24 in cynomolgus monkeys treated with 1 mg / kg (filled circles) or 10 mg / kg (open circles). [Figure 9] Figures 9A-9B show the binding activity of Fabs, including deimmunized / optimized variants of hPD-L1 MAB-2(1.1). ELISA binding curves are plotted for Fab variants hPD-L1 MAB-2B, hPD-L1 MAB-2D, and hPD-L1 MAB-2F (Figure 9A), and hPD-L1 MAB-2A, hPD-L1 MAB-2C, and hPD-L1 MAB-2E (Figure 9B). [Figure 10] Figures 10A-10B show the ability of CD137xTA binding molecules containing deimmunized or optimized PD-L1 binding domains to bind PD-L1 expressed on the cell surface of engineered CHO cells. Binding curves are plotted for DART-A1, DART-A4, and the anti-PD-L1 antibody hPD-L1 MAB-2(1.1) (Figure 10A), TRIDENT-A, TRIDENT-A4, and the negative control hIgG1 (Figure 10B). [Figure 11]Figures 11A-11C show the ability of CD137xTA binding molecules containing deimmunized and / or optimized PD-L1 binding domains to block PD-L1 / PD-1 interaction in a PD-L1 reporter assay. Activity curves are plotted for DART-A1, DART-A4, and the anti-PD-L1 antibody hPD-L1 MAB-2(1.1) (Figure 11A), TRIDENT-A, TRIDENT-A4, and the negative control hIgG1 (Figure 11B), and DART-A4, DART-A7, DART-A8, DART-A9, and the negative control hIgG1 (Figure 11C). [Figure 12] Figures 12A-12B show the ability of CD137xTA binding molecules containing deimmunized CD137-binding domains and / or deimmunized / optimized PD-L1-binding domains to bind to CD137 expressed on the surface of engineered CHO cells. Binding curves are plotted for DART-A4, DART-A5, and DART-A6 (Figure 12A), and TRIDENT-A4, TRIDENT-A5, and TRIDENT-A6 (Figure 12B). Also plotted in both figures are the comparator r-urelumab and the negative control hIgG1. [Figure 13] Figures 13A-13B show the ability of CD137xTA binding molecules comprising deimmunized CD137-binding domains and / or deimmunized / optimized PD-L1-binding domains to mediate target-dependent signaling in CD137 reporter assays performed with N87 target cells, which express low PD-L1 (Figure 13A), or JIMT-1 target cells, which express moderate PD-L1 (Figure 13B). The activities of DART-A4, DART-A5, DART-A6, TRIDENT-A4, TRIDENT-A5, TRIDENT-A6, comparator r-urelumab, and negative control hIgG1 are plotted. [Figure 14]Figures 14A-14B show the ability of CD137xTA binding molecules comprising a deimmunized CD137 binding domain and a deimmunized / optimized PD-L1 binding domain to mediate target-dependent release of the cytokines INF-γ (Figure 14A) and IL-2 (Figure 14B) in a primary T cell cytokine release assay. The activities of DART-A4, DART-A5, DART-A6, TRIDENT-A4, TRIDENT-A5, TRIDENT-A6, comparator r-urelumab, and negative control hIgG1 are plotted. [Figure 15] Figures 15A-15B show the ability of CD137xTA binding molecules containing parental or deimmunized / optimized PD-L1 and / or CD137 binding domains to bind to PD-L1 (Figure 15A) and CD137 (Figure 15B) expressed on the cell surface of engineered CHO cells. Binding curves are plotted for DART-A, DART-A4, DART-A6, DART-A7, DART-A10, the anti-PD-L1 antibody hPD-L1 MAB-2(1.1), and r-atezolizumab, and the negative control hIgG1 (Figure 15A), and for DART-A, DART-A4, DART-A6, DART-A7, DART-A10, r-urelumab, and the negative control hIgG1 (Figure 15B). [Figure 16] Figures 16A-16B show the ability of CD137xTA binding molecules containing parental or deimmunized / optimized PD-L1 and / or CD137 binding domains to block the PD-L1 / PD-1 interaction in a PD-L1 reporter assay. Results for the tetravalent molecules DART-A, DART-A4, DART-A6, DART-A7, and DART-A10 are plotted in Figure 16A, and results for the trivalent molecules TRIDENT-A, TRIDENT-A4, and TRIDENT-A6 are plotted in Figure 16B. Also plotted in both figures are the anti-PD-L1 antibodies hPD-L1 MAB-2F and r-atezolizumab, as well as the negative control hIgG1. [Figure 17]Figures 17A-17B show the ability of CD137xTA binding molecules containing parental or deimmunized / optimized PD-L1 and / or CD137 binding domains to mediate target-dependent signaling in a CD137 reporter assay performed in the presence (Figure 17A) or absence (Figure 17B) of JIMT-1 target cells, which have moderate PD-L1 expression. The activities of DART-A, DART-A4, DART-A6, DART-A7, DART-A10, TRIDENT-A, TRIDENT-A4, TRIDENT-A6, comparator r-urelumab, and negative control hIgG1 are plotted. [Figure 18] Figures 18A-18B show the ability of CD137xTA binding molecules containing parental or deimmunized / optimized PD-L1 and / or CD137 binding domains to mediate target-dependent release of the cytokines INF-γ (Figure 18A) and IL-2 (Figure 18B). The activity of DART-A, DART-A4, DART-A6, DART-A7, DART-A10, TRIDENT-A, TRIDENT-A4, TRIDENT-A6, the combination of r-atezolizumab and r-urelumab (r-atezo+r-ure combo), and the negative control hIgG1 are plotted. [Figure 19] Figures 19A-19C show the ability of several representative PD-L1xCD137 bispecific molecules: DART-A (Figure 19A), TRIDENT-A (Figure 19B), or TRIDENT-A4 (Figure 19C) in combination with a representative TAxCD3 bispecific molecule (5T4xCD3 diabody) to prevent or inhibit tumor growth or pathogenesis of RKO colon cancer cells in vivo in a mouse PBMC-reconstituted xenograft model compared to the TAxCD3 bispecific molecule alone or vehicle control. [Figure 20]Figures 20A-20B show the ability of several representative PD-L1xCD137 bispecific molecules: DART-A6 (Figure 20A), or TRIDENT-A6 (Figure 20B), in combination with a representative TAxCD3 bispecific molecule (5T4xCD3 diabody) to prevent or inhibit tumor growth or pathogenesis of RKO colon cancer cells in vivo in a mouse PBMC-reconstituted xenograft model, compared to the TAxCD3 bispecific molecule alone or vehicle control. [Figure 21] Figures 21A-21B show the ability of several representative PD-L1xCD137 bispecific molecules containing the VH / VL of the CD137 MAB-6 binding domain: TRIDENT-A, TRIDENT-A6, or comparator molecules containing the VH / VL of a different CD137 binding domain: TRIDENT-2, DUO-1, combined with a representative TAxCD3 bispecific molecule (5T4xCD3 diabody) to prevent or inhibit tumor growth or pathogenesis of RKO colon cancer cells in vivo in a mouse PBMC-reconstituted xenograft model compared to vehicle control. Representative data from the first study are plotted in Figure 21A, and representative data from the second study are plotted in Figure 21B. [Figure 22] Figures 22A-22B show the ability of CD137xTA binding molecules, containing CD137-binding domains and HER2-binding domains, to mediate target-dependent signaling in CD137 reporter assays performed with JIMT-1 cells, which have moderate HER2 expression (Figure 22A), or N87 target cells, which have high HER2 expression (Figure 22B). The activities of DART-B1, DART-B2, TRIDENT-B1, TRIDENT-B2, the parental hHER2 MAB-1 (1.3), and CD137 MAB-6 (1.1) antibodies, as well as the negative controls DART-4, DART-5, TRIDENT-3, and TRIDENT-4, are plotted. [Figure 23]Figures 23A-23D show the ability of CD137xTA binding molecules, which contain CD137-binding domains and HER2-binding domains, to mediate target-dependent release of the cytokines INF-γ (Figures 23A-23B) and IL-2 (Figures 23C-23D) in primary T cell cytokine release assays performed with JIMT-1 cells, which have moderate HER2 expression (Figures 22A-23C), or N87 target cells, which have high HER2 expression (Figures 22B-23D). The activities of DART-B1, DART-B2, TRIDENT-B1, TRIDENT-B2, the parental hHER2 MAB-1 (1.3) and CD137 MAB-6 (1.1) antibodies, and the negative controls DART-4, DART-5, TRIDENT-3, and TRIDENT-4 are plotted. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention is directed to CD137-binding molecules, such as monospecific antibodies and molecules comprising epitope-binding fragments thereof, capable of binding to an epitope on CD137. The present invention is further directed to multispecific CD137-binding molecules (e.g., bispecific antibodies, bispecific diabodies, BiTEs, trivalent binding molecules, etc.) capable of binding to both an epitope on CD137 and an epitope on a second antigen, particularly a tumor antigen ("TA") (e.g., "CD137xTA binding molecules"). The present invention also provides novel PD-L1-binding molecules, such as monospecific antibodies and molecules comprising epitope-binding fragments thereof, capable of binding to an epitope on PD-L1, as well as derivatives thereof and uses thereof. The present invention is also directed to pharmaceutical compositions comprising the above-described molecules. The present invention also includes the use of the above-described molecules in the treatment of disease, particularly cancer, or diseases or conditions associated with or characterized by the presence of a suppressed immune system.

[0059] I. Antibodies and Other Binding Molecules The CD137xTA binding molecules of the present invention may be antibodies or may be derivable from antibodies (e.g., by fragmenting, cleaving, etc., of antibody polypeptides, or by using a polynucleotide (or sequence thereof) encoding the amino acid sequence of an antibody molecule, or such an amino acid sequence).

[0060] A. Antibodies An antibody is an immunoglobulin molecule that can specifically bind to an "epitope" of a molecule such as a carbohydrate, polynucleotide, lipid, or polypeptide (an "antigen") via at least one "epitope binding site" located in the variable region of the immunoglobulin molecule. As used herein, the terms "antibody" and "antibodies" encompass monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFvs), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFvs), intrabodies, and epitope-binding fragments of any of the foregoing. In particular, the term "antibody" includes immunoglobulin molecules and immunologically active fragments of immunoglobulins, i.e., molecules that contain an epitope-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. An antibody can "immunospecifically bind" to a polypeptide or protein or non-protein molecule by virtue of the presence of a particular domain, portion, or conformation ("epitope") on said molecule. As used herein, an "epitope-binding fragment of an antibody" is intended to refer to a portion of an antibody that is capable of immunospecifically binding to an epitope. As used herein, the term also includes fragments of diabodies (e.g., Fab, Fab', F(ab')2Fv) and single chain (scFv), as well as epitope-binding fragments. As used herein, an antibody or epitope-binding fragment thereof is said to "immunospecifically" bind to a region (i.e., epitope) of another molecule if it reacts or associates with that epitope more frequently, rapidly, for longer periods, and / or with greater affinity or avidity than with another epitope. It is understood in reading this definition that, for example, an antibody or epitope-binding fragment thereof that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target. Epitope-containing molecules possess immunogenic activity, thereby eliciting an antibody response in an animal; such molecules are referred to as "antigens." While naturally occurring antibodies can only bind to one epitope species (i.e., they are "monospecific"), naturally occurring antibodies can bind to multiple copies of that species (i.e., they exhibit "bivalency" or "multivalency").

[0061] The term "monoclonal antibody" refers to a homogeneous population of antibodies, which are composed of amino acids (naturally occurring or non-naturally occurring) involved in the selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single epitope (or antigenic site). The term "monoclonal antibody" includes intact and full-length monoclonal antibodies as well as fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single chain (scFv), variants thereof, and the like. The term "antibody" encompasses antibodies, fusion proteins containing antibody portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of an immunoglobulin molecule containing an antigen recognition site with the required specificity and ability to bind to the antigen. No limitation is intended as to the source of the antigen or the method by which the antigen is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes whole immunoglobulins and fragments such as those mentioned above in the definition of "antibody." Methods for making monoclonal antibodies are known in the art. One method that may be employed is the method of Kohler, G. et al. (1975) "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity," Nature 256:495-497, or a modification thereof. Typically, monoclonal antibodies are made from mouse, rat, or The antibodies are generated by injecting the animals with an immunogenic amount of the antibody containing the desired epitope. Antibodies are produced by immunizing with cells, cell extracts, or protein preparations of the present invention. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancer cells, proteins, peptides, nucleic acids, or tissues. Alternatively, existing monoclonal antibodies and other equivalent antibodies with immunospecificity for a desired pathogenic epitope can be recombinantly sequenced and produced by any means known in the art. In one embodiment, such antibodies are sequenced, and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest is maintained in the vector in a host cell, which can then be expanded and frozen for future use. Such antibody polynucleotide sequences may be used for genetic engineering to improve the affinity or other characteristics of the antibody by generating monospecific or multispecific (e.g., bispecific, trispecific, and tetraspecific) molecules of the present invention, as well as affinity-optimized, chimeric, humanized, and / or caninized antibodies, as described in more detail below. The general principle of antibody humanization involves retaining the base sequence of the epitope-binding portion of the antibody while replacing the non-human remainder of the antibody with humanized antibody sequences.

[0062] In recent decades, there has been a resurgence of interest in the therapeutic potential of antibodies, and they have become one of the major classes of biotechnology-derived drugs: over 200 antibody-based drugs are approved for use or are in development.

[0063] I. General Structural Attributes of Antibodies The basic structural unit of naturally occurring immunoglobulins (e.g., IgG) is a tetramer composed of two short "light chains" complexed with two long "heavy chains," usually represented as a glycoprotein of about 150,000 Da. Each chain consists of an amino-terminal ("N-terminal") portion containing a "variable domain" and a carboxy-terminal ("C-terminal") portion containing at least one "constant domain." An IgG light chain is composed of a single "light chain variable domain" ("VL") and a single "light chain constant domain" ("CL"). Thus, the structure of the light chain of an IgG molecule is n-VL-CL-c (where n and c represent the N- and C-termini of the polypeptide, respectively). An IgG heavy chain is composed of a single "heavy chain variable domain" ("VH"), three "heavy chain constant domains" ("CH1," "CH2," and "CH3"), and a "hinge" region ("H") located between the CH1 and CH2 domains. Thus, the structure of an IgG heavy chain is n-VH-CH1-H-CH2-CH3-c, where n and c represent the N- and C-termini of the polypeptide, respectively. The ability of an intact, unmodified antibody (e.g., an IgG antibody) to bind to an epitope of an antigen depends on the presence and sequence of variable domains.

[0064] a) Constant Domain (1) Light chain constant domain A representative CL domain is a human IgG CLκ domain. The amino acid sequence of a representative human CLκ domain is (SEQ ID NO: 1): RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC is.

[0065] Alternatively, a representative CL domain is a human IgG CLλ domain. The amino acid sequence of a representative human CLλ domain is (SEQ ID NO: 2): QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTPSKQS NNKYAASSYL SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS is.

[0066] (2) Heavy chain CH1 domain A representative CH1 domain is the human IgG1 CH1 domain. The amino acid sequence of a representative human IgG1 CH1 domain is (SEQ ID NO: 3): ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRV is.

[0067] Another exemplary CH1 domain is the human IgG2 CH1 domain. The amino acid sequence of a representative human IgG2 CH1 domain is (SEQ ID NO:4): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSNFGTQT YTCNVDHKPS NTKVDKTV is.

[0068] Another exemplary CH1 domain is the human IgG3 CH1 domain. The amino acid sequence of a representative human IgG3 CH1 domain is (SEQ ID NO:5): ASTKGPSVFP LAPCSRSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YTCNVNHKPS NTKVDKRV is.

[0069] Another exemplary CH1 domain is the human IgG4 CH1 domain. The amino acid sequence of a representative human IgG4 CH1 domain is (SEQ ID NO:6): ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRV is.

[0070] (3) Heavy chain hinge region A representative hinge region is a human IgG1 hinge region. The amino acid sequence of a representative human IgG1 hinge region is (SEQ ID NO:7): EPKSCDKTHT CPPCP is.

[0071] Another exemplary hinge region is the human IgG2 hinge region. The amino acid sequence of a representative human IgG2 hinge region is (SEQ ID NO:8): ERKCCVECPP CP is.

[0072] Another exemplary hinge region is the human IgG3 hinge region. The amino acid sequence of a representative human IgG3 hinge region is (SEQ ID NO:9): ELKTPLGDTT HTCPRCPEPK SCDTPPPCPR CPEPKSCDTP PPCPRCPEPK SCDTPPPCPR CP is.

[0073] Another exemplary hinge region is the human IgG4 hinge region. The amino acid sequence of a representative human IgG4 hinge region is (SEQ ID NO: 10): ESKYGPPCPS CP is.

[0074] As described herein, the IgG4 hinge region may contain stabilizing mutations such as the S228P substitution (as numbered by the EU index as set forth in Kabat). The amino acid sequence of one exemplary stabilized IgG4 hinge region is (SEQ ID NO: 11): ESKYGPPCP P CP is.

[0075] (4) Heavy chain CH2 and CH3 domains The CH2 and CH3 domains of the two heavy chains interact to form the "Fc region" of an IgG antibody, which is the domain recognized by cellular Fc receptors, including but not limited to Fcγ receptors (FcγRs). As used herein, the term "Fc region" is used to define the C-terminal region of an IgG heavy chain. A portion of the Fc region (including a portion encompassing the entire Fc region) is referred to herein as an "Fc domain." An Fc region is said to be of a particular IgG isotype, class, or subclass if its amino acid sequence is more homologous to that IgG isotype than to other IgG isotypes. In addition to their known uses in diagnostics, antibodies have also been shown to be useful as therapeutic agents.

[0076] The amino acid sequence of a representative human IgG1 CH2-CH3 domain is (SEQ ID NO: 12): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X which are numbered according to the EU index as set forth in Kabat, where X is lysine (K) or absent.

[0077] The amino acid sequence of a representative human IgG2 CH2-CH3 domain is (SEQ ID NO: 13): 231 240 250 260 270 280 APPVA-GPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTF RVVSVLTVVH QDWLNGKEYK CKVSNKGLPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDISVE 390 400 410 420 430 WESNGQPENN YKTTPPMLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSPG X which are numbered according to the EU index as set forth in Kabat, where X is lysine (K) or absent.

[0078] The amino acid sequence of a representative human IgG3 CH2-CH3 domain is (SEQ ID NO: 14): 231 240 250 260 270 280 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD 290 300 310 320 330 GVEVHNAKTK PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 340 350 360 370 380 PIEKTISKTK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESSGQPENN YNTTPMLDS DGSFFLYSKL TVDKSRWQQG NIFSCSVMHE 440 447 ALHNRFTQKS LSLSPG X which are numbered according to the EU index as set forth in Kabat, where X is lysine (K) or absent.

[0079] The amino acid sequence of a representative human IgG4 CH2-CH3 domain is (SEQ ID NO: 15): 231 240 250 260 270 280 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD 290 300 310 320 330 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS 340 350 360 370 380 SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE 390 400 410 420 430 WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE 440 447 ALHNHYTQKS LSLSLG X which are numbered according to the EU index as set forth in Kabat, where X is lysine (K) or absent.

[0080] Throughout this specification, the numbering of residues in the constant region of the IgG heavy chain is based on Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, NH1, MD (1991) ("Kabat"), which is expressly incorporated herein by reference. The term "EU index as in Kabat" refers to the numbering of the constant domains of the human IgG1 EU antibody.

[0081] Polymorphisms have been observed at many different positions within antibody constant regions (e.g., Fc positions including, but not limited to, positions 270, 272, 312, 315, 356, and 358, numbered according to the EU index as set forth in Kabat), and therefore slight differences may exist between the sequences presented herein and those of the prior art. Polymorphic forms of human immunoglobulins have been well characterized. Currently, 18 Gm allotypes are known: G1m(1, 2, 3, 17) or G1m(a, x, f, z), G2m(23) or G2m(n), G3m(5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m(b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) (Lefranc, et al., The human IgG subclasses: molecular analysis of structure, function and regulation. Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-211). In particular, the antibody of the present invention is Any allotype, isoallotype, or haplotype of an immunoglobulin gene can be incorporated, and is not intended to be limited to the allotype, isoallotype, or haplotype of the sequences presented herein. Furthermore, depending on the expression system, the C-terminal amino acid residue of the CH3 domain (bold above) can be removed post-translationally. Thus, the C-terminal residue of the CH3 domain can be any amino acid residue in the molecules of the invention. Specifically encompassed by the invention are molecules of the invention lacking the C-terminal residue of the CH3 domain. Also specifically encompassed by the invention are molecules containing a C-terminal lysine residue of the CH3 domain.

[0082] b) Variable Domain The variable domain of an IgG molecule consists of three complementarity determining regions ("CDRs") that contain the amino acid residues of the antibody that will contact the epitope, and intervening non-CDR segments called framework regions ("FRs"), which generally enable such contacts by maintaining the structure and positioning of the CDR loops (although certain framework residues may also contact the epitope). The VL and VH domains thus have the structure n-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-c. The amino acid sequences of the CDRs determine whether an antibody can bind to a particular epitope. The interaction of the antibody light chain and heavy chain, and in particular the interaction of these VL and VH domains, which are present on separate polypeptides, forms the epitope-binding site of the antibody.

[0083] Amino acids from the mature heavy and light chain variable domains of immunoglobulins are designated by the position of the amino acid within the chain. Kabat (Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, NH1, MD (1991)) describes numerous amino acid sequences for antibodies, identifies amino acid consensus sequences for each subgroup, assigns residue numbers to each amino acid, and identifies CDRs and FRs as defined by Kabat (Chothia, C. & Lesk, A. M. (1987) "Canonical Structures For The Hypervariable Regions Of Immunoglobulins,” J.Mol. Biol. 196:901‐917 ) defined by H 1 will be understood to start 5 residues earlier) The Kabat numbering scheme can be extended for antibodies not included in the compendium by aligning the antibody with one of the Kabat consensus sequences with reference to conserved amino acids. This method of assigning residue numbers has become standard in the art, and amino acids at identical positions in different antibodies, including chimeric or humanized variants, are readily identified. For example, amino acid 50 of a human antibody light chain occupies the same position as amino acid 50 of a murine antibody light chain. Thus, the positions within the VL and VH domains where these CDRs begin and end are well defined and can be confirmed by inspection of the sequences of the VL and VH domains (see, e.g., Martin, CR (2010) "Protein Sequence Analysis of Human Antibodies"). and Structure Analysis of Antibody Variable Domains,”In: Antibody Engineering Vol. 2 (Kontermann, R. and Dubel, S. (eds.), Springer-Verlag Berlin Heidelberg, See Chapter 3 (pages 33-51).

[0084] Polypeptides that are (or can function as) the first, second, and third CDRs of the light chain of an antibody are referred to herein as CDRs 1, 2, and 3, respectively. L 1 domain, CDR L 2 domains and CDRs L Similarly, the first, second and third domains of an antibody heavy chain are called Polypeptides which are (or can function as) the first, second, and third CDRs of are referred to herein as CDRs 1, 2, and 3, respectively. H 1 domain, CDR H 2 domains and CDRs H 3 domains and Therefore, CDR L 1 domain, CDR L 2 domains, CDR L 3 domains, CD R H 1 domain, CDR H 2 domains and CDRs H The term 3-domain refers to a The present invention is directed to polypeptides that, when incorporated into a protein, enable the protein to bind to a particular epitope, whether that protein is an antibody having a light and heavy chain, or a diabody, or a single-chain binding molecule (e.g., scFv, BiTe, etc.), or another type of protein. Thus, as used herein, the term "epitope binding fragment" refers to a fragment of a molecule that is capable of immunospecifically binding to an epitope. An epitope-binding fragment may contain one, two, three, four, or five of the CDR domains of an antibody, or may contain all six of the CDR domains of an antibody, and may be capable of immunospecifically binding to such an epitope, but may not be directed to an epitope that is different from the epitope of such an antibody. The epitope-binding fragment may exhibit different isomerism, affinity, or selectivity. Typically, however, an epitope-binding fragment will contain all six of the CDR domains of such an antibody. An epitope-binding fragment of an antibody may be a single polypeptide chain (e.g., an scFv) or may comprise two or more polypeptide chains, each having an amino terminus and a carboxy terminus (e.g., a diabody, an Fab fragment, an Fab2 fragment, etc.). Unless otherwise specified, the term "epitope-binding fragment" as used herein refers to a fragment of an antibody that is a single polypeptide chain (e.g., an scFv), or may comprise two or more polypeptide chains, each having an amino terminus and a carboxy terminus (e.g., a diabody, an Fab fragment, an Fab2 fragment, etc.). The domain order of the protein molecule described is from "N-terminal to C-terminal" direction.

[0085] Epitope-binding sites may comprise complete variable domains fused to constant domains, or only the complementarity-determining regions (CDRs) of such variable domains grafted onto appropriate framework regions. Epitope-binding sites may be wild-type or modified by one or more amino acid substitutions.

[0086] c) Antibody humanization The present invention particularly encompasses binding molecules (including antibodies and diabodies) comprising the VL and / or VH domains of a humanized antibody. The term "humanized" antibody refers to a chimeric molecule, generally prepared using recombinant techniques, that contains an epitope-binding site of an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The polynucleotide sequences of the variable domains of such antibodies can be used for genetic engineering to generate derivatives and improve the affinity or other characteristics of the antibody. Both heavy and light chain variable domains are known to contain three CDRs flanked by four FRs that vary in response to the antigen of interest and determine binding capacity; the framework regions are relatively conserved in a given species and are presumed to provide a scaffold for the CDRs. When preparing non-human antibodies against a particular antigen, the variable domains can be "reshaped" or "humanized." The general principle of antibody humanization involves retaining the nucleotide sequence of the epitope-binding portion of the antibody while replacing the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanize a monoclonal antibody: (1) determining the nucleotide and predicted amino acid sequences of the light and heavy chain variable domains of the starting antibody; (2) designing the humanized or caninized antibody, i.e., determining the antibody framework regions to be used during the humanization or caninization process; (3) the actual humanization or caninization method / technique; and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

[0087] A number of humanized antibody molecules containing epitope-binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies with rodent or modified rodent variable domains and their associated CDRs fused to human constant domains (see, e.g., Lobuglio et al. (1989) "Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989)). Other references The literature describes rodent CDRs that are grafted into human supporting framework regions (FRs) before fusion with appropriate human antibody constant domains (e.g., Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; and (See Jones et al. (1986) "Replacing the Complementarity-Determining Regions in a Human Antibody with Those from a Mouse," Nature 321:522-525). The reference describes rodent CDRs supported by recombinantly veneered rodent framework regions. See, e.g., European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize undesirable immunological responses to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic applications of these moieties in human recipients. Other methods that may be utilized to humanize antibodies are described by Daugherty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And "Rapid Expression of a Murine Monoclonal Antibody Directed Against the CD18 Component of Leukocyte Integrins," Nucl. Acids Res. 19:2471-2476 and U.S. Patent No. 6 Nos. 6,054,297; and 5,997,867. In some embodiments, humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibody). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered to differ in sequence relative to the original antibody.

[0088] 2.CD137-binding domain The present invention is directed to CD137-binding molecules, such as monospecific antibodies capable of binding to epitopes of CD137 and molecules comprising epitope-binding fragments thereof. A novel human monoclonal antibody CD137-binding domain, "CD137 MAB-6," is provided below. The present invention specifically relates to the VL and / or VH domains of CD137 MAB-6, and / or the CDRs of the VL region. L and / or a VH domain. CDR d a CD137-binding molecule comprising one, two or all three of the following: This includes and encompasses multispecific CD137 binding molecules (e.g., bispecific antibodies, bispecific diabodies, BiTEs, trivalent binding molecules, etc.), such as 137xTA binding molecules, or any of the variants thereof provided below.

[0089] a) Human CD137 MAB-6 CD137 MAB-6 is a novel human monoclonal antibody. The amino acid sequence of the VH domain of CD137 MAB-6 (CD137 MAB-6 VH1) (SEQ ID NO: 46) is (CDR H Residues are underlined): QVQLQESGPG LVKPSETLSL TCTVSGGSIS SYYWS WIRQP PGKGLEWIG R IYTSGSTNYN PSLKS RVTMS VDTSKNQFSL KLSSVTAADT AVYYCAR DGW YDEDYNYYGM DV WGQGTTVT VSS is.

[0090] CD137 MAB‐6 VH1 CDR H The amino acid sequence of is: CDR H 1 (SEQ ID NO: 47): SYYWS CDR H 2 (SEQ ID NO: 48): RIYTSGSTNYNPSLKS CDR H 3 (SEQ ID NO: 49): DGWYDEDYNYYGMDV is.

[0091] The amino acid sequence of the VL domain of CD137 MAB-6 (CD137 MAB-6 VL1) is (SEQ ID NO: 50) (CDR L Residues are underlined): EIVMTQSPAT LSLTPGERAT LSC RASQSVS SNYLS WFQQI PGQAPRLLIY GASTRAT GIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYC Q QDYDLPWT FG QGTKVEIK is.

[0092] CD137 MAB‐6 VL1 CDR L The amino acid sequence of is: CDR L 1 (SEQ ID NO: 51): RASQSVSSNYLS CDR L 2 (SEQ ID NO: 52): GASTRAT CDR L 3 (SEQ ID NO: 53): QQDYDLPWT is.

[0093] b) Deimmunized CD137 MAB-6 Deimmunize the VL domain of CD137 MAB-6 as described in the example below. As a result, the amino acid sequence of SEQ ID NO: 54 (CDR L residues are underlined) : EIVMTQSPAT LSLX1PGERAT LSC RASQSVS SNYLS WX2QQX3PGQAPRLLIY GASTRAT GIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYC Q QDYDLPWT FG QGTKVEIK wherein X1, X2, and X3 are independently selected, and X1 is S or T; X2 is F or X3 is I or K) and designated "CD137 MAB-6 VLx" The VL domain named

[0094] In certain embodiments, X1 is S; X2 is Y; and X3 is K; or X1 is S; X2 is F; and X3 is K.

[0095] The amino acid sequences of variants of the CD137 MAB-6 VL domain, designated CD137 MAB-6 VL2 and CD137 MAB-6 VL3, are provided below. Any of these variant VL domains can be paired with a VH domain. Molecules containing a particular combination of CD137 MAB-6 VH / VL domains are referred to by reference to that particular VH / VL domain, e.g., binding domain CD137 MAB-6 A molecule comprising VH1 and CD137 MAB-6 VL3 is specifically referred to as "CD137 MAB-6(1.3)."

[0096] The amino acid sequence of variant CD137 MAB-6 VL2 is (SEQ ID NO: 55) (CDR L Residues are underlined): EIVMTQSPAT LSLSPGERAT LSC RASQSVS SNYLS WYQQK PGQAPRLLIY GASTRAT GIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYC Q QDYDLPWT FG QGTKVEIK is.

[0097] The amino acid sequence of variant CD137 MAB-6 VL3 is (SEQ ID NO: 56) (CDR L Residues are underlined): EIVMTQSPAT LSLSPGERAT LSC RASQSVS SNYLS WFQQK PGQAPRLLIY GASTRAT GIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYC Q QDYDLPWT FG QGTKVEIK is.

[0098] The CDRs, VL domains, and / or VH domains of the fully human and / or variant VH and VL CD137 MAB-6 domains described above, including any encompassed by one or more of the generic sequences of the CD137 MAB-6 VL domains presented above, can be used to form antibodies, diabodies, or binding molecules capable of binding to CD137. In specific embodiments, CD137 binding molecules of the invention, including CD137xTA binding molecules, comprise CD137 MAB-6 VH1 and CD137 MAB-6 VL3.

[0099] B. Bispecific Antibodies, Multispecific Diabodies, and Trivalent Molecules As mentioned above, natural antibodies can only bind to one epitope species, but can bind to multiple copies of that species. The ability of an antibody to bind to an epitope of an antigen depends on the presence and amino acid sequence of the antibody's VL and VH domains. The interaction between the antibody's light and heavy chains, particularly the interaction between its VL and VH domains, forms one of the two epitope-binding domains of a natural antibody such as IgG. Natural antibodies can only bind to one epitope species (i.e., they are monospecific), but they can bind to multiple copies of that species (i.e., they exhibit bivalency or multivalency).

[0100] Antibody functionality can be enhanced by generating multispecific antibody-based molecules that can simultaneously bind to two distinct and different antigens (or different epitopes of the same antigen) and / or by generating antibody-based molecules with greater valency (i.e., three or more binding domains) for the same epitope and / or antigen.

[0101] A variety of recombinant bispecific antibody formats have been developed for the production of such bispecific antibodies in order to provide molecules with greater potency than natural antibodies.

[0102] Most such approaches use linker peptides to fuse additional binding domains (e.g., scFv, VL, VH, etc.) to or within an antibody core (IgA, IgD, IgE, IgG, or IgM), or to fuse multiple antigen-binding moieties (e.g., two Fab fragments or scFvs) to each other. An alternative format uses linker peptides to fuse a binding protein (e.g., scFv, VL, VH, etc.) to a dimerization domain, such as a CH2-CH3 domain or alternative polypeptide (WO 2005 / 070966, WO 2006 / 107786A, WO 2006 / 107617A, WO 2007 / 046893). WO 2013 / 174873, WO 2011 / 133886, and WO 2010 / 136172 disclose multispecific antibodies in which the CL and CH1 domains have been switched from their natural positions, WO 2008 / 027236 and WO 2010 / 108127 disclose antibodies in which the VL and VH domains have been diversified to enable binding to more than one antigen, and WO 2010 / 028797, WO 2010028796, and WO 2010 / 028795 disclose recombinant antibodies in which the Fc region has been replaced with additional VL and VH domains to form trivalent binding molecules. WO 2003 / 025018 and WO 2003 / 012069 disclose recombinant diabodies in which each chain contains an scFv domain. WO 2013 / 006544 discloses multivalent fab molecules that are synthesized as single polypeptide chains and then subjected to proteolytic degradation to obtain heterodimeric structures. Thus, the molecules disclosed in these documents exchange all or some of their ability to mediate effector functions for the ability to bind additional antigen species.International Publication Nos. WO 2014 / 022540, WO 2013 / 003652, WO 2012 / 162583, WO 2012 / 156430, WO 2011 / 086091, WO 2008 / 024188, WO 2007 / 024715, WO 2007 / 075270, WO 1998 / 002463, WO 1992 / 022583 and WO 1991 / 003493 disclose adding additional binding domains or functional groups to antibodies or antibody portions (e.g., adding a diabody to the light chain of an antibody, or adding additional VL and VH domains to the light and heavy chains of an antibody, or adding heterologous fusion proteins to each other or linking multiple Fab domains to each other).

[0103] The art has further noted the possibility of generating diabodies that differ from such natural antibodies in that they can bind two or more different epitope species (i.e., can exhibit bispecificity or multispecificity in addition to bivalency or multivalency) (see, e.g., Holliger et al. (1993) "'Diabodies': Small Bivalent And Bispecific Antibody Fragments," Proc. Natl. Acad. Sci. (USA) 90:6444-6448; U.S. Patent Publication No. 2004 / 00584). 00 (Hollinger et al.); U.S. Patent Publication No. 2004 / 0220388 (Mertens et al. al.);Alt et al. (1999) FEBS Lett. 454(1‐2):90‐94;Lu, D. etal. (2005) “A Fully Human Recombinant IgG‐Like Bispecific Antibody To Both The Epidermal Growth FactorReceptor And The Insulin‐Like Growth Factor ReceptorFor Enhanced Ant itumor Activity,” J. Biol.Chem. 280(20):19665‐19672;Olafsen, T. et al. (2004) “Covalent Disulfide‐Linked Anti‐CEA Diabody Allows Site‐Specific Conjugation And Radiolabeling For Tumor TargetingApplications,” Protein Eng Des Sel. 17(1):21‐27;Baeuerle, PA et al. al. (2009) “Bispecific T cell Engaging Antibodies For Cancer Therapy,” Cancer Res. 69(12):4941-4944).

[0104] The provision of non-monospecific "diabodies" offers a key advantage over antibodies: the ability to co-ligate and colocalize cells expressing different epitopes. Bispecific diabodies therefore have a wide range of applications, including therapeutics and immunodiagnostics. Bispecificity allows for great flexibility in the design and engineering of diabodies for a variety of applications, thereby providing increased avidity for multimeric antigens, cross-linking of different antigens, and directed targeting to specific cell types based on the presence of both target antigens.

[0105] The formation of such non-monospecific diabodies requires the successful assembly of two or more distinct and different polypeptides (i.e., it requires that the diabody be formed by heterodimerization of different polypeptide chain species). Despite this challenge, the art has successfully developed stable, covalent heterodimeric, non-monospecific diabodies (e.g., Chichili, GR et al. (2015) "A CD3xCD123 Bispecific DART For Redirecting Host T Cells To Myelogenous Leukemia: Preclinical Activity And Safety In Nonhuman Primates," Sci. Transl. Med. 7(289):289ra82; Veri, MC et al. (2010) "Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIB (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold," Arthritis Rheum. 62(7):1933-1943; Moore, PA et al. (2011) "Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T Cell Killing Of B Cells," Arthritis Rheum. -Cell Lymphoma,” Blood 117(17):4542-4551; U.S. Patent Publication No. 2007 / 00049 (See U.S. Patent Publication Nos. 2009 / 0060910; 2010 / 0174053; 20130295121; 2014 / 0099318; 2015 / 0175697; 2016 / 0017038; 2016 / 0194396; 2016 / 0200827; and 2017 / 0247452.) Such diabodies comprise two or more covalently conjugated polypeptides and involve the incorporation of one or more cysteine ​​residues into each of the employed polypeptide species. For example, the addition of a cysteine ​​residue to the C-terminus of such a structure has been shown to allow disulfide bonding between the polypeptide chains, which stabilizes the resulting heterodimer without interfering with the binding properties of the divalent molecule.

[0106] C. Components of Representative CD137xTA Binding Molecules of the Invention The CD137xTA binding molecules of the invention are composed of polypeptides and may be composed of two, three, four, or more polypeptide chains. As used herein, the term "composed of" is intended to be open-ended; thus, a CD137xTA binding molecule of the invention that is composed of two polypeptide chains may have an additional polypeptide chain. Such a chain may have the same sequence as another polypeptide chain of the binding molecule, or may have a different sequence from any other polypeptide chain of the binding molecule.

[0107] 1. Representative "linker" peptides The polypeptides of the CD137xTA binding molecules of the invention comprise domains that are preceded by, followed by, and / or linked together by "linker" peptides, such as linker 1, linker 2, linker 3, etc. Although specific "linker" peptides are utilized, in light of the teachings provided herein, alternative linkers can be readily identified and employed to obtain CD137xTA binding molecules.

[0108] The length of linker 1 separating the VL and VH domains of a polypeptide chain is selected to substantially or completely prevent the VL and VH domains from binding to one another (e.g., a length of 12 amino acid residues or less). Thus, the VL1 and VH2 domains of a first polypeptide chain are substantially or completely unable to bind to one another and do not form an epitope-binding site capable of substantial binding to a first or second antigen. Similarly, the VL2 and VH1 domains of a second polypeptide chain are substantially or completely unable to bind to one another and do not form an epitope-binding site capable of substantial binding to a first or second antigen. A representative intervening linker peptide (linker 1) has the amino acid sequence (SEQ ID NO: 16): GGGSGGGG, which, in contrast to the longer intervening linker peptides (e.g., GGGGSGGGGSGGGGS (SEQ ID NO: 17)) employed in the generation of scFv molecules, separate the VL and VH domains of the same polypeptide chain. It's too short to be combined with the main part.

[0109] One purpose of Linker 2 is to separate the VH Domain of a polypeptide chain from any Heterodimer-Promoting Domains of that polypeptide chain. Any of a variety of linkers can be used for this purpose. Exemplary sequences for such Linker 2 include the amino acid sequence GGCGGG (SEQ ID NO: 18), which has a cysteine ​​residue that can be used to covalently link the first and second polypeptide chains together via a disulfide bond, or ASTKG (SEQ ID NO: 19), which is the IgG CH1 domain. Linker 2, ASTKG (SEQ ID NO: 19), contains such cysteines. Since the use of such a Linker 2 is typically associated with the use of a cysteine-containing Heterodimer-Promoting Domain, such as the E coil of SEQ ID NO:39 or the K coil of SEQ ID NO:40 (see below).

[0110] One purpose of Linker 3 is to separate the Heterodimer-Promoting Domain of a polypeptide chain from the Fc Domain of that polypeptide chain. A second purpose is to provide a cysteine-containing polypeptide domain. Any of a variety of linkers can be used for this purpose. A representative sequence for such a Linker 3 comprises the amino acid sequence: DKTHTCPPCP (SEQ ID NO: 20). Another representative sequence for Linker 3 comprises the amino acid sequence: GGGDKTHTCPPCP (SEQ ID NO: 21). Yet another representative sequence for Linker 3 comprises the amino acid sequence: String: LEPKSADKTHTCPPCP (SEQ ID NO: 30), or LEPKSSDKTHTCPPCP (SEQ ID NO: 31).

[0111] One purpose of linker 4 is to separate the CH2-CH3 domain of the Fc region ("Fc domain") from the N-terminus of the VL domain. Any of a variety of linkers can be used for this purpose. Exemplary sequences for such linkers 4 include the amino acid sequence APSSS (SEQ ID NO: 22), the amino acid sequence APSSSPME (SEQ ID NO: 23), the amino acid sequence GGGSGGSGGGG (SEQ ID NO: 24), or the amino acid sequence GGGGSGGGSGGG (SEQ ID NO: 25).

[0112] The Fc Region-containing molecules of the present invention may include an additional intervening linker peptide (linker); generally, such a linker will be incorporated between the Heterodimer-Promoting Domain (e.g., E coil or K coil) and the CH2-CH3 Domain, and / or between the CH2-CH3 Domain and the Variable Domain (i.e., VH or VL). Typically, such additional linkers contain 3-20 amino acid residues and may optionally include all or a portion of an IgG hinge region (preferably the cysteine-containing portion of the IgG hinge region). Linkers that can be employed in the bispecific Fc Region-containing diabody molecules of the present invention include: GGC, GGG, ASTKG (SEQ ID NO: 19), DKTHTCPPCP (SEQ ID NO: 20), APSSS (SEQ ID NO: 22), APSSSPME (SEQ ID NO: 23), GGGSGGGSGGG (SEQ ID NO: 24), GGGGSGGGSGGG (SEQ ID NO: 25), and GGGGSGGGSGGG (SEQ ID NO: 26). SEQ ID NO: 25), LGGGSG (SEQ ID NO: 26), GGGS (SEQ ID NO: 27), LEPKSS (SEQ ID NO: 28), VEPKSADKTHTCPPCP (SEQ ID NO: 29), LEPKSADKTHTCPPCP (SEQ ID NO: 30), and LEPKSSDKTHTCPPCP (SEQ ID NO: 31). For ease of cloning, LEPKSS (SEQ ID NO: 28) may be used in place of GGG or GGC. Additionally, the amino acids GGG, or LEPKSS ( Alternative linkers can be obtained by immediately following DKTHTCPPCP (SEQ ID NO: 28) with the following linkers: GGGDKTHTCPPCP (SEQ ID NO: 21); and LEPKSSDKTHTCPPCP (SEQ ID NO: 31). The bispecific Fc region-containing molecules of the present invention can incorporate an IgG hinge region, or a portion thereof, of a human IgG1, IgG2, IgG3, or IgG4 antibody.

[0113] 2. Representative Heterodimer-Promoting Domains As discussed above, the formation of the CD137xTA binding molecules of the present invention involves the assembly (i.e., heterodimerization) of two or more different polypeptide chains. Heterodimer formation of the first and second polypeptide chains can be promoted by the inclusion of a "Heterodimer-Promoting Domain." A Heterodimer-Promoting Domain is a domain in the hinge region of IgG (i.e., for example, GVEPKSC (SEQ ID NO: 32), VEPKSC (SEQ ID NO: 33)) or AEPKS C (SEQ ID NO: 34) on one of the polypeptide chains. 4)), and a CL domain on the other polypeptide chain (i.e., a polypeptide derived from the hinge region (i.e., a polypeptide derived from the hinge region, e.g ... , a polypeptide derived from the CL domain) (US Patent Publication No. 2007 / 0004909).

[0114] Alternatively, Heterodimer-Promoting Domains of the invention may comprise tandemly repeated coil domains with opposite charges, such as an "E-coil" helical domain (SEQ ID NO: 37) with glutamic acid residues that form a negative charge at pH 7. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E The other Heterodimer-Promoting Domain will contain four tandem "K coil" domains (SEQ ID NO: 38: ) with lysine residues that form a positive charge at pH 7. K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL KThe presence of such a charged domain promotes association between the first and second polypeptides, thereby promoting heterodimerization. In another embodiment, the Heterodimer-Promoting Domain is engineered to contain a Heterodimer-Promoting Domain in which one of the four tandem "E-coil" helical domains of SEQ ID NO:37 has been modified to contain a cysteine ​​residue: E VAA CE K‐ E VAAL E K‐ E VAAL E K - E VAAL E Similarly, in another embodiment, 4 of SEQ ID NO: 38 is utilized. Heterodimer-Promoting Domain, in which one of the two tandem "K-coil" helical domains has been modified to contain a cysteine ​​residue: K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40) is utilized.

[0115] 3. Covalent bonding of polypeptide chains The CD137xTA binding molecules of the invention are engineered so that a pair of its polypeptide chains are covalently linked to one another via one or more cysteine ​​residues positioned along their length to form a covalent molecular complex. Such cysteine ​​residues can be introduced into the intervening linker separating the VL and VH domains of the polypeptides. Optionally, or alternatively, Linker 2 or Linker 3, or another linker, may contain a cysteine ​​residue. Optionally, or alternatively, one or more coil domains of the coil-containing heterodimer-promoting domains contain amino acid substitutions incorporating a cysteine ​​residue, such as SEQ ID NO:39 or SEQ ID NO:40.

[0116] 4. Representative Fc domains The Fc domain of the Fc-bearing CD137xTA binding molecules of the invention may comprise a complete Fc region (e.g., a complete IgG Fc region), or only a fragment of a complete Fc region. Thus, the Fc domain of the Fc-bearing CD137xTA binding molecules of the invention may comprise only a fragment of the CH2 domain of a complete Fc region. The Fc Domain of the bispecific Fc diabodies of the present invention may comprise a portion or all of a CH2 and / or CH3 Domain, or may comprise a variant CH2 and / or variant CH3 sequence (which may comprise, for example, one or more insertions and / or one or more deletions relative to the CH2 or CH3 Domain of a complete Fc Region). The Fc Domain of the bispecific Fc diabodies of the present invention may comprise a non-Fc polypeptide portion, or may comprise a portion of a non-native complete Fc Region, or may comprise a non-naturally occurring orientation of the CH2 and / or CH3 Domain (e.g., two CH2 Domains or two CH3 Domains; or a CH2 Domain linked to a CH3 Domain in an N-terminal to C-terminal direction, etc.).

[0117] The Fc domain of the Fc-bearing CD137xTA binding molecules of the invention may comprise the amino acid sequence of a naturally occurring Fc domain; however, it may be desirable to include one or more substitutions in the CH2-CH3 domains forming such Fc domain such that the resulting Fc domain exhibits reduced binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (e.g., less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the binding exhibited by such a molecule when having an Fc domain with the amino acid sequence of a naturally occurring Fc region), or substantially undetectable (compared to the binding exhibited by the wild-type Fc region). Fc variants and mutant forms capable of mediating such altered binding are known in the art and include amino acid substitutions at one or more positions selected from the group consisting of 234, 235, 265, and 297, where the numbering is that of the EU index as described in Kabat (see, e.g., U.S. Pat. No. 5,624,821). In one embodiment, the CH2-CH3 domain of the first and / or third polypeptide chain of the Fc-bearing molecule of the invention comprises any one, two, three, or four of the following substitutions: L234A, L235A, D265A, N297Q, and N297G. Alternatively, the CH2-CH3 domain of a naturally occurring Fc region is utilized that has naturally reduced (or substantially no) binding to FcγRIIIA (CD16a) and / or naturally reduced effector function (compared to the binding and effector function exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 12)). In a specific embodiment, the Fc-bearing molecule of the present invention comprises an IgG2 Fc region (SEQ ID NO: 13) or an IgG4 Fc region (SEQ ID NO: 15). When an IgG4 Fc region is used, the present invention also encompasses the introduction of a stabilizing mutation, such as the hinge region S228P substitution described above (see, for example, SEQ ID NO: 11).

[0118] In one exemplary embodiment, the IgG1 CH2-CH3 domain of the Fc-bearing CD137xTA binding molecule of the invention employs a substitution with alanine at position 234 and a substitution with alanine at position 235 (wherein the numbering is that of the EU index as set forth in Kabat) (SEQ ID NO: 41): APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X wherein X is lysine (K) or is absent.

[0119] The serum half-life of a protein comprising an Fc region can be extended by increasing the binding affinity of the Fc region for FcRn. As used herein, the term "half-life" refers to a pharmacokinetic property of a molecule that is a measure of the average survival time of the molecule after administration. Half-life can be expressed as the time required for 50 percent (50%) of a known amount of a molecule to be cleared from a subject's body (e.g., a human patient or other mammal) or a particular body cavity thereof, as measured in serum (i.e., circulating half-life) or other tissues. Generally, an increase in half-life is achieved by increasing the mean residence time (MRT) in the circulation of an administered molecule. This leads to an extension of the

[0120] In some embodiments, the Fc-bearing CD137xTA binding molecules of the invention comprise a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region such that the half-life of the molecule is increased (compared to a molecule comprising a wild-type Fc region). The variant Fc region comprises an Fc region, and the variant Fc region comprises an amino acid substitution that extends half-life. Many amino acid substitutions that can extend the half-life of Fc-bearing molecules are known in the art, see, for example, the amino acid substitutions described in U.S. Patent Nos. 6,277,375, 7,083,784, 7,217,797, 8,088,376, U.S. Patent Publication Nos. 2002 / 0147311, 2007 / 0148164, and 2011 / 0081347. The Fc-bearing CD137xTA binding molecules with enhanced half-life may comprise two or more substitutions selected from: T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, H435K, and Y436I, wherein the numbering is that of the EU index as set forth in Kabat.

[0121] In particular, the CH2-CH3 domain employed may contain the following substitutions: (A) M252Y, S254T and T256E; (B) M252Y and S254T; (C) M252Y and T256E; (D) T250Q and M428L; (E) T307Q and N434A; (F) A378V and N434A; (G) N434A and Y436I; (H) V308P and N434A; (I) K288D and H435K; or (J)M428L and N434S where the numbering is that of the EU index as set forth in Kabat.

[0122] Representative sequences of the CH2 and CH3 domains include triple amino acid substitutions: M252Y / S254T / T256E (YTE), which significantly enhance serum half-life, such as SEQ ID NO: 42 or SEQ ID NO: 43, which are variants of the IgG1 CH2-CH3 domain, or SEQ ID NO: 44, which is a variant of the IgG4 CH2-CH3 domain ((Dall'Acqua, WF et al. (2006) "Properties of Human IgGs Engineered for Enhanced Binding to the Neonatal Fc Receptor (FcRn)," J. Biol. Chem. 281(33):23514-23524): SEQ ID NO:42: APELLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X (wherein X is lysine (K) or absent) SEQ ID NO:43: APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X (wherein X is lysine (K) or absent) SEQ ID NO:44: APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X (wherein X is lysine (K) or absent)

[0123] The present invention also encompasses Fc-bearing CD137xTA binding molecules comprising variant Fc domains that exhibit altered effector function, such as when assessed in NK-dependent or macrophage-dependent assays, altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function. Fc domain modifications that have been identified to alter effector function are known in the art and include modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and modifications that decrease binding to inhibitory receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, JB et al. (2007) "Fc Optimization of Therapeutic Antibodies Enhances Their Ability to Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors," Cancer Res. 57(18):8882-8890). Exemplary variants of human IgG1 Fc domains with reduced binding to CD32B and / or increased binding to CD16A contain L235V, F243L, R292P, Y300L, V305I, or P296L substitutions. These amino acid substitutions may be present in any combination within the human IgG1 Fc domain. In one embodiment, the human IgG1 The Fc domain variant contains F243L, R292P, and Y300L substitutions, where the numbering is that of the EU index as set forth in Kabat. In another embodiment, the human IgG1 Fc domain variant contains F243L, R292P, Y300L, V305I, and P296L substitutions, where the numbering is that of the EU index as set forth in Kabat. In another embodiment, the human IgG1 Fc domain variant contains L235V, F243L, R292P, Y300L, and P396L substitutions, where the numbering is that of the EU index as set forth in Kabat.

[0124] The CH2 and / or CH3 domains of the CD137xTA binding molecules of the present invention need not be identical in sequence and are advantageously modified to promote heterodimerization between two CH2-CH3-bearing polypeptide chains. For example, amino acid substitutions (preferably with amino acids containing bulky side groups, e.g., tryptophan, that form "knobs") can be introduced into the CH2 or CH3 domains to prevent interaction with similarly mutated domains through steric hindrance, allowing the altered domains to pair with domains containing complementary or adaptive mutations (e.g., glycine substitutions), i.e., "holes," to promote heterodimerization. Such mutations can be introduced into any pair of polypeptides comprising the bispecific Fc-bearing diabody molecule and into any portion of the polypeptide chains of the pair. Methods for engineering proteins to discourage homodimerization and promote heterodimerization are known in the art, particularly for the engineering of immunoglobulin-like molecules, and are encompassed herein (see, e.g., Ridgway et al. (1996) "'Knobs-Into -Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,”Protein Engr. 9:617-621; Atwell et al. (1997) “Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35; and Xie et al. (2005) "A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J. Immunol. Methods 296:95-101, each of which is incorporated herein by reference in its entirety. In one embodiment, the knob is a CHCl-CH of the first polypeptide chain. In one embodiment, a knob is introduced into the CH2-CH3 domain of the third polypeptide chain, and a hole is introduced into the CH2-CH3 domain of the third polypeptide chain. The knob thus serves to prevent two molecules of the first polypeptide chain from homodimerizing via their CH2 and / or CH3 domains. Because the third polypeptide chain in this embodiment contains a hole substitution, it has the ability to heterodimerize with the first polypeptide chain and homodimerize with itself (although such homodimerization does not form a molecule with an epitope-binding site). An exemplary knob is created by modifying a native IgG Fc domain to contain the modification T366W, where the numbering is that of the EU index as set forth in Kabat. An exemplary hole is created by modifying a native IgG Fc domain to contain the modifications T366S, L368A, and Y407V, where the numbering is that of the EU index as set forth in Kabat. To aid in the purification of the third polypeptide chain homodimer from the final bispecific Fc-bearing diabody comprising the first and third polypeptide chain heterodimer, the Protein A binding site in the CH2 and CH3 domains of the third polypeptide chain is preferably mutated by an amino acid substitution at position 435 (H435R), where the numbering is that of the EU index as set forth in Kabat. This renders the third polypeptide chain homodimer incapable of binding Protein A, while a properly assembled bispecific Fc-bearing diabody retains the ability to bind Protein A via the Protein A binding site on the first polypeptide chain.

[0125] SEQ ID NO: 45, SEQ ID NO: 146 and SEQ ID NO: 147 provide representative sequences of "knob-bearing" CH2 and CH3 domains that can be used in the CD137xTA binding molecules of the invention: SEQ ID NO:45: APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W C L VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL Y SKL TVDKSRWQQG NVFSCSVMHE ALHN H YTQKS LSLSPGX (wherein X is lysine (K) or absent) SEQ ID NO:146: APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL W C L VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG X (wherein X is lysine (K) or absent) SEQ ID NO:147: APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSL WC L VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLG X (wherein X is lysine (K) or absent)

[0126] SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150 provide representative sequences of "hole-bearing" CH2 and CH3 domains that can be used in the CD137xTA binding molecules of the invention: SEQ ID NO:148: APE AA GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL S C A VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS LSLSPG X (wherein X is lysine (K) or absent) SEQ ID NO:149: APE AA GGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSL SC A VK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SKL TVDKSRWQQG NVFSCSVMHE ALHN R YTQKS LSLSPG X (wherein X is lysine (K) or absent) SEQ ID NO:150: APEFLGGPSV FLFPPKPKDT L Y I T R E PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSL S CA V K GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFL V SRL TVDKSRWQEG NVFSCSVMHE ALHN R YTQKS LSLSLG X (wherein X is lysine (K) or absent)

[0127] The CH2-CH3 domains of SEQ ID NOs: 47 and 50 are IgG4 domains, and the CH2-CH3 domains of SEQ ID NOs: 45, 146, 148, and 149 are IgG1 domains. Note that SEQ ID NOs: 45, 146, 148, and 149 contain an alanine substitution at position 234 and an alanine substitution at position 235, thereby forming Fc domains with reduced (or substantially no) binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) compared to the binding exhibited by the wild-type Fc region (SEQ ID NO: 12). The present invention specifically encompasses CD137xTA-binding molecules comprising CH2-CH3 domains derived from any class of human IgG containing the substitutions described herein (e.g., M252Y / S254T / T256E; T366W; T366S / L368A / Y407V; and / or H435R). Furthermore, the present invention specifically encompasses CD137xTA-binding molecule structures that do not contain the C-terminal lysine residue described above.

[0128] In the above-described embodiment, the first polypeptide chain will have a "knob-bearing" CH2-CH3 sequence, such as the sequences of SEQ ID NOs: 45, 146, and 147, and the third polypeptide chain will have a "hole-bearing" CH2-CH3 sequence, such as the sequences of SEQ ID NOs: 148, 149, and 150. However, it will be appreciated that a "hole-bearing" CH2-CH3 domain (e.g., SEQ ID NO: 48) may be employed in the first polypeptide chain, in which case a "knob-bearing" CH2-CH3 domain (e.g., SEQ ID NO: 45) would be employed in the third polypeptide chain.

[0129] 5. Representative tumor antigens (TAs) and representative variable domains The CD137xTA binding molecules of the invention comprise at least one epitope-binding site specific for an epitope of a tumor antigen. Exemplary tumor antigens ("TAs") to which the CD137xTA binding molecules of the invention can bind include, but are not limited to, those presented in Table 1, which may be referred to herein by their common names, abbreviations, and / or gene names.

[0130] [Table 1] JPEG2025186381000003.jpg236164 JPEG2025186381000004.jpg238161 JPEG2025186381000005.jpg167161

[0131] Antibodies that recognize TA are known in the art or can be generated using known methods, including those described herein. Representative antibodies that contain VL and VH domains capable of binding to TA and whose sequences or polypeptide chains can therefore be employed in constructing CD137xTA binding molecules of the invention are listed in Table 2. Representative VH and VL domains of antibodies capable of binding to several tumor antigens are provided below.

[0132] [Table 2] JPEG2025186381000007.jpg233164 JPEG2025186381000008.jpg236165 JPEG2025186381000009.jpg236166 JPEG2025186381000010.jpg237166 JPEG2025186381000011.jpg28167

[0133] a) PD-L1 binding domain PD-L1 (also known as CD274 and B7-H1) is a 40-kDa transmembrane protein commonly expressed on the surface of T lymphocytes, B lymphocytes, DCs, macrophages, and non-hematological cells. Furthermore, PD-L1 is abnormally highly expressed in tumor cells, which is thought to be a major factor promoting tumor immune evasion. Binding of PD-L1 to its receptor PD-1 on T cells activates downstream signaling of the PD-1 receptor, delivering signals that inhibit T cell proliferation, cytokine production and release, and cytotoxicity. Antibodies that block PD-L1 / PD-1 disrupt the PD-1 axis, thereby reversing T cell suppression and enhancing endogenous antitumor immunity. CD137×TA binding molecules that bind to PD-L1 can co-link PD-L1-expressing tumor cells with CD137-expressing immune cells. Without being limited to any particular method, such co-localization can stimulate immune cells while also attenuating or blocking the inhibition of the immune system that occurs upon binding of PD-L1 and PD-1.

[0134] Any anti-PD-L1 antibody epitope-binding site may be used in accordance with the present invention, and the principles of the present invention will be described with reference to the PD-L1 tumor antigen. Representative antibodies that bind to human PD-L1 include atezolizumab, avelumab, and durvalumab, each of which has recently been approved for use in humans. Atezolizumab (marketed as TECENTRIQ®; CAS Registry Number 1380723-44-3; see U.S. Patent No. 9,873,740) is a humanized monoclonal antibody with modified IgG1 and kappa constant regions. Avelumab (marketed as BAVENCIO®; CAS Registry Number 1537032-82-8; see U.S. Patent No. 9,873,740) is a fully human monoclonal antibody with IgG1 / lambda constant regions. Durvalumab (commercially available as IMFINZI®; CAS Registry Number 1428935-60-7; see U.S. Patent No. 8,779,108) is a fully human monoclonal antibody with modified IgG1 and kappa constant regions. The complete heavy and light chain amino acid sequences of atezolizumab (WHO Drug Information, 2015, Recommended INN: List 74, 29(3):387), durvalumab (WHO Drug Information, 2015, Recommended INN: List 74, 29(3):393-394), and avelumab (WHO Drug Information, 2016, Recommended INN: List 74, 30(1):100-101) are known in the art. Additional anti-PD-L1 antibodies are also provided herein, including the humanized anti-PD-L1 antibody "hPD-L1 MAB-2" and optimized variants thereof.

[0135] (1) hPD-L1 MAB-2 The amino acid sequence of the VH domain of hPD-L1 MAB-2 (hPD-L1 MAB-2 VH1) is (SEQ ID NO: 57) (CDR H Residues are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMSWVRQA PGKGLEWVA Y ISIGGGTTYY PDTVKG RFTI SRDNAKNTLY LQMNSLKTED TAVYYCAR QG LPYYFDY WGQ GTLVTVSS is.

[0136] The amino acid sequence of the VL domain of hPD-L1 MAB-2 (hPD-L1 MAB-2 VL1) is (SEQ ID NO: 58) (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDVN TAVA WYQQKP GKAPKLLIY W ASTRHT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ HYNTPLT FGQ GTKVEIK is.

[0137] (2) Deimmunized and optimized hPD-L1 MAB-2 As described in the examples below, hPD-L1 MAB-2 was deimmunized and optimized for binding and expression to yield a variant VH domain designated "hPD-L1 MAB-2 VHx," and a VL domain designated "hPD-L1 MAB-2 VLx." The amino acid sequences of certain deimmunized and optimized variant VH and VL domains are provided below, and additional variants are provided in the examples.

[0138] The amino acid sequence of hPD-L1 MAB-2 VHx is (SEQ ID NO: 59) (CDR H Residue Groups are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIX 4 GGTTYY PDTVKG RFTI SRDNAKNX5LY LQMNSLX6X7ED TAVYYCAR X 8 G LPYYX 9 DY WGQ GTLVTVSS and X4, X5, X6, X7, X8, and X9 are independently selected; X4 is G or K; X5 is S or T; X6 is K or R; X7 is A or T; X8 is A or Q;

[0139] In a specific embodiment: a) X4 is G; X5 is S; X6 is R; X7 is A; and X8 is Q ;Is X9 an F?; b) X4 is K; X5 is S; X6 is R; X7 is A; and X8 is Q ;Is X9 G?; c) X4 is G; X5 is S; X6 is R; X7 is A; and X8 is A ;Is X9 an F?; d) X4 is K; X5 is S; X6 is R; X7 is A; and X8 is A. ;Is X9 an F?; e) X4 is G; X5 is S; X6 is R; X7 is A; and X8 is A ;X9 is G; or f) X4 is K; X5 is S; X6 is R; X7 is A; and X8 is Q ;X9 is F.

[0140] CDRs of hPD-L1 MAB-2 VHx H The amino acid sequence of is: CDR H 1 (SEQ ID NO: 60): SYTMS CDR H 2 (SEQ ID NO: 61): YISIX4GGTTYYPDTVKG CDR H 3 (SEQ ID NO: 62): X8GLPYYX9DY wherein: X4 is G or K; X8 is A or Q; and X9 is F or G.

[0141] The amino acid sequence of hPD-L1 MAB-2 VLx is (SEQ ID NO: 63) (CDR L Residue Groups are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDVN X 10 AVA WYQQKP GKAPKLLIY W ASTRHT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ HYNTPLT FGQ GTKVEIK and X 10 is E or T.

[0142] In a specific embodiment, X 10 is E.

[0143] CDRs of PD‐L1 MAB‐2 VLx L The amino acid sequence of is: CDR L 1 (SEQ ID NO: 64): KASQDVNX 10 AVA CDR L 2 (SEQ ID NO: 65): WASTRHT CDR L 3 (SEQ ID NO: 66): QQHYNTPLT and X 10 is E or T.

[0144] The amino acid sequences of five variant VH domains designated herein as "hPD-L1 MAB-2 VH2," "hPD-L1 MAB-2 VH3," "hPD-L1 MAB-2 VH4," "hPD-L1 MAB-2 VH5," and "hPD-L1 MAB-2 VH6," and one variant VL domain designated herein as "hPD-L1 MAB-2 VL2," are provided below. Any of the variant hPD-L1 MAB-2 VH domains disclosed herein can be paired with any of the hPD-L1 MAB-2 VL domains. Molecules comprising particular combinations of PD-L1 MAB-2 VH / VL domains will be referred to by reference to those particular VH / VL domains, for example, a molecule comprising the binding domains PD-L1 MAB-2 VH3 and hPD-L1 MAB-2 VL2 will be specifically referred to as "PD-L1 MAB-2(3.2)." The amino acid sequences of these variant VH and VL domains are provided below, with substitutions within the CDRs compared to VH1 or VL1 double underlined.

[0145] The amino acid sequence of hPD-L1 MAB-2 VH2 is (SEQ ID NO: 67) (CDR H Residue Groups are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIGGGTTYY PDTVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR QG LPYYFDY WGQ GTLVTVSS is.

[0146] The amino acid sequence of hPD-L1 MAB-2 VH3 is (SEQ ID NO: 68) (CDR H Residue Groups are shown underlined and substitutions are double underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIKGGTTYY PDTVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR QG LPYYGDY WGQ GTLVTVSS is.

[0147] The amino acid sequence of hPD-L1 MAB-2 VH4 is (SEQ ID NO: 69) (CDR H Residue Groups are shown underlined and substitutions are double underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIGGGTTYY PDTVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR AG LPYYFDY WGQ GTLVTVSS is.

[0148] The amino acid sequence of hPD-L1 MAB-2 VH5 is (SEQ ID NO: 70) (CDR H Residue Groups are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIKGGTTYY PDTVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR AG LPYYFDY WGQ GTLVTVSS is.

[0149] The amino acid sequence of hPD-L1 MAB-2 VH6 is (SEQ ID NO: 71) (CDR H Residue Groups are underlined): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMS WVRQA PGKGLEWVA Y ISIGGGTTYY PDTVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR AG LPYYGDY WGQ GTLVTVSS is.

[0150] The amino acid sequence of hPD-L1 MAB-2 VL2 is (SEQ ID NO: 72) (CDR L Residue Groups are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDVN EAVA WYQQKP GKAPKLLIY W ASTRHT GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QQ HYNTPLT FGQ GTKVEIK is.

[0151] CDRs of optimized hPD-L1 MAB-2 variants H 2. CDR H 3, and CDR L 1 of A Note that the amino acid sequences are different from those present in the parent molecule. These different CDRs are summarized below, with the parts that differ from VH1 and VL1 double underlined.

[0152] [Table 3]

[0153] The present invention specifically relates to the VL and / or VH domains, and / or CDRs of the VL region, of atezolizumab, avelumab, durvalumab, hPD-L1 MAB-2 and variants thereof, or any of the other anti-PD-L1 antibodies provided herein. L brain one, two, or all three of the CDRs of the VH domain H One of , two, or all three, more typically the CDRs of the VL region of an anti-PD-L1 monoclonal antibody as described above. L One, two, or all three of the above, and / or V H domain CDR H CD137×P, which has one, two, or all three of the following: D-L1 binding molecules are also encompassed.

[0154] b) HER2-binding domain HER2 is a 185 kDa receptor originally identified as the product of a transforming gene from neuroblastoma cells in chemically treated rats. HER2 has been extensively studied because of its function in many human carcinomas, including breast and gastric cancers.

[0155] Any anti-HER2 antibody epitope binding site may be used in accordance with the present invention, and the principles of the present invention will be described with reference to the HER2 tumor antigen. Representative antibodies that bind to human HER2 include margetuximab, trastuzumab, and pertuzumab. Margetuximab (also known as MGAH22; CAS Registry Number 1350624-75-7, see, e.g., U.S. Patent No. 8,802,093) is an Fc-optimized monoclonal antibody that binds to HER2 and mediates enhanced ADCC activity. Trastuzumab (also known as rhuMAB4D5, commercially available as Herceptin®; CAS Registry Number 180288-69-1; see, U.S. Patent No. 5,821,337) is a humanized version of the antibody 4D5 with an IgG1 / κ constant region. Pertuzumab (also known as rhuMAB2C4, commercially available as Perjet®; CAS Registry Number 380610-2 7-5; see, for example, WO 2001 / 000245) is a humanized version of the antibody 2C4, with an IgG1 / κ constant region. Margetuximab (WHO Drug Information, 2014, Recommended INN: List 70, 28(1):93-94) and trastuzumab (Trastuzumab) are also available. The complete heavy and light chain amino acid sequences of stuzumab emtansine (see WHO Drug Information, 2011, RecommendedINN: List 65, 25(1):89-90), and the Fab domain of pertuzumab (Protein Data Bank Accession No. 117i) are known in the art. Additional anti-HER2 antibodies, including HER2 MAB-1 and humanized variants thereof, are also provided herein.

[0156] (1) hHER2 MAB-1 The antibody hHER2 MAB-1 is a humanized anti-HER2 monoclonal antibody that binds to an epitope on HER2 that is distinct from the epitope recognized by margetuximab, trastuzumab, and pertuzumab (see, e.g., WO 2018 / 156740).

[0157] The amino acid sequence of the VH domain of the humanized antibody (hHER2 MAB-1 VHx) is (SEQ ID NO: 78) (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W INTNIGEPTY TEEFKG RVTM TRDTSISTAY MELSRLRSDD TAVYYCAR DX 1 X 2 YGNRVSY WG QGTLVTVSS wherein X1 is D or E and X2 is G or I.

[0158] The amino acid sequence of the VL domain of the humanized antibody (hHER2 MAB-1 VLx) is (SEQ ID NO: 79) (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDIX 3 X 4 YLS WFQQKP GKAPKTLIY R ANRLX 5 X 6GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC LQ HDEFPWT FGQ GTKLEIK and X3 is N or S; X4 is S, T or N; X5 is V or Q; and X6 is D, E or S.

[0159] Three mutant hHER2 MAB-1 VH domains were isolated: hHER2MAB-1 VH1, hHER2 MAB-1 VH2, and hHER2 MAB-1 VH3. The amino acid sequences of these mutant hHER2 MAB-1 VH domains are presented below.

[0160] The amino acid sequence of hHER2 MAB-1 VH1 is (SEQ ID NO: 80) (CDR H residue are underlined; H The second and third residues of 3 are D and and G): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W INTNIGEPTY TEEFKG RVTM TRDTSISTAY MELSRLRSDD TAVYYCAR DD GYGNRVSY WG QGTLVTVSS is.

[0161] The amino acid sequence of hHER2 MAB-1 VH2 is (SEQ ID NO: 81) (CDR H residue are underlined; H The second and third residues of 3 are E and and G): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMN WVRQA PGQGLEWMG W INTNIGEPTY TEEFKG RVTM TRDTSISTAY MELSRLRSDD TAVYYCAR DE GYGNRVSY WG QGTLVTVSS is.

[0162] The amino acid sequence of hHER2 MAB-1 VH3 is (SEQ ID NO: 82) (CDR H residue are underlined; H The second and third residues of 3 are D and and I): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLEWMG W INTNIGEPTY TEEFKG RVTM TRDTSISTAY MELSRLRSDD TAVYYCAR DD IYGNRVSY WG QGTLVTVSS is.

[0163] Three mutant hHER2 MAB-1 VL domains were isolated: hHER2MAB-1 VL1, hHER2 MAB-1 VL2, and hHER2 MAB-1 VL3. The amino acid sequences of these mutant hHER2 MAB-1 VL domains are presented below.

[0164] The amino acid sequence of hHER2 MAB-1 VL1 is (SEQ ID NO: 83) (CDR L residue are underlined; L The seventh and eighth residues of 1 are N and N, respectively. and S, CDR L The sixth and seventh residues of SEQ ID NO:2 are V and D, respectively): DIQMTQSPSS LSASVGDRVT ITC KASQDIN SYLS WFQQKP GKAPKTLIY R ANRLVD GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC LQ HDEFPWT FGQ GTKLEIK is.

[0165] The amino acid sequence of hHER2 MAB-1 VL2 is (SEQ ID NO: 84) (CDR L residue are underlined; L The seventh and eighth residues of 1 are N and N, respectively. and T, and CDR L The sixth and seventh residues of 2 are V and E, respectively): DIQMTQSPSS LSASVGDRVT ITC KASQDIN TYLS WFQQKP GKAPKTLIY R ANRLVE GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC LQ HDEFPWT FGQ GTKLEIK is.

[0166] The amino acid sequence of hHER2 MAB-1 VL3 is (SEQ ID NO: 85) (CDR L residue are underlined; L The seventh and eighth residues of 1 are S and and N, and CDR L The sixth and seventh residues of SEQ ID NO:2 are Q and S, respectively): DIQMTQSPSS LSASVGDRVT ITC KASQDIS NYLS WFQQKP GKAPKTLIY R ANRLQS GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC LQ HDEFPWT FGQ GTKLEIK is.

[0167] Any of the humanized VH and VL hHER2 MAB-1 domains described above, including any encompassed by one or more of the generic sequences of the hHER2 MAB-1 VH and / or VL domains presented above, can be used to form antibodies, diabodies, or binding molecules capable of binding to HER2.

[0168] (2) Other HER2-binding domains In addition to the HER-binding domains identified above, the present invention provides the following anti-HER-2 binding domains: 1.44.1; 1.140; 1.43; 1.14.1; 1.100.1; 1.96; 1.18.1; 1.20; 1.39; 1.24; and 1.71.3 (U.S. Pat. No. 8,350,011; U.S. Pat. No. 8,858,942; and WO 2008 / 019290); F5 and C1 (U.S. Pat. No. 7,892,554; U.S. Pat. No. 8,173,424; U.S. Pat. No. 8,974,792; and WO 99 / 55367); and U.S. Pat. No. 2011 / 0097323, U.S. Pat. No. 2013 / 017114 No. 2014 / 0328836, U.S. Patent Publication No. 2016 / 0130360, and U.S. Patent Publication No. 2016 / 0257761, and the anti-HER2 binding molecules of WO 2011 / 147986 are contemplated.

[0169] The present invention specifically relates to: margetuximab, trastuzumab, pertuzumab, hHER2 The VL and / or VH domains, and / or CDRs of the VL region of MAB-1 or any of the other anti-HER2 antibodies provided herein. L One or two of At least three CDRs of the VH domain and / or H One, two or all three of and more typically, the CDRs of the VL region of such anti-HER2 monoclonal antibodies. L and / or the CDRs of the VH domain. H The present invention also includes and encompasses CD137xHER2 binding molecules having one, two or all three of the following:

[0170] c) EphA2 binding domain The receptor tyrosine kinase, ephrin type A receptor 2 (EphA2), is normally expressed at cell-cell contact sites in adult epithelial tissues. However, recent studies have shown that EphA2 is also overexpressed in various types of epithelial cancers, with the highest levels of EphA2 expression observed in metastatic lesions. High expression levels of EphA2 have been confirmed in a wide range of cancers and numerous tumor cell lines, including prostate cancer, breast cancer, non-small cell lung cancer, and melanoma. EphA2 does not appear to be a simple cancer marker, but appears to be persistently overexpressed and functionally altered in many human cancers. Any epitope-binding site of an anti-EphA2 antibody can be used in accordance with the present invention. Presented below are some representative anti-EphA2 antibodies that can be used to generate the molecules of the present invention.

[0171] (1) EphA2 MAB-1 Antibody EphA2 MAB-1 is a murine anti-EphA2 monoclonal antibody. The amino acid sequence of the VH domain of EphA2 MAB-1 is (SEQ ID NO: 86) (CDR residues are underlined): QVQLKESGPG LVAPSQSLSI TCTVSGFSLS RYSVH WVRQP PGKGLEWLG M IWGGGSTDYN SALKS RLSIS KDNSKSQVFL KMNSLQTDDT AMYYCAR KHG NYYTMDY WGQ GTSVTVSS is.

[0172] The amino acid sequence of the VL domain of EphA2 MAB-1 is (SEQ ID NO:87) (CDR residues are underlined): DIQMTQTTSS LSASLGDRIT ISC RASQDIS NYLN WYQQKP DGTVKLLIY Y TSRLHS GVPS RFSGSGSGTD YSLTISNLEQ EDIATYFC QQ GYTLYTFGGG TKLEIK is.

[0173] (2) EphA2 MAB-2 The antibody EphA2 MAB-2 is a murine anti-EphA2 monoclonal antibody. The amino acid sequence of the VH domain of EphA2 MAB-2 is (SEQ ID NO: 88) (CDR residues are underlined): QIQLVQSGPE LKKPGETVKI SCKASGFTFT NYGMN WVKQA PGKGLKWMG W INTYIGEPTY ADDFKG RFVF SLETSASTAY LQINNLKNED MATYFCAR EL GPYYFDY WGQ GTTLTVSS is.

[0174] The amino acid sequence of the VL domain of EphA2 MAB-2 is (SEQ ID NO:89) (CDR residues are underlined): DVVMTQTPLS LPVSLGDQAS ISC RSSQSLV HSSGNTYLH W YLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFC SQSTHVP T FGSGTKLEI K is.

[0175] (3) EphA2 MAB-3 The antibody EphA2 MAB-3 is a murine anti-EphA2 monoclonal antibody. The amino acid sequence of the VH domain of EphA2 MAB-3 is (SEQ ID NO: 90) (CDR residues are underlined): EVQLVESGGG SVKPGGSLKL SCAASGFTFT DHYMY WVRQT PEKRLEWVA T ISDGGSFTSY PDSVKGRFTI SRDIAKNNLY LQMSSLKSED TAMYYCTR DE SDRPFPY WGQ GTLVTVSS is.

[0176] The amino acid sequence of the VL domain of EphA2 MAB-3 is (SEQ ID NO: 91) (CDR residues are underlined): DIVLTQSHRS MSTSVGDRVN ITC KASQDVT TAVA WYQQKP GQSPKLLIF W ASTRHA GVPD RFTGSGSGTD FTLTISSVQA GDLALYYC QQ HYSTPYT FGG GTKLEIK is.

[0177] (4) Other EphA2-binding domains In addition to the EphA2 binding domains identified above, the present invention contemplates the use of any of the epitope binding sites of any of the following anti-EphA2 antibodies: SPL1, LUCA19, SG5, or LUCA40 (see WO 2006 / 084226); B13 (see U.S. Patent No. 7,101,976); D7 (see U.S. Patent No. 7,192,698); B-233, and EA2 (see WO 2003 / 094859).

[0178] Specifically, the present invention relates to the VL and / or VH domains and / or CDRs of the VL region of anti-EphA2 monoclonal antibodies EphA2 MAB-1, EphA2 MAB-2, or EphA2 MAB-3. L One, two or all three of the VH domains and / or Main CDR H CD137×EphA2, including one, two, or all three of the following: It includes and encompasses binding molecules.

[0179] d) 5T4 binding domain The oncofetal protein 5T4 is a tumor-associated protein expressed on the cell membrane of many carcinomas, including kidney, colon, prostate, and lung cancers, and in acute lymphoblastic leukemia. Any epitope-binding site of an anti-5T4 antibody can be used in accordance with the present invention. Provided below are two representative anti-5T4 antibodies: humanized "5T4 MAB-1" and murine "5T4 MAB-2." Additional anti-5T4 antibodies have been described in the art (see, e.g., U.S. Patent Nos. 8,084,249; 8,409,577; 8,759,495; 8,409,577; WO 2013 / 041687; WO 2014 / 137931; and WO 2016 / 022939).

[0180] (1) 5T4 MAB-1 The amino acid sequence of the VH domain of 5T4 MAB-1 is (SEQ ID NO: 92) (CDR residues: are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SFWMH WVRQA PGQGLEWMG R IDPNRGGTEY NEKAKS RVTM TADKSTSTAY MELSSLRSED TAVYYCAG GN PYYPMDY WGQ GTTVTVSS is.

[0181] The amino acid sequence of the VL domain of 5T4 MAB-1 is (SEQ ID NO:93) (CDR residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASQGIS NYLA WFQQKP GKAPKSLIY R ANRLQS GVPS RFSGSGSGTD FTLTISSLQP EDVATYYC LQ YDDFPWT FGQ GTKLEIK is.

[0182] (2) 5T4 MAB-2 The amino acid sequence of the VH domain of 5T4 MAB-2 is (SEQ ID NO:94) (CDR residues are underlined): QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYWIT WVKQR PGQGLEWIG D IYPGSGRANY NEKFKS KATL TVDTSSSTAY MQLSSLTSED SAVYNCAR YG PLFTTVVDPN SYAMDY WGQG TSVTVSS is.

[0183] The amino acid sequence of the VL domain of 5T4 MAB-2 is (SEQ ID NO:95) (CDR residues are underlined): DVLMTQTPLS LPVSLGDQAS ISC RSSQSIV YSNGNTYLE W YLQKPGQSPK LLIY KVSNRF S GVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYC FQGSHVP FT FGSGTKLE IK is.

[0184] The present invention specifically relates to the anti-5T4 monoclonal antibody 5T4 MAB-1 or 5T4 The VL and / or VH domains, and / or CDRs of the VL region, of MAB-2 or of any of the anti-5T4 antibodies provided in WO 2007 / 106744, WO 2013 / 041687, or WO 2015 / 184203. L and / or the CDRs of the VH domain. H One of the It includes and encompasses CD137x5T4 binding molecules, including two or all three.

[0185] e) B7-H3 binding domain B7-H3 is a tumor antigen overexpressed in a wide variety of solid tumors and a member of the B7 family of molecules involved in immune regulation. In particular, multiple independent studies have shown that human malignant tumor cells (e.g., neuroblastoma, gastric cancer, ovarian cancer, and non-small cell lung cancer tumor cells) exhibit significantly increased expression of B7-H3 protein, and that this increased expression is associated with increased disease severity, suggesting that B7-H3 is utilized by tumors as an immune evasion pathway.

[0186] The epitope binding site of any anti-B7-H3 antibody can be used in accordance with the present invention. One representative humanized antibody that binds to human B7-H3 is "enoblitutuzumab." Enoblituzumab (also known as MGA271; CAS Registry Number 1353485-38-7; see, e.g., U.S. Patent No. 8,802,091) is an Fc-optimized monoclonal antibody that binds to B7-H3 and mediates enhanced ADCC activity. The complete heavy and light chain amino acid sequences of enoblituzumab (WHO Drug Information, 2017, Recommended INN: List 77, 31(1):149) are known in the art. Further representative anti-B7-H3 antibodies are presented.

[0187] (1) hBRCA69D Representative VH and VL domains of the humanized anti-B7-H3 antibody "hBRCA69D" are provided below. Two humanized VH domains, i.e., hBRCA69D VH1 and hBRCA69D VH2; and two humanized VL domains, i.e., hBRCA69D VL1 and hBRCA69D VL2, are provided below, which can be used in any VH / VL combination to obtain a functional humanized binding domain.

[0188] The amino acid sequence of the VH domain of hBRCA69D VH1 is (SEQ ID NO: 96) (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQ WVRQA PGQGLEWMG T IYPGDGDTRY TQKFKG RVTI TADKSTSTAY MELSSLRSED TAVYYCAR RG IPRLWYFDV W GQGTTVTVSS is.

[0189] The amino acid sequence of the VH domain of hBRCA69D VH2 is (SEQ ID NO: 97) (CDR H Residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT SYWMQ WVRQA PGQGLEWMG T IYPGGGDTRY TQKFQG RVTI TADKSTSTAY MELSSLRSED TAVYYCAR RG IPRLWYFDV W GQGTTVTVSS is.

[0190] The amino acid sequence of the VL domain of hBRCA69D VL1 is (SEQ ID NO: 98) (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASQDIS NYLN WYQQKP GKAPKLLIY Y TSRLHS GVPS RFSGSGSGTD FTLTISSLQP EDIATYYC QQ GNTLPPT FGG GTKLEIK is.

[0191] The amino acid sequence of the VL domain of hBRCA69D VL2 is (SEQ ID NO: 99) (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASQSIS SYLN WYQQKP GKAPKLLIY Y TSRLQS GVPS RFSGSGSGTD FTLTISSLQP EDIATYYC QQ GNTLPPT FGG GTKLEIK is.

[0192] (2) hPRCA157 Another representative humanized anti-B7-H3 antibody is "hPRCA157." The amino acid sequence of the VH domain of hPRCA157 VH1 is (SEQ ID NO: 100) (CDR H Residues are below (shown with a line): EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYGMS WVRQA PGKGLEWVA T INSGGSNTYY PDSLKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR HD GGAMDY WGQG TTVTVSS is.

[0193] The amino acid sequence of the VL domain of hPRCA157 VL1 is (SEQ ID NO: 101) (CDR L Residues are underlined): DIQMTQSPSS LSASVGDRVT ITC RASESIY SYLA WYQQKP GKAPKLLVY N TKTLPEGVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYGTPPWT FG QGTRLEIK is.

[0194] (3) Other B7-H3 binding domains In addition to the B7-H3 binding domains identified above, the present invention also provides the following anti-B7-H3 antibodies: LUCA1; BLA8; PA20; or SKN2 (see U.S. Pat. No. 7,527,969; U.S. Pat. No. 8,779,098 and WO 2004 / 001381); M30; cM30; M30-H1-L1; M30-H1-L2; M30-H1-L3; M30-H1-L4; M30-H1-L5; M30-H1-L6; M30-H1-L7; M30-H4-L1; M30-H4-L2; M30-H4-L3; and M30-H4-L4 (U.S. Pat. No. 6,527,969; U ... and WO 2004 / 001381). and 8H9 (see U.S. Patent Application Publication Nos. 7,666,424; 7,737,258; 7,740,845; 8,148,154; 8,414,892; 8,501,471; 9,062,110; U.S. Patent Application Publication No. 2010 / 0143245; and WO 2008 / 116219).

[0195] The present invention specifically relates to the VL and / or VH domains, and / or CDRs of the VL region, of humanized BRCA69D, PRCA157, humanized PRCA157, or enoblituzumab, or any of the other anti-B7-H3 antibodies provided herein. L One of one, two, or all three, and / or the CDRs of the VH domain H One or two of , or all three, more typically the CDRs of the VL region of an anti-B7-H3 monoclonal antibody as described above. L One, two, or all three of the VH domains In CDR H CD137×B7‐H, which has one, two, or all three of the following: 3 binding molecules, and also encompasses these.

[0196] f) GpA33 binding domain The 43 kD transmembrane glycoprotein A33 (gpA33) is expressed in >95% of all colorectal cancers. Any epitope-binding site of an anti-gpA33 antibody can be used in accordance with the present invention. A representative humanized anti-gpA33 antibody ("gpA33 MAB-1") is provided below.

[0197] The amino acid sequence of the VH domain of gpA33 MAB-1 is (SEQ ID NO: 102) (CDR residues are underlined): QVQLVQSGAE VKKPGASVKV SCKASGYTFT GSWMN WVRQA PGQGLEWIG R IYPGDGETNY NGKFKD RVTI TADKSTSTAY MELSSLRSED TAVYYCAR IY GNNVYFDV WG QGTTVTVSS is.

[0198] The amino acid sequence of the VL domain of gpA33 MAB-1 is (SEQ ID NO: 103) (CDR residues are underlined): DIQLTQSPSF LSASVGDRVT ITC SARSSIS FMY WYQQKPG KAPKLLIY DT SNLAS GVPSR FSGSGSGTEF TLTISSLEAE DAATYYC QQW SSYPLT FGQG TKLEIK is.

[0199] The present invention specifically relates to the anti-gpA33 monoclonal antibody gpA33 MAB-1, or the anti-gpA33 monoclonal antibody provided in WO 2015 / 026894. VL and / or VH domains, and / or CDRs of the VL region of any of the L and / or the CDRs of the VH domain. H The present invention also includes and encompasses CD137xgpA33 binding molecules comprising one, two or all three of:

[0200] g) CEACAM5 and CEACAM6 binding domains Carcinoembryonic antigen-related cell adhesion molecules 5 (CEACAM5) and 6 (CEACAM6) have been shown to be associated with various types of cancer, including thyroid cancer, colorectal cancer, pancreatic cancer, hepatocellular carcinoma, gastric cancer, lung cancer, head and neck cancer, bladder cancer, prostate cancer, uterine cancer, endometrial cancer, breast cancer, hematopoietic cancer, leukemia, and ovarian cancer, and in particular, colorectal cancer, gastrointestinal cancer, pancreatic cancer, non-small cell lung cancer (NSCL), breast cancer, thyroid cancer, stomach cancer, ovarian cancer, and uterine carcinoma. The epitope-binding site of any anti-CEACAM5 / CEACAM6 antibody can be used in accordance with the present invention. Representative anti-CEACAM5 / CEACAM6 antibodies are provided below.

[0201] (1)16C3 The amino acid sequence of the VH domain of humanized anti-CEACAM5 / CEACAM6 antibody 16C3 (EP 2585476) is (SEQ ID NO: 104) (CDR residues are underlined): QVQLQQSGPE VVRPGVSVKI SCKGSGYTFT DYAMH WVKQS HAKSLEWIG L ISTYSGDTKY NQNFKG KATM TVDKSASTAY MELSSLRSED TAVYYCAR GD YSGSRYWFAY WGQGTLVTVS S is.

[0202] The amino acid sequence of the VL domain of humanized anti-CEACAM5 / CEACAM6 antibody 16C3 (EP 2585476) is (SEQ ID NO: 105) (CDR residues are underlined): DIQMTQSPSS LSASVGDRVT ITC GASENIY GALN WYQRKP GKSPKLLIW G ASNLAD GMPS RFSGSGSGRQ YTLTISSLQP EDVATYY CQN VLSSPYT FGG GTKLEIK is.

[0203] (2) hMN15 The amino acid sequence of the VH domain of the humanized anti-CEACAM5 / CEACAM6 antibody hMN15 (U.S. Pat. No. 8,287,865) is (SEQ ID NO: 106) (CDR residues are underlined): QVQLVESGGG VVQPGRSLRL SCSSSGFALT DYYMS WVRQA PGKGLEWLG F IANKANGHTT DYSPSVKG RF TISRDNSKNT LFLQMDSLRP EDTGVYFCAR DMGIRWNFDV WGQGTPVTVS S is.

[0204] The amino acid sequence of the VL domain of the humanized anti-CEACAM5 / CEACAM6 antibody hMN15 (U.S. Pat. No. 8,287,865) is (SEQ ID NO: 107) (CDR residues are underlined): DIQLTQSPSS LSASVGDRVT MTC SASSRVS YIH WYQQKPG KAPKRWIY GT STLAS GVPAR FSGSGSGTDF TFTISSLQPE DIATYYC QQW SYNPPT FGQG TKVEIKR is.

[0205] Specifically, the present invention relates to the anti-CEACAM5 / CEACAM6 monoclonal antibody 16C3 or the VL and / or VH domains and / or CDRs of the VL region of hMN15 L and / or the CDRs of the VH domain. H One of the It includes and encompasses CD137xCEACAM5 / CEACAM6 binding molecules, including two or all three.

[0206] h) CD19-binding domain CD19 (B-lymphocyte surface antigen B4, Genbank accession number: M28170) is a component of the B-cell receptor (BCR) complex and a positive regulator of B-cell signaling, modulating the threshold for B-cell activation and humoral immunity. CD19 is one of the most ubiquitously expressed antigens in the B-cell lineage and is expressed in >95% of B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL). Notably, CD19 expression persists in B-cell lymphomas that have become resistant to anti-CD20 therapy. CD19 has also been suggested as a target for treating autoimmune diseases.

[0207] The epitope-binding site of any anti-CD19 antibody can be used in accordance with the present invention. An exemplary humanized antibody that binds to human CD19 and may be employed in the present invention is the anti-CD19 antibody disclosed in WO 2016 / 048938 (referred to herein as "CD19 MAB-1").

[0208] The amino acid sequence of the VH domain of CD19 MAB-1 is (SEQ ID NO: 108) (CDR H Residues are underlined): QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMGVG WIR QPPGKALEWL A HIWWDDDKR YNPALKS RLT ISKDTSKNQV FLTMTNMDPV DTATYYCAR M ELWSYYFDY W GQGTTVTVSS is.

[0209] The amino acid sequence of the VL domain of CD19 MAB-1 is (SEQ ID NO: 109) (CDR L Residues are underlined): ENVLTQSPAT LSVTPGEKAT ITC RASQSVS YMH WYQQKPG QAPRLLIY DA SNRAS GVPSR FSGSGSGTDH TLTISSLEAE DAATYYC FQG SVYPF TFGQG TKLEIK is.

[0210] The present invention specifically relates to the anti-CD19 monoclonal antibody CD19 MAB-1, or blinatumomab (BLINCYTO®; WHO Drug Information, 2009, Recommended INN: List 62, 23(3):240-241), as disclosed in U.S. Pat. No. 7,112,324. and duvortuxizumab (also known as MGD011; amino acid sequence found in WHO Drug Information, 2016, Proposed INN: List 116, 30(4):627-629). The VL and / or VH domains and / or CDRs of the VL region of any of the anti-CD19 antibodies present in the L and / or one, two or all three of VH domain CDRs H CD137×CD containing one, two or all three of the following: 19 binding molecules, including and encompassing these.

[0211] i) CD123-binding domain CD123 (interleukin-3 receptor) is a 40 kDa molecule containing a unique α chain, IL-3Ra. Interleukin-3 (IL-3) promotes the early differentiation of pluripotent stem cells into erythroid, myeloid, and lymphoid progenitor cells. CD123 has been reported to be overexpressed on malignant cells in a wide range of hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Overexpression of CD123 is associated with the poor prognosis of AML. Relevant to prognosis.

[0212] The epitope-binding site of any anti-CD123 antibody can be used in accordance with the present invention. An exemplary humanized antibody that binds to human CD123 and can be employed in the present invention is "CD123 MAB-1" (see, e.g., WO 2015 / 026892).

[0213] The amino acid sequence of the VH domain of CD123 MAB-1 is (SEQ ID NO: 110) (CDR H Residues are underlined): EVQLVQSGAE LKKPGASVKV SCKASGYTFT DYYMK WVRQA PGQGLEWIG D IIPSNGATFY NQKFKG RVTI TVDKSTSTAY MELSSLRSED TAVYYCAR SH LLRASWFAY W GQGTLVTVSS is.

[0214] The amino acid sequence of the VL domain of CD123 MAB-1 is (SEQ ID NO: 111) (CDR L Residues are underlined): DFVMTQSPDS LAVSLGERVT MSC KSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIY WASTR ES GVPDRFSG SGSGTDFTLT ISSLQAEDVA VYYC QNDYSY PYT FGQGTKL EIK is.

[0215] The present invention specifically relates to the VL and / or VH domains, and / or CDRs of the VL region, of the anti-CD123 monoclonal antibody CD123 MAB-1, or any of the anti-CD123 antibodies disclosed in U.S. Patent No. 2017 / 081424 and WO 2016 / 036937, or present in JNJ-63709178 (Johnson & Johnson, see also WO 2016 / 036937) and XmAb14045 (Xencor, see also U.S. Patent No. 2017 / 081424). L brain one, two, or all three of the CDRs of the VH domain H One or two of or all three, including, but not limited to, CD137xCD123 binding molecules.

[0216] j) IL13Rα2 Interleukin-13 receptor alpha 2 (IL13Rα2) is overexpressed in a variety of cancers, including glioblastoma, colorectal cancer, cervical cancer, pancreatic cancer, multiple melanoma, osteosarcoma, leukemia, lymphoma, prostate cancer, and lung cancer. Antibodies that immunospecifically bind to IL13Rα2 are commercially available and have been previously described in the art (see, e.g., WO 2008 / 146911). Exemplary humanized antibodies that bind to human IL13Rα2 include "hu08" (see, e.g., WO 2014 / 072888).

[0217] The amino acid sequence of the VH domain of hu08 (SEQ ID NO: 112) is shown below (CDR residues are underlined): EVQLVESGGG LVQPGGSLRL SCAAS GFTFS RNGMS WVRQA PGKGLEWVA T VSSGGSYIYY ADSVKG RFTI SRDNAKNSLY LQMNSLRAED TAVYYCAR QG TTALATRFFD V WGQGTLVTV SS

[0218] The amino acid sequence of the VL domain of hu08 (SEQ ID NO: 113) is shown below (CDR residues are underlined): DIQMTQSPSS LSASVGDRVT ITC KASQDVG TAVA WYQQKP GKAPKLLIY S ASYRST GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC QH HYSAPWT FGG GTKVEIK

[0219] Specifically, the present invention relates to the VL and / or VH domains and / or CDRs of the VL region of the anti-IL13Rα2 monoclonal antibody hu08. L One, two or three of All and / or the CDRs of the VH domain H C, including one, two or all three of D137×IL13Rα2 binding molecules, and also encompasses these.

[0220] k) ROR1 Receptor Tyrosine Kinase-Like Orphan Receptor 1 (Receptor Tyrosine Kinase Kinase-Like Orphan Receptor 1 (ROR1) is a type I membrane protein belonging to the ROR subfamily of cell surface receptors. ROR1 is an oncoembryonic antigen expressed by many tissues during embryogenesis, absent from most mature tissues. It is expressed in numerous hematological and solid malignancies, including ovarian, colon, lung, lymphoma, skin, pancreas, testis, bladder, uterus, prostate, adrenal gland, breast, and B-cell malignancies, as well as in some cancer stem cells. ROR1 expression is associated with less differentiated, more aggressive tumors and correlates with poor clinical outcomes. Any anti-ROR1 antibody epitope-binding site can be used in accordance with the present invention. Provided below are representative humanized / optimized anti-ROR1 antibodies that can be used to generate the molecules of the present invention; variations of these antibodies are described in WO 2017 / 142928.

[0221] (1) Anti-ROR1 The amino acid sequence of a representative VH of an anti-ROR1 antibody is (SEQ ID NO: 114) (CDR residues are underlined): QEQLVESGGG LVQPGGSLRL SCAASGFTFS DYYMS WXRQA PGKGLEWVA T IYPSSGKTYY ADSAKG RLTI SSDNAKDSLY LQMNSLRAED TAVYYCTR DS YADDAALFDI WGQGTTVTVS S where X is I or V.

[0222] The amino acid sequence of a representative VL of an anti-ROR1 antibody is (SEQ ID NO: 115) (CDR residues are underlined): QLVLTQSPSA SASLGSSVKL TC TLSSGHKT DTID WYQQQP GKAPRYLMK L EGSGSY NKGS GVPDRFSGSS SGADWYLTIS SLQSEDEADY YC GTDYPGNY LFGGGTQLTV LG is.

[0223] (2) Other ROR1-binding domains In addition to the ROR1 binding domains identified above, the present invention contemplates the use of any of the epitope binding sites of any of the following anti-ROR1 antibodies: 4A5 (see U.S. Patent No. 8,212,009); R11, R12, and Y31 (see U.S. Patent No. 9,758,586); and A1-A14 (see, e.g., U.S. Patent No. 9,228,023).

[0224] The present invention specifically includes and encompasses CD137xROR1 binding molecules that comprise the VL and / or VH domains, and / or one, two, or all three of the CDRLs of the VL region and / or one, two, or all three of the CDRHs of the VH domain, of any of the anti-ROR1 monoclonal antibodies provided herein.

[0225] D. CD137xTA Binding Molecules of the Invention The present invention is particularly directed to Fc-bearing tetravalent and trivalent CD137xTA binding molecules capable of simultaneously binding CD137 and TA, as well as other Fc-bearing CD137xTA binding molecules capable of simultaneously binding CD137 and TA. The present invention is further directed to the use of such molecules in the treatment of cancer and other diseases and conditions.

[0226] 1. Tetravalent CD137xTA Fc-bearing diabody The present invention specifically encompasses a variety of Fc-bearing diabodies capable of simultaneously binding to CD137 and TA. Representative CD137xTA Fc-bearing diabodies are described below.

[0227] Such tetravalent Fc-bearing diabodies will comprise two polypeptide chains, the first of which can contain, from N- to C-terminal direction, an N-terminus, an antibody light chain variable domain (VL) (VL1) capable of binding to an epitope on a "first" antigen (CD137 or TA), an antibody heavy chain variable domain (VH) (VH2) capable of binding to an epitope on a "second" antigen (TA if VL1 is selected to bind to CD137, or CD137 if VL1 is selected to bind to an epitope on TA), a cysteine-containing domain, one or more additional domains provided in more detail below, and a C-terminus. The second of the polypeptide chains can comprise, from N- to C-terminal, an N-terminus, an antibody light chain variable domain (VL) (VL2) capable of binding to an epitope on the "second" antigen (TA if the first antigen was CD137, or CD137 if the first antigen was TA), an antibody heavy chain variable domain (VH) (VH2) capable of binding to an epitope on the "second" antigen (TA if VL2 is selected to bind to CD137, or CD137 if VL2 is selected to bind to an epitope on TA), a cysteine-containing domain, one or more additional domains provided in more detail below, and a C-terminus. An intervening linker peptide (Linker 1) separates the light chain variable domain (VL1 or VL2) from the heavy chain variable domain (VH1 or VH2).

[0228] In certain embodiments, Fc-bearing diabodies of the invention are covalent tetravalent diabodies having four epitope-binding sites comprising four polypeptide chains, with the general structure shown in Figure 1A. The first and third polypeptide chains of such diabodies contain, from N- to C-terminal, (i) a VL1-containing Domain; (ii) a VH2-containing Domain; (iii) a Heterodimer-Promoting Domain; and (iv) a Domain containing a CH2-CH3 sequence. The second and fourth polypeptide chains contain: (i) a VL2-containing Domain; (ii) a VH1-containing Domain; and (iii) a Heterodimer-Promoting Domain, which promotes dimerization and covalent association of the first / third polypeptide chain with the second / fourth polypeptide chain. The VH Domain is linked to the Heterodimer-Promoting Domain by an intervening linker peptide (Linker 2), which may contain a cysteine ​​residue. Optionally, or in addition, the Heterodimer-Promoting Domain may contain a cysteine ​​residue. In a representative CD137xTA bispecific Fc-bearing diabody embodiment, the C-terminus of the Heterodimer-Promoting Domain of the first polypeptide chain is linked to the CH2-CH3 Domain by a linker peptide (Linker 3). The VL and / or VH Domains of the third and fourth polypeptide chains and the VL and / or VH Domains of the first and second polypeptide chains can be the same or different, allowing for monospecific, bispecific, or tetravalent binding. In Table 3 below, the notation "VL3" and "VH3" refer to the light chain variable domain and heavy chain variable domain, respectively, that bind the "third" epitope of such a diabody. Similarly, the notation "VL4" and "VH4" refer to the variable light chain domain and variable heavy chain domain, respectively, that bind the "fourth" epitope of such a diabody. The general structures of the polypeptide chains of representative four-chain bispecific Fc Region-containing diabodies of the invention are provided in Table 3.

[0229] [Table 4]

[0230] In certain embodiments, the CD137xTA-binding molecules of the invention are bispecific, tetravalent (i.e., having four epitope-binding sites), Fc-bearing diabodies composed of a total of four polypeptide chains (Figures 1A-1C). The CD137xTA-binding molecules of the invention are bispecific, tetravalent, Fc-containing diabodies that include two epitope-binding sites immunospecific for CD137 (which can bind to the same epitope on CD137 or different epitopes on CD137) and two epitope-binding sites immunospecific for a tumor antigen (which can bind to the same epitope on one TA, or different epitopes on one TA, or different epitopes on different TAs).

[0231] In further embodiments, Fc domain-containing diabodies of the invention may comprise three polypeptide chains. The first polypeptide of such diabodies often contains three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The second polypeptide of such diabodies often contains: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a domain that promotes heterodimerization and covalent bonding with the first polypeptide chain of the diabody. The third polypeptide of such diabodies often contains a CH2-CH3 sequence. Thus, the first and second polypeptide chains of such diabodies often associate together to form a VL1 / VH1 epitope-binding domain capable of binding the first or second epitope, and a VL2 / VH2 epitope-binding domain capable of binding the other of these epitopes. The first and second polypeptides are often linked to each other by disulfide bonds involving cysteine ​​residues in their respective third domains. In particular, the first and third polypeptide chains often complex with each other to form a disulfide-stabilized Fc domain. Figure ID shows a representative structure of such a diabody.

[0232] In each of the above-described embodiments, the light chain variable domain (VL1) of the first polypeptide chain is coordinately selected to interact with the heavy chain variable domain (VH1) of the second polypeptide chain to form a functional epitope-binding site capable of immunospecifically binding to an epitope of the first antigen (i.e., TA or CD137). Similarly, the light chain variable domain (VL2) of the second polypeptide chain is coordinately selected to interact with the heavy chain variable domain (VH2) of the first polypeptide chain to form a functional epitope-binding site capable of immunospecifically binding to an epitope of the second antigen (i.e., TA or CD137). Thus, the selection of the light chain variable domain and the heavy chain variable domain are coordinated so that the two polypeptide chains collectively contain epitope-binding sites capable of binding to CD137 and TA. can be.

[0233] Additional Fc-bearing diabodies of the present invention comprise five polypeptide chains, as shown in FIG. 2. The first polypeptide chain of such a diabody contains: (i) a VH1-containing domain; (ii) a CH1-containing domain; and (iii) a domain containing a CH2-CH3 sequence. The first polypeptide chain may be a heavy chain of an antibody containing a VH1 and a heavy chain constant region. The second and fifth polypeptide chains of such a diabody contain: (i) a VL1-containing domain; and (ii) a CL-containing domain. The second and / or fifth polypeptide chains of such a diabody may be a light chain of an antibody containing a VL1 that is complementary to the VH1 of the first / third polypeptide chain. The first, second, and / or fifth polypeptide chains may be isolated from naturally occurring antibodies or may be recombinantly constructed. In one embodiment, the second and fifth polypeptide chains have identical amino acid sequences. The third polypeptide chain of such diabodies contains: (i) a VH1-containing Domain, (ii) a CH1-containing Domain, (iii) a Domain containing a CH2-CH3 sequence, (iv) a VL2-containing Domain, (v) a VH3-containing Domain, and (vi) a Heterodimer-Promoting Domain, where the Heterodimer-Promoting Domain promotes dimerization between the third chain and the fourth chain. The fourth polypeptide of such diabodies contains: (i) a VL3-containing Domain, (ii) a VH2-containing Domain, and (iii) a Domain that promotes heterodimerization and covalent bonding with the third polypeptide chain of the diabody. The C-terminus of the VH3-containing Domain or VH2-containing Domain of the third and fourth polypeptide chains is linked to the Heterodimer-Promoting Domain by an intervening linker peptide (Linker 2), and the C-terminus of the CH2-CH3 Domain of the third polypeptide chain is linked to the VL2-containing Domain by an intervening linker peptide (Linker 4).

[0234] Thus, the first and second polypeptide chains and the third and fifth polypeptide chains of such a diabody are linked together to form two VL1 / VH1 binding sites capable of binding to a first epitope. The third and fourth polypeptide chains of such a diabody are linked together to form a single diabody binding domain, including a VL2 / VH2 binding site capable of binding to a second epitope and a VL3 / VH3 binding site capable of binding to a third epitope. The first and third polypeptides are linked together by disulfide bonds involving cysteine ​​residues in their respective constant regions. In particular, the first and third polypeptide chains complex with each other to form an Fc region. Such bispecific diabodies have enhanced potency. Figure 2 shows the structure of such a diabody. It will be understood that the VL1 / VH1, VL2 / VH2, and VL3 / VH3 domains can be identical or different, thereby enabling monospecific, bispecific, or trispecific binding. However, as presented herein, these domains are selected to bind CD137 and TA.

[0235] The VL and VH domains of the polypeptide chains are selected to form a VL / VH binding site specific for a desired epitope. The VL / VH binding sites formed by the linkage of the polypeptide chains can be identical or different, thereby enabling tetravalent binding that is monospecific, bispecific, trispecific, or tetraspecific. In particular, the VL and VH domains can be selected such that a bispecific diabody contains two binding sites for a first epitope and two binding sites for a second epitope, or three binding sites for a first epitope and one binding site for a second epitope, or (as shown in Figure 2) two binding sites for a first epitope, one binding site for a second epitope, and one binding site for a third epitope. The general structures of the polypeptide chains of representative five-chain Fc Region-containing diabodies of the invention are shown in Table 4.

[0236] [Table 5]

[0237] In a specific embodiment, a CD137xTA-binding molecule of the invention is a bispecific, tetravalent (i.e., having four epitope-binding sites), Fc-bearing diabody composed of a total of five polypeptide chains, having two epitope-binding sites immunospecific for CD137 (which can bind the same epitope on CD137 or different epitopes on CD137) and two epitope-binding sites immunospecific for a TA (which can bind the same epitope on a TA, or different epitopes on a TA, or different epitopes on different TAs). In another embodiment, a CD137xTA-binding molecule of the invention is a bispecific, tetravalent, Fc-bearing diabody comprising three epitope-binding sites immunospecific for CD137 (which can bind the same epitope on CD137 or two or three different epitopes on CD137) and one epitope-binding site specific for a TA.

[0238] 2. Trivalent CD137×TA binding molecule In one embodiment, the CD137xTA binding molecule of the invention is trivalent and comprises a first epitope-binding site (e.g., VL1 and VH1), a second epitope-binding site (e.g., VL2 and VH2), and a third epitope-binding site (e.g., VL3 and VH3), and is thus capable of binding to an epitope of TA, an epitope of CD137, and a third epitope, wherein the third epitope is: (a) the same or different epitopes of the above TA; (b) the same or a different epitope of CD137; or (c) Epitopes of different TAs It may be.

[0239] In certain embodiments, such a "trivalent CD137xTA binding molecule" of the present invention comprises two epitope-binding sites for epitopes of CD137 (which epitopes may be the same or different) and one epitope-binding site for an epitope of TA.

[0240] Generally, such trivalent CD137xTA binding molecules of the invention are composed of three, four, five, or six or more polypeptide chains that form a covalent molecular complex comprising a "diabody-type binding domain" and a "non-diabody-type binding domain" through one or more disulfide bonds between pairs of such polypeptides.

[0241] A "diabody-type binding domain" is an epitope-binding domain of a diabody, particularly a DART® diabody. The terms "diabody" and "DART® diabody" are described above. A "non-diabody-type" binding domain is intended to refer to a binding domain that does not have the structure of a diabody-type binding domain. Typically, a non-diabody-type binding domain is a Fab-type binding domain or an ScFv-type binding domain. As used herein, the term "Fab-type binding domain" refers to an epitope-binding domain formed by the interaction of the VL domain of an immunoglobulin light chain with the VH domain of a complementary immunoglobulin heavy chain. A Fab-type binding domain differs from a diabody-type binding domain in that the two polypeptide chains that form a Fab-type binding domain contain only a single epitope-binding domain, whereas the two polypeptide chains that form a diabody-type binding domain contain at least two epitope-binding domains. ScFv-type binding domains differ from diabody-type binding domains in that the VL and VH domains of the same polypeptide chain interact to form an epitope-binding domain, and therefore, as used herein, Fab-type binding domains and ScFv-type binding domains are distinct from diabody-type binding domains.

[0242] Thus, the trivalent CD137xTA binding molecules of the invention: (I) a "first" epitope-binding domain capable of immunospecifically binding to a "first" epitope; (II) a "second" epitope-binding domain capable of immunospecifically binding to the "second" epitope; (III) a "third" epitope binding domain capable of immunospecifically binding to the "third" epitope; and (IV) Fc domain, formed by the association of two CH2-CH3 domains with each other. Including, (A) The "first" epitope-binding domain and the "second" epitope-binding domain are both "diabody-type binding domains"; (B) the "third" epitope binding domain is a non-diabody-type binding domain; (C) One of the "first," "second," or "third" epitope-binding domains binds to an epitope of TA, and another of the "first," "second," or "third" epitope-binding domains binds to an epitope of CD137.

[0243] The epitopes bound by the remaining epitope binding domains can be any desired epitope, for example an epitope of CD137, which may be the same or different from the CD137 epitope bound by the other epitope binding domains of the molecule.

[0244] Figures 3A-3C provide schematic diagrams of the domains of a representative trivalent CD137xTA-binding molecule. Figure 3A shows a schematic diagram of the domains of a representative trivalent CD137xTA-binding molecule, which is composed of a covalent complex of four polypeptide chains and has one non-diabody-type binding site (VL3 / VH3; thus, monovalent for such epitopes) and two diabody-type binding sites (VL1 / VH1 and VL2 / VH2; thus, monovalent for each such epitope). Figures 3B-3C show a schematic diagram of the domains of a representative trivalent CD137xTA-binding molecule, which is composed of a covalent complex of three polypeptide chains and has one non-diabody-type binding site (VL3 / VH3; thus, monovalent for such epitopes) and two diabody-type binding sites (VL1 / VH1 and VL2 / VH2; thus, monovalent for each such epitope). The non-diabody-type binding sites are Fab-type binding domains in Figures 3A-3B and scFv-type binding domains in Figure 3C. As provided below, the VL / VH binding sites formed by association of the polypeptide chains can be the same or different, thus allowing for monospecific, bispecific, or trispecific trivalent binding.

[0245] II. Representative CD137×TA binding molecules The present invention provides CD137xTA binding molecules, which are bispecific tetravalent Fc diabodies capable of simultaneously and specifically binding to CD137 and a TA. As described above, the CD137xTA binding molecules of the present invention may comprise three, four, or five polypeptide chains. Representative polypeptide chains of CD137xTA binding molecules capable of binding to CD137 and a TA, PD-L1, or HER2 are provided below (designated "DART-A," "DART-A1," "DART-A2," "DART-A3," "DART-A4," "DART-A5," "DART-A6," "DART-A7," "DART-A8," "DART-A9," "DART-A10," "DART-B1," and "DART-B2"). The present invention also provides CD137xTA binding molecules, which are bispecific trivalent binding molecules capable of simultaneously and specifically binding to CD137 and a TA. As described above, the trivalent CD137xTA binding molecules of the present invention may comprise four polypeptide chains. Representative polypeptide chains of trivalent CD137xTA binding molecules capable of binding to CD137 and TA, PD-L1, or HER2 are presented below (designated "TRIDENT-A," "TRIDENT-A4," "TRIDENT-A5," "TRIDENT-A6," "TRIDENT-B1," and "TRIDENT-B1").

[0246] A. Tetravalent CD137×TA binding molecule 1. DART-A DART-A is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 1B). DART-A contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (1.1).

[0247] The first and third polypeptide chains of DART-A are arranged in an N-terminal to C-terminal direction, with an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1, and a C-terminus of a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1.PD‐L1 ) (hPD-L1 MAB-2 VL1 (SEQ ID NO: 58)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E-coil) domain ( E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39)), linker (LEPKSADKTHTCPPCP (SEQ ID NO: 30)), L234A / L235A / M252Y / S254T / T25 It includes the CH2-CH3 domain of a representative human IgG1 (SEQ ID NO: 43, where X is absent) containing a 6E substitution, and the C-terminus.

[0248] Thus, the first and third polypeptide chains of DART-A are composed of: SEQ ID NO:58-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:30-SEQ ID NO:43.

[0249] The amino acid sequence of the first and third polypeptide chains of DART-A is (SEQ ID NO: 116): DIQMTQSPSS LSASVGDRVT ITCKASQDVN TAVAWYQQKP GKAPKLLIYW ASTRHTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYNTPLTFGQ GTKVEIKGGG SGGGGQVQLQ ESGPGLVKPS ETLSLTCTVS GGSISSYYWS WIRQPPGKGL EWIGRIYTSG STNYNPSLKS RVTMSVDTSK NQFSLKLSSV TAADTAVYYC ARDGWYDEDY NYYGMDVWGQ GTTVTVSSGG CGGGEVAA C E KEVAALEKEV AALEKEVAAL EKLEPKSADK THTCPPCPAP EAAGGPSVFL FPPKPKDTLY ITREPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPG is.

[0250] Heterodimer-promoting (E-coil) domain containing no cysteine ​​residues ( E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E Alternative DART-A first and third polypeptide chains may be employed that comprise the sequences SEQ ID NO:58-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43. Further alternative DART-A first and third polypeptide chains may be employed in which the amino acid residues of SEQ ID NO:58 (hPD-L1 MAB-2 VL1) are replaced with the amino acid residues of SEQ ID NO:72 (hPD-L1 MAB-2 VL2). Alternative molecules comprising multiple such polypeptide chains are described below.

[0251] The second and fourth polypeptide chains of DART-A are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, CD137 (CD13 7 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH1, SEQ ID NO: 57)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0252] Thus, the second and fourth polypeptide chains of DART-A are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:57-SEQ ID NO:18-SEQ ID NO:40.

[0253] The amino acid sequence of the second and fourth polypeptide chains of DART-A is (SEQ ID NO: 117): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIG GGTTYYPDTV KGRFTISRDN AKNTLYLQMN SLKTEDTAVY YCARQGLPYY FDYWGQGTLV TVSSGGCGGG KVAA C KEKVA ALKEKVAALK EKVAALKE is.

[0254] Heterodimer-promoting (K-coil) domains containing no cysteine ​​residues (e.g. K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K Alternative DART-A second and fourth polypeptide chains may be employed that include SEQ ID NO:E (SEQ ID NO:38). Such alternative DART-A second and fourth polypeptide chains may optionally be composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:57-SEQ ID NO:18-SEQ ID NO:38. Further alternative DART-A first and third polypeptide chains may be employed in which the amino acid residue of SEQ ID NO:50 (CD137 MAB-6 VL1) is replaced with the amino acid residue of SEQ ID NO:55 (CD137 MAB-6 VL2) or SEQ ID NO:56 (CD137 MAB-6 VL3), and / or the amino acid residue of SEQ ID NO:57 (hPD-L1 MAB-2 VH1) is replaced with the amino acid residue of SEQ ID NO:67 (hPD-L1 MAB-2 VH2), SEQ ID NO:68 (hPD-L1 MAB-2 VH3), SEQ ID NO:69 (hPD-L1 MAB-2 VH4), SEQ ID NO:70 (hPD-L1 MAB-2 VH5), or SEQ ID NO:72 (hPD-L1 MAB-2 VH6). Alternatively, the VL / VH domain of PD-L1 may be replaced with the VL / VH domain of a TA-binding molecule that binds to a different epitope of PD-L1 or that binds to a different TA. Alternative molecules containing such multiple polypeptide chains are described below.

[0255] 2. DART‐A1 DART-A1 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A1 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A1 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (2.1).

[0256] The first and third polypeptide chains of DART-A1 are arranged in an N-terminal to C-terminal direction, with an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1, and a C-terminus of a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1. PD‐L1 ) (hPD-L1 MAB-2 VL1 (SEQ ID NO: 58)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker -peptide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E coil) domain ( E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39)), a linker (LEPKSADKTHTCPPCP (SEQ ID NO: 30)), the CH2-CH3 domain of a representative IgG1 containing L234A / L235A / M252Y / S254T / T256E substitutions (SEQ ID NO: 43), and a C-terminus.

[0257] Thus, the first and third polypeptide chains of DART-A1 are composed of: SEQ ID NO:58-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:30-SEQ ID NO:43.

[0258] The amino acid sequence of the first and third polypeptide chains of DART-A1 is (SEQ ID NO: 118): DIQMTQSPSS LSASVGDRVT ITCKASQDVN TAVAWYQQKP GKAPKLLIYW ASTRHTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYNTPLTFGQ GTKVEIKGGG SGGGGQVQLQ ESGPGLVKPS ETLSLTCTVS GGSISSYYWS WIRQPPGKGL EWIGRIYTSG STNYNPSLKS RVTMSVDTSK NQFSLKLSSV TAADTAVYYC ARDGWYDEDY NYYGMDVWGQ GTTVTVSSGG CGGGEVAA C E KEVAALEKEV AALEKEVAAL EKLEPKSADK THTCPPCPAP EAAGGPSVFL FPPKPKDTLY ITREPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK is.

[0259] The second and fourth polypeptide chains of DART-A1 are arranged in the N-terminal to C-terminal direction, with an N-terminus containing a VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 (CD1 37 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1(hPD-L1 MAB-2 VH2, SEQ ID NO: 67)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0260] Thus, the second and fourth polypeptide chains of DART-A1 are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:67-SEQ ID NO:18-SEQ ID NO:40.

[0261] The amino acid sequence of the second and fourth polypeptide chains of DART-A1 is (SEQ ID NO: 119): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIG GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARQGLPYY FDYWGQGTLV TVSSGGCGGG KVAA C KEKVA ALKEKVAALK EKVAALKE is.

[0262] Alternative first / third and second / fourth polypeptide chains of DART-A1 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ EVAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ DART-A1 first / third polypeptide chains, optionally consisting of: SEQ ID NO:58-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A1 second / fourth chains, optionally consisting of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:67-SEQ ID NO:18-SEQ ID NO:38.

[0263] 3. DART‐A2 DART-A2 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A2 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A2 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (2.2).

[0264] The first and third polypeptide chains of DART-A2 are arranged in an N-terminal to C-terminal direction, with an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1, and a C-terminus of a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1. PD‐L1 ) (hPD-L1 MAB-2 VL2 (SEQ ID NO: 72)), an intervening linker peptide (linker 1 GGGSGGGG (SEQ ID NO: 16)), the VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker -peptide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E coil) domain ( E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39)), a linker (LEPKSADKTHTCPPCP (SEQ ID NO: 30)), the CH2-CH3 domain of a representative IgG1 containing L234A / L235A / M252Y / S254T / T256E substitutions (SEQ ID NO: 43), and a C-terminus.

[0265] Thus, the first and third polypeptide chains of DART-A2 are composed of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:30-SEQ ID NO:43.

[0266] The amino acid sequence of the first and third polypeptide chains of DART-A2 is (SEQ ID NO: 120): DIQMTQSPSS LSASVGDRVT ITCKASQDVN EAVAWYQQKP GKAPKLLIYW ASTRHTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYNTPLTFGQ GTKVEIKGGG SGGGGQVQLQ ESGPGLVKPS ETLSLTCTVS GGSISSYYWS WIRQPPGKGL EWIGRIYTSG STNYNPSLKS RVTMSVDTSK NQFSLKLSSV TAADTAVYYC ARDGWYDEDY NYYGMDVWGQ GTTVTVSSGG CGGGEVAACE KEVAALEKEV AALEKEVAAL EKLEPKSADK THTCPPCPAP EAAGGPSVFL FPPKPKDTLY ITREPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK

[0267] The second and fourth polypeptide chains of DART-A2 are identical to the second and fourth polypeptide chains of DART-A1 (SEQ ID NO: 119).

[0268] Alternative first / third and second / fourth polypeptide chains of DART-A2 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ the sequences SEQ ID NO:E (SEQ ID NO:38). Such alternative DART-A2 first / third polypeptide chains are optionally comprised of SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and such alternative DART-A2 second / fourth chains are optionally comprised of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:67-SEQ ID NO:18-SEQ ID NO:38.

[0269] 4. DART‐A3 DART-A3 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A3 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A3 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (3.1).

[0270] The first and third polypeptide chains of DART-A3 are identical to the first and third polypeptide chains of DART-A1 (SEQ ID NO: 118).

[0271] The second and fourth polypeptide chains of DART-A3 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, and CD137 (CD1 37 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH3, SEQ ID NO: 68)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0272] Thus, the second and fourth polypeptide chains of DART-A3 are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:40.

[0273] The amino acid sequence of the second and fourth polypeptide chains of DART-A3 is (SEQ ID NO: 121): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIK GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARQGLPYY GDYWGQGTLV TVSSGGCGGG KVAA C KEKVA ALKEKVAALK EKVAALKE is.

[0274] Alternative first / third and second / fourth polypeptide chains of DART-A3 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ DART-A3 first / third polypeptide chains, optionally consisting of: SEQ ID NO:58-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A3 second / fourth chains, optionally consisting of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:38.

[0275] 5. DART‐A4 DART-A4 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A4 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A4 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (3.2).

[0276] The first and third polypeptide chains of DART-A4 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0277] The second and fourth polypeptide chains of DART-A4 are identical to the second and fourth polypeptide chains of DART-A3 (SEQ ID NO: 121).

[0278] Alternative first / third and second / fourth polypeptide chains of DART-A4 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is specifically contemplated that such alternative first / third polypeptide chains of DART-A4 may optionally employ: SEQ ID NO: 72 - SEQ ID NO: E (SEQ ID NO: 38) SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:30 - SEQ ID NO:43, and the second / fourth strand of such an alternative DART-A4 is optionally composed of: SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:68 - SEQ ID NO:18 - SEQ ID NO:38.

[0279] 6. DART‐A5 DART-A5 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A5 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A5 contains the binding domains of CD137 MAB-6 (1.2) and hPD-L1 MAB-2 (3.2).

[0280] The first and third polypeptide chains of DART-A5 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0281] The second and fourth polypeptide chains of DART-A5 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, and CD137 (CD1 37 MAB-6 VL2 (SEQ ID NO: 55)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH2, SEQ ID NO: 68)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0282] Thus, the second and fourth polypeptide chains of DART-A5 are composed of: SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:40.

[0283] The amino acid sequence of the second and fourth polypeptide chains of DART-A5 is (SEQ ID NO: 122): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWYQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIK GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARQGLPYY GDYWGQGTLV TVSSGGCGGG KVAACKEKVA ALKEKVAALK EKVAALKE is.

[0284] Alternative first / third and second / fourth polypeptide chains of DART-A5 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL KIt is also specifically contemplated to employ DART-A5 first / third polypeptide chains, optionally consisting of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A5 second / fourth chains, optionally consisting of: SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:38.

[0285] 7. DART‐A6 DART-A6 is a tetravalent CD137×CD137×TA×TA binding molecule that has two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A6 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A6 contains the binding domains of CD137 MAB-6 (1.3) and hPD-L1 MAB-2 (3.2).

[0286] The first and third polypeptide chains of DART-A6 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0287] The second and fourth polypeptide chains of DART-A6 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, and CD137 (CD1 37 MAB-6 VL3 (SEQ ID NO: 56)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH3, SEQ ID NO: 68)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ KVAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0288] Thus, the second and fourth polypeptide chains of DART-A6 are composed of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:40.

[0289] The amino acid sequence of the second and fourth polypeptide chains of DART-A6 is (SEQ ID NO: 123): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWFQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIK GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARQGLPYY GDYWGQGTLV TVSSGGCGGG KVAACKEKVA ALKEKVAALK EKVAALKE is.

[0290] Alternative first / third and second / fourth polypeptide chains of DART-A6 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐K VAAL K It is also specifically contemplated to employ DART-A6 first / third polypeptide chains, optionally consisting of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A6 second / fourth chains, optionally consisting of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:38.

[0291] 8. DART‐A7 DART-A7 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A7 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A7 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (4.2).

[0292] The first and third polypeptide chains of DART-A7 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0293] The second and fourth polypeptide chains of DART-A7 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, CD137 (CD1 37 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH4, SEQ ID NO: 69)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL KE‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0294] Thus, the second and fourth polypeptide chains of DART-A7 are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:69-SEQ ID NO:18-SEQ ID NO:40.

[0295] The amino acid sequence of the second and fourth polypeptide chains of DART-A7 is (SEQ ID NO: 124): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIG GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARAGLPYY FDYWGQGTLV TVSSGGCGGG KVAACKEKVA ALKEKVAALK EKVAALKE is.

[0296] Alternative first / third and second / fourth polypeptide chains of DART-A7 (e.g., α-DART-A7) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAALK E‐ K VAAL K It is also specifically contemplated to employ DART-A7 first / third polypeptide chains, optionally consisting of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A7 second / fourth chains, optionally consisting of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:69-SEQ ID NO:18-SEQ ID NO:38.

[0297] 9. DART‐A8 DART-A8 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A8 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A8 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (5.2).

[0298] The first and third polypeptide chains of DART-A8 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0299] The second and fourth polypeptide chains of DART-A8 are arranged in the N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, and CD137 (CD1 37 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH5, SEQ ID NO: 70)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ KVAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0300] Thus, the second and fourth polypeptide chains of DART-A8 are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:70-SEQ ID NO:18-SEQ ID NO:40.

[0301] The amino acid sequence of the second and fourth polypeptide chains of DART-A8 is (SEQ ID NO: 125): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIK GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARAGLPYY FDYWGQGTLV TVSSGGCGGG KVAACKEKVA ALKEKVAALK EKVAALKE

[0302] Alternative first / third and second / fourth polypeptide chains of DART-A8 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAALK E‐ K VAAL K It is also specifically contemplated to employ DART-A8 first / third polypeptide chains, optionally consisting of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A8 second / fourth chains, optionally consisting of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:70-SEQ ID NO:18-SEQ ID NO:38.

[0303] 10. DART‐A9 DART-A9 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A9 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A9 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (6.2).

[0304] The first and third polypeptide chains of DART-A9 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0305] The second and fourth polypeptide chains of DART-A9 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, CD137 (CD1 37 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH6, SEQ ID NO: 71)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ KVAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0306] Thus, the second and fourth polypeptide chains of DART-A9 are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:71-SEQ ID NO:18-SEQ ID NO:40.

[0307] The amino acid sequence of the second and fourth polypeptide chains of DART-A9 is (SEQ ID NO: 126): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIG GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARAGLPYY GDYWGQGTLV TVSSGGCGGG KVAACKEKVA ALKEKVAALK EKVAALKE is.

[0308] Alternative first / third and second / fourth polypeptide chains of DART-A9 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐K VAAL K E‐ K VAAL K It is also specifically contemplated to employ DART-A9 first / third polypeptide chains, optionally consisting of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-A9 second / fourth chains, optionally consisting of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:71-SEQ ID NO:18-SEQ ID NO:38.

[0309] 11. DART‐A10 DART-A10 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for PD-L1, a representative TA. DART-A10 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 3B). DART-A10 contains the binding domains of CD137 MAB-6 (1.3) and hPD-L1 MAB-2 (4.2).

[0310] The first and third polypeptide chains of DART-A10 are identical to the first and third polypeptide chains of DART-A2 (SEQ ID NO: 120).

[0311] The second and fourth polypeptide chains of DART-A10 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, CD137 (CD 137 MAB-6 VL3 (SEQ ID NO: 56)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to PD-L1 (VH PD‐L1 (hPD-L1 MAB-2 VH4, SEQ ID NO: 69)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0312] Thus, the second and fourth polypeptide chains of DART-A10 are composed of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:69-SEQ ID NO:18-SEQ ID NO:40.

[0313] The amino acid sequence of the second and fourth polypeptide chains of DART-A10 is (SEQ ID NO: 139): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWFQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGEVQL VESGGGLVQP GGSLRLSCAA SGFTFSSYTM SWVRQAPGKG LEWVAYISIG GGTTYYPDTV KGRFTISRDN AKNSLYLQMN SLRAEDTAVY YCARAGLPYY FDYWGQGTLV TVSSGGCGGG KVAACKEKVA ALKEKVAALK EKVAALKE is.

[0314] Alternative first / third and second / fourth polypeptide chains of DART-A10 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ KVAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ such alternatives. The first / third polypeptide chain of such alternative DART-A10 is optionally composed of: SEQ ID NO:72-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and the second / fourth chain of such alternative DART-A10 is optionally composed of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:69-SEQ ID NO:18-SEQ ID NO:38.

[0315] 12. DART‐B1 DART-B1 is a bivalent CD137×TA binding molecule with one CD137-binding site and one binding site for HER2, a representative TA. DART-B1 is composed of three polypeptide chains, where the first, second, and third polypeptide chains are distinct (see Figure 1D). DART-B1 contains the binding domains of CD137 MAB-6 (1.1) and hHER2 MAB-1 (1.3).

[0316] The first polypeptide chain of DART-B1 is arranged in an N-terminal to C-terminal direction, containing at its N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 (CD137 M AB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to HER2 (VH HER2 (hHER2 MAB-1 VH1, SEQ ID NO: 80)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (E-coil) domain ( E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL EK (SEQ ID NO: 37)), an intervening linker peptide (GGGDKTHTCPPCP (SEQ ID NO: 21), "knob-bearing" CH2 and CH3 domains containing L234A / L235A / M252Y / S254T / T256E substitutions (SEQ ID NO: 146), and a C-terminus.

[0317] Thus, the first polypeptide chain of DART-B1 is composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:80-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:21-SEQ ID NO:146.

[0318] The amino acid sequence of the first polypeptide chain of DART-B1 is (SEQ ID NO: 143): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL VQSGAEVKKP GASVKVSCKA SGYTFTNYGM NWVRQAPGQG LEWMGWINTN IGEPTYTEEF KGRVTMTRDT SISTAYMELS RLRSDDTAVY YCARDDGYGN RVSYWGQGTL VTVSSGGCGG GEVAALEKEV AALEKEVAAL EKEVAALEKG GGDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLWCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK is.

[0319] The second polypeptide chain of DART-B1 is arranged in an N-terminal to C-terminal direction, containing an N-terminal VL domain (VL) of a monoclonal antibody capable of binding to HER2. HER2 (hHER2 MAB-1 VL3, SEQ ID NO: 85)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAAL K E - K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 38)), and the C-terminus.

[0320] Thus, the second polypeptide chain of DART-B1 is: SEQ ID NO:85 - SEQ ID NO:16 - SEQ ID NO: Includes sequence number 46 - sequence number 18 - sequence number 38.

[0321] The amino acid sequence of the second polypeptide chain of DART-B1 is (SEQ ID NO: 144): DIQMTQSPSS LSASVGDRVT ITCKASQDIS NYLSWFQQKP GKAPKTLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ HDEFPWTFGQ GTKLEIKGGG SGGGGQVQLQ ESGPGLVKPS ETLSLTCTVS GGSISSYYWS WIRQPPGKGL EWIGRIYTSG STNYNPSLKS RVTMSVDTSK NQFSLKLSSV TAADTAVYYC ARDGWYDEDY NYYGMDVWGQ GTTVTVSSGG CGGGKVAALK EKVAALKEKV AALKEKVAAL KE is.

[0322] The third polypeptide chain of DART-B1 comprises, from N- to C-terminus, a linker DKTHTCPPCP (SEQ ID NO: 20) and "hole-bearing" CH2 and CH3 domains containing L234A / L235A / M252Y / S254T / T256E / H435R substitutions (SEQ ID NO: 149).

[0323] Thus, the third polypeptide chain of DART-B1 is composed of: SEQ ID NO:20-SEQ ID NO:149.

[0324] It will be appreciated that the third polypeptide chain of DART-B does not contain any epitope binding domain and therefore can be employed in a variety of CD137xTA binding molecules having the diabody structure shown in Figure 1D.

[0325] The third polypeptide chain of DART-B1 has the amino acid sequence of SEQ ID NO: 145: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNRYTQKS LSLSPGK It has.

[0326] 12. DART‐B2 DART-B2 is a tetravalent CD137xCD137xTAxTA binding molecule with two CD137-binding sites and two binding sites for HER2, a representative TA. DART-B1 is composed of four polypeptide chains, of which the first and third polypeptide chains are identical, and the second and fourth polypeptide chains are identical (see Figure 1B). DART-B1 contains the binding domains of CD137 MAB-6 (1.1) and hHER2 MAB-1 (1.3).

[0327] The first and third polypeptide chains of DART-B2 are arranged in the N-terminal to C-terminal direction, with an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to HER2, HER2 ) (hHER2 MAB-1 VL3 (SEQ ID NO: 85)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide tide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E-coil) domain ( E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39)), a linker (LEPKSADKTHTCPPCP (SEQ ID NO: 30)), the CH2-CH3 domain of a representative IgG1 containing the L234A / L235A / M252Y / S254T / T256E substitutions (SEQ ID NO: 43, where X is absent), and a C-terminus.

[0328] Thus, the first and third polypeptide chains of DART-B2 are composed of: SEQ ID NO:85-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:30-SEQ ID NO:43.

[0329] The amino acid sequence of the first and third polypeptide chains of DART-B2 is (SEQ ID NO: 151): DIQMTQSPSS LSASVGDRVT ITCKASQDIS NYLSWFQQKP GKAPKTLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ HDEFPWTFGQ GTKLEIKGGG SGGGGQVQLQ ESGPGLVKPS ETLSLTCTVS GGSISSYYWS WIRQPPGKGL EWIGRIYTSG STNYNPSLKS RVTMSVDTSK NQFSLKLSSV TAADTAVYYC ARDGWYDEDY NYYGMDVWGQ GTTVTVSSGG CGGGEVAACE KEVAALEKEV AALEKEVAAL EKLEPKSADK THTCPPCPAP EAAGGPSVFL FPPKPKDTLY ITREPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK is.

[0330] The second and fourth polypeptide chains of DART-B2 are arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, CD137 (CD1 37 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to HER2 (VH HER2(hHER2 MAB-1 VH1, SEQ ID NO: 80)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), a heterodimer-promoting (K-coil) domain ( K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 40), and the C-terminus.

[0331] Thus, the second and fourth polypeptide chains of DART-B2 are composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:80-SEQ ID NO:18-SEQ ID NO:40.

[0332] The amino acid sequence of the second and fourth polypeptide chains of DART-B2 is (SEQ ID NO: 152): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL VQSGAEVKKP GASVKVSCKA SGYTFTNYGM NWVRQAPGQG LEWMGWINTN IGEPTYTEEF KGRVTMTRDT SISTAYMELS RLRSDDTAVY YCARDDGYGN RVSYWGQGTL VTVSSGGCGG GKVAACKEKV AALKEKVAAL KEKVAALKE is.

[0333] Alternative first / third and second / fourth polypeptide chains of DART-B2 (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains that do not contain cysteine ​​residues. E VAAL E K‐ E VAAL E K‐ E VAALE K‐ E VAAL E K (SEQ ID NO: 37) and K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ DART-B2 first / third polypeptide chains, optionally consisting of: SEQ ID NO:85-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:30-SEQ ID NO:43, and DART-B2 second / fourth chains, optionally consisting of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:80-SEQ ID NO:18-SEQ ID NO:38.

[0334] B. Trivalent CD137×TA binding molecule 1. TRIDENT‐A TRIDENT-A is a trivalent CD137xCD137xTA binding molecule with two CD137-binding sites and one binding site for PD-L1, a representative TA. TRIDENT-A is composed of four polypeptide chains (see Figure 3A, VL1 / VH1 (site A) is identical to VL2 / VH2 (site B) and binds to CD137, and VL3 / VH3 (site C) binds to PD-L1). TRIDENT-A binds to CD137. It contains the binding domains of MAB-6(1.1) and hPD-L1 MAB-2(1.1).

[0335] The first polypeptide chain of TRIDENT-A is arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137, CD137 )(CD13 7 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide tide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E-coil) domain ( E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37)), an intervening linker peptide (GGGDKTHTCPPCP (SEQ ID NO: 21)), L234A / L235A / M252Y / S254T / It comprises "knob-bearing" CH2 and CH3 domains (SEQ ID NO: 146) containing the T256E substitution, and a C-terminus.

[0336] Thus, the first polypeptide chain of TRIDENT-A is composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:21-SEQ ID NO:146.

[0337] The amino acid sequence of the first polypeptide chain of TRIDENT-A is (SEQ ID NO: 127): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL QESGPGLVKP SETLSLTCTV SGGSISSYYW SWIRQPPGKG LEWIGRIYTS GSTNYNPSLK SRVTMSVDTS KNQFSLKLSS VTAADTAVYY CARDGWYDED YNYYGMDVWG QGTTVTVSSG GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLYIT REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK is.

[0338] The second polypeptide chain of TRIDENT-A contains, in an N-terminal to C-terminal direction, an N-terminal VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 (CD137 MAB-6 VL1 (SEQ ID NO: 50)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide (Linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (K-coil) domain (e.g. K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 38)), and the C-terminus.

[0339] Thus, the second polypeptide chain of TRIDENT-A is composed of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38.

[0340] The amino acid sequence of the second polypeptide chain of TRIDENT-A is (SEQ ID NO: 128): EIVMTQSPAT LSLTPGERAT LSCRASQSVS SNYLSWFQQI PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL QESGPGLVKP SETLSLTCTV SGGSISSYYW SWIRQPPGKG LEWIGRIYTS GSTNYNPSLK SRVTMSVDTS KNQFSLKLSS VTAADTAVYY CARDGWYDED YNYYGMDVWG QGTTVTVSSG GCGGGKVAAL KEKVAALKEK VAALKEKVAA LKE is.

[0341] Heterodimer-promoting (E-coil and K-coil) domains containing cysteine ​​residues (e.g. E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39) and K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL KAlternatively, alternative TRIDENT-A first and second polypeptide chains may be employed comprising a CD137 MAB-6 VL1 domain pair (SEQ ID NO: 16-SEQ ID NO: 46-SEQ ID NO: 18-SEQ ID NO: 39-SEQ ID NO: 21-SEQ ID NO: 146) and a second polypeptide chain may be optionally comprised of SEQ ID NO: 50-SEQ ID NO: 16-SEQ ID NO: 46-SEQ ID NO: 18-SEQ ID NO: 40. Further alternative TRIDENT-A first and second polypeptide chains may be employed in which the amino acid residues of SEQ ID NO: 50 (CD137 MAB-6 VL1) are replaced with the amino acid residues of SEQ ID NO: 55 (CD137 MAB-6 VL2) or SEQ ID NO: 56 (CD137 MAB-6 VL3). It is also specifically contemplated that one CD137 VL / VH domain pair may be replaced with the VL / VH pair of a TA-binding molecule. Such alternative molecules comprising multiple polypeptide chains are described below.

[0342] The third polypeptide chain of TRIDENT-A is arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VH domain (VH) of a monoclonal antibody capable of binding to PD-L1, PD‐L1 (hPD-L1 MAB-2 VH1, SEQ ID NO:57), a human IgG1 CH1 domain (SEQ ID NO:3), a human IgG1 hinge region (SEQ ID NO:7), and "hole-bearing" CH2 and CH3 domains containing the L234A / L235A / M252Y / S254T / T256E / H435R substitutions (SEQ ID NO:149).

[0343] Thus, the third polypeptide chain of TRIDENT-A is composed of: SEQ ID NO:57-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149.

[0344] The amino acid sequence of the third polypeptide chain of TRIDENT-A is (SEQ ID NO: 129): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMSWVRQA PGKGLEWVAY ISIGGGTTYY PDTVKGRFTI SRDNAKNTLY LQMNSLKTED TAVYYCARQG LPYYFDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLYITREPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLSCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLLSPGK is.

[0345] The fourth polypeptide chain of TRIDENT-A contains, in an N-terminal to C-terminal direction, an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to PD-L1. PD‐L1 ) (hPD-L1 MAB-1 VL1 (SEQ ID NO: 58)), a human IgG CLκ domain (SEQ ID NO: 1), and a C-terminus.

[0346] Thus, the fourth polypeptide chain of TRIDENT-A is composed of: SEQ ID NO:69-SEQ ID NO:1.

[0347] The amino acid sequence of the fourth polypeptide chain of TRIDENT-A is (SEQ ID NO: 130): DIQMTQSPSS LSASVGDRVT ITCKASQDVN TAVAWYQQKP GKAPKLLIYW ASTRHTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYNTPLTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC is.

[0348] Alternative TRIDENT-A third and fourth polypeptide chains may be employed in which the amino acid residues of SEQ ID NO:57 (hPD-L1 MAB-2 VH1) are replaced with amino acid residues of SEQ ID NO:67 (hPD-L1 MAB-2 VH2), SEQ ID NO:68 (hPD-L1 MAB-2 VH3), SEQ ID NO:69 (hPD-L1 MAB-2 VH4), SEQ ID NO:70 (hPD-L1 MAB-2 VH5), or SEQ ID NO:72 (hPD-L1 MAB-2 VH6), and / or the amino acid residues of SEQ ID NO:58 (hPD-L1 MAB-2 VL1) are replaced with amino acid residues of SEQ ID NO:72 (hPD-L1 MAB-2 VL2). Alternatively, the VL / VH domain of PD-L1 may be replaced with the VL / VH domains of a TA-binding molecule that binds to a different epitope of PD-L1 or that binds to a different TA. It is also specifically contemplated that where a TA binding site is formed by association of a first polypeptide chain with a second polypeptide chain, the VL / VH domains of the third and fourth polypeptide chains may be replaced with any of the CD137 MAB-6 VL / VH domains provided herein. Alternative molecules comprising such multiple polypeptide chains are described below.

[0349] 2. TRIDENT‐A4 TRIDENT-A4 is a trivalent CD137xCD137xTA binding molecule with two CD137-binding sites and one binding site for PD-L1, a representative TA. TRIDENT-A4 is composed of four polypeptide chains (see Figure 3A; VL1 / VH1 (site A) is identical to VL2 / VH2 (site B) and binds CD137, and VL3 / VH3 (site C) binds PD-L1). TRIDENT-A4 contains the binding domains of CD137 MAB-6 (1.1) and hPD-L1 MAB-2 (3.2).

[0350] The first polypeptide chain of TRIDENT-A4 is identical to the first polypeptide chain of TRIDENT-A (SEQ ID NO: 127).

[0351] The second polypeptide chain of TRIDENT-A4 is identical to the second polypeptide chain of TRIDENT-A (SEQ ID NO: 128).

[0352] Alternative TRIDENT-A4 first and second polypeptide chains (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains containing cysteine ​​residues. E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39) and K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ TRIDENT-A4 variants in which the first polypeptide chain is optionally comprised of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:21-SEQ ID NO:146, and the second polypeptide chain is optionally comprised of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40.

[0353] The third polypeptide chain of TRIDENT-A4 is arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VH domain (VH) of a monoclonal antibody capable of binding to PD-L1, PD‐L1 (hPD-L1 MAB-2 VH3, SEQ ID NO: 68)), a human IgG1 CH1 domain (SEQ ID NO: 3), a human IgG1 hinge region (SEQ ID NO: 7), and "hole-bearing" CH2 and CH3 domains containing the L234A / L235A / M252Y / S254T / T256E / H435R substitutions (SEQ ID NO: 149).

[0354] Thus, the third polypeptide chain of TRIDENT-A4 is composed of: SEQ ID NO:68-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149.

[0355] The amino acid sequence of the third polypeptide chain of TRIDENT-A4 is (SEQ ID NO: 131): EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYTMSWVRQA PGKGLEWVAY ISIKGGTTYY PDTVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARQG LPYYGDYWGQ GTLVTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPEAAGGPS VFLFPPKPKD TLYITREPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLSCAV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLVSK LTVDKSRWQQ GNVFSCSVMH EALHNRYTQK SLLSPGK is.

[0356] The fourth polypeptide chain of TRIDENT-A4 is composed of, in an N-terminal to C-terminal direction, an N-terminus, a VL domain of a monoclonal antibody capable of binding to PD-L1 (VL PD‐L1 ) (hPD-L1 MAB-1 VL2 (SEQ ID NO: 72)), a human IgG CLκ domain (SEQ ID NO: 1), and a C-terminus.

[0357] Thus, the fourth polypeptide chain of TRIDENT-A4 is composed of: SEQ ID NO:72-SEQ ID NO:1.

[0358] The amino acid sequence of the fourth polypeptide chain of TRIDENT-A is (SEQ ID NO: 132): DIQMTQSPSS LSASVGDRVT ITCKASQDVN EAVAWYQQKP GKAPKLLIYW ASTRHTGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ HYNTPLTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC is.

[0359] 3. TRIDENT‐A5 TRIDENT-A5 is a trivalent CD137xCD137xTA binding molecule with two CD137-binding sites and one binding site for PD-L1, a representative TA. TRIDENT-A5 is composed of four polypeptide chains (see Figure 3A; VL1 / VH1 (site A) is identical to VL2 / VH2 (site B) and binds to CD137, and VL3 / VH3 (site C) binds to PD-L1). TRIDENT-A5 contains the binding domains of CD137 MAB-6 (1.2) and hPD-L1 MAB-2 (3.2).

[0360] The first polypeptide chain of TRIDENT-A5 is arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 )(CD1 37 MAB-6 VL2 (SEQ ID NO: 55)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E coil) domain ( E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37)), an intervening linker peptide (GGGDKTHTCPPCP (SEQ ID NO: 21)), L234A / L235A / M252Y / S254T / T256E substitution (SEQ ID NO: 146), and a "knob-bearing" CH2 and CH3 domains, and a C-terminus.

[0361] Thus, the first polypeptide chain of TRIDENT-A5 is composed of: SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:21-SEQ ID NO:146.

[0362] The amino acid sequence of the first polypeptide chain of TRIDENT-A5 is (SEQ ID NO: 133): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWYQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL QESGPGLVKP SETLSLTCTV SGGSISSYYW SWIRQPPGKG LEWIGRIYTS GSTNYNPSLK SRVTMSVDTS KNQFSLKLSS VTAADTAVYY CARDGWYDED YNYYGMDVWG QGTTVTVSSG GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLYIT REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK is.

[0363] The second polypeptide chain of TRIDENT-A5 contains, in an N-terminal to C-terminal direction, an N-terminal VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 (CD13 7 MAB-6 VL2 (SEQ ID NO: 55)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide tide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (K-coil) domain (e.g. K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 38)), and the C-terminus.

[0364] Thus, the second polypeptide chain of TRIDENT-A5 is composed of: SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38.

[0365] The amino acid sequence of the second polypeptide chain of TRIDENT-A5 is (SEQ ID NO: 134): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWYQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL QESGPGLVKP SETLSLTCTV SGGSISSYYW SWIRQPPGKG LEWIGRIYTS GSTNYNPSLK SRVTMSVDTS KNQFSLKLSS VTAADTAVYY CARDGWYDED YNYYGMDVWG QGTTVTVSSG GCGGGKVAAL KEKVAALKEK VAALKEKVAA LKE is.

[0366] May contain heterodimer-promoting (E-coil and K-coil) domains containing cysteine ​​residues , alternative TRIDENT-A5 first and second polypeptide chains (e.g. E VAA CE K‐ E VAAL EK‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39) and K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ TRIDENT-A5 variants having the sequences SEQ ID NO:E (SEQ ID NO:40). In such alternative TRIDENT-A5 variants, the first polypeptide chain optionally consists of: SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:21-SEQ ID NO:146, and the second polypeptide chain optionally consists of: SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40.

[0367] The third polypeptide chain of TRIDENT-A5 is identical to the third polypeptide chain of TRIDENT-A4 (SEQ ID NO: 131).

[0368] The fourth polypeptide chain of TRIDENT-A5 is identical to the fourth polypeptide chain of TRIDENT-A4 (SEQ ID NO: 132).

[0369] 4. TRIDENT‐A6 TRIDENT-A6 is a trivalent CD137xCD137xTA binding molecule with two CD137-binding sites and one binding site for PD-L1, a representative TA. TRIDENT-A6 is composed of four polypeptide chains (see Figure 3A; VL1 / VH1 (site A) is identical to VL2 / VH2 (site B) and binds to CD137, and VL3 / VH3 (site C) binds to PD-L1). TRIDENT-A6 contains the binding domains of CD137 MAB-6 (1.3) and hPD-L1 MAB-2 (3.2).

[0370] The first polypeptide chain of TRIDENT-A6 is arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137.CD137 )(CD1 37 MAB-6 VL3 (SEQ ID NO: 56)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (E coil) domain ( E VAAL E K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 37)), an intervening linker peptide (GGGDKTHTCPPCP (SEQ ID NO: 21)), L234A / L235A / M252Y / S254T / T256E substitution (SEQ ID NO: 146), and a "knob-bearing" CH2 and CH3 domains, and a C-terminus.

[0371] Thus, the first polypeptide chain of TRIDENT-A6 is composed of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:37-SEQ ID NO:21-SEQ ID NO:146.

[0372] The amino acid sequence of the first polypeptide chain of TRIDENT-A6 is (SEQ ID NO: 135): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWFQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL QESGPGLVKP SETLSLTCTV SGGSISSYYW SWIRQPPGKG LEWIGRIYTS GSTNYNPSLK SRVTMSVDTS KNQFSLKLSS VTAADTAVYY CARDGWYDED YNYYGMDVWG QGTTVTVSSG GCGGGEVAAL EKEVAALEKE VAALEKEVAA LEKGGGDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLYIT REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPS REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK is.

[0373] The second polypeptide chain of TRIDENT-A6 is arranged in an N-terminal to C-terminal direction, containing at its N-terminus, a VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 (CD13 7 MAB-6 VL3 (SEQ ID NO: 56)), an intervening linker peptide (Linker 1; GGGSGGGG (SEQ ID NO: 16)), a VH domain of a monoclonal antibody capable of binding to CD137 (VH CD137 ) (CD137 MAB-6 VH1 (SEQ ID NO: 46)), an intervening linker peptide tide (linker 2; GGCGGG (SEQ ID NO: 18)), heterodimer-promoting (K-coil) domain (e.g. K VAAL K E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K E (SEQ ID NO: 38)), and the C-terminus.

[0374] Thus, the second polypeptide chain of TRIDENT-A6 is composed of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38.

[0375] The amino acid sequence of the second polypeptide chain of TRIDENT-A6 is (SEQ ID NO: 136): EIVMTQSPAT LSLSPGERAT LSCRASQSVS SNYLSWFQQK PGQAPRLLIY GASTRATGIP ARFSGSGSGT DFTLTISSLQ PEDFAVYYCQ QDYDLPWTFG QGTKVEIKGG GSGGGGQVQL QESGPGLVKP SETLSLTCTV SGGSISSYYW SWIRQPPGKG LEWIGRIYTS GSTNYNPSLK SRVTMSVDTS KNQFSLKLSS VTAADTAVYY CARDGWYDED YNYYGMDVWG QGTTVTVSSG GCGGGKVAAL KEKVAALKEK VAALKEKVAA LKE

[0376] Alternative TRIDENT-A6 first and second polypeptide chains (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains containing cysteine ​​residues. E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39) and K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL KIt is also specifically contemplated to employ TRIDENT-A6 (SEQ ID NO:E (SEQ ID NO:40)). In such alternative TRIDENT-A6, the first polypeptide chain optionally consists of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:21-SEQ ID NO:146, and the second polypeptide chain optionally consists of: SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40.

[0377] The third polypeptide chain of TRIDENT-A6 is identical to the third polypeptide chain of TRIDENT-A4 (SEQ ID NO: 131).

[0378] The fourth polypeptide chain of TRIDENT-A6 is identical to the fourth polypeptide chain of TRIDENT-A4 (SEQ ID NO: 132).

[0379] 5. TRIDENT‐B1 TRIDENT-B1 is a trivalent CD137xCD137xTA binding molecule with two CD137-binding sites and one binding site for HER2, a representative TA. TRIDENT-B1 is composed of four polypeptide chains (see Figure 3A; VL1 / VH1 (site A) is identical to VL2 / VH2 (site B) and binds to CD137, and VL3 / VH3 (site C) binds to HER2). TRIDENT-B1 contains the binding domains of CD137 MAB-6 (1.1) and hHER2 MAB-1 (1.3).

[0380] The first polypeptide chain of TRIDENT-B1 is identical to the first polypeptide chain of TRIDENT-A (SEQ ID NO: 127).

[0381] The second polypeptide chain of TRIDENT-B1 is identical to the second polypeptide chain of TRIDENT-A (SEQ ID NO: 128).

[0382] Alternative TRIDENT-B1 first and second polypeptide chains (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains containing cysteine ​​residues.E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39) and K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ TRIDENT-B1 having a first polypeptide chain optionally consisting of: SEQ ID NO:85-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:21-SEQ ID NO:146, and a second polypeptide chain optionally consisting of: SEQ ID NO:85-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40.

[0383] The third polypeptide chain of TRIDENT-B1 is arranged in an N-terminal to C-terminal direction, containing an N-terminus, a VH domain of a monoclonal antibody capable of binding to HER2 (VH HER2 (hHER2 MAB-1 VH1, SEQ ID NO: 80)), a human IgG1 CH1 domain (SEQ ID NO: 3), a human IgG1 hinge region (SEQ ID NO: 7), and "hole-bearing" CH2 and CH3 domains containing L234A / L235A / M252Y / S254T / T256E / H435R substitutions (SEQ ID NO: 149).

[0384] Thus, the third polypeptide chain of TRIDENT-B1 is composed of: SEQ ID NO:80-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149.

[0385] The amino acid sequence of the third polypeptide chain of TRIDENT-B1 is (SEQ ID NO: 153): QVQLVQSGAE VKKPGASVKV SCKASGYTFT NYGMNWVRQA PGQGLEWWMGW INTNIGEPTY TEEFKGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARDD GYGNRVSYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLYITREPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPSREEM TKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLVS KLTVDKSRWQ QGNVFSCSVM HEALHNRYTQ KSLSLSPGK is.

[0386] The fourth polypeptide chain of TRIDENT-B1 contains, in the N-terminal to C-terminal direction, an N-terminal VL domain (VL) of a monoclonal antibody capable of binding to HER2. HER2 )(hHER2 MAB-1 VL3 (SEQ ID NO: 85), human IgG CLκ domain (SEQ ID NO: 1), and C-terminus.

[0387] Thus, the fourth polypeptide chain of TRIDENT-B1 is composed of: SEQ ID NO:85-SEQ ID NO:1.

[0388] The amino acid sequence of the fourth polypeptide chain of TRIDENT-B1 is (SEQ ID NO: 154): DIQMTQSPSS LSASVGDRVT ITCKASQDIS NYLSWFQQKP GKAPKTLIYR ANRLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCLQ HDEFPWTFGQ GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC is.

[0389] 6. TRIDENT‐B2 TRIDENT-B2 is a trivalent CD137xCD137xTA binding molecule with two CD137-binding sites and one binding site for HER2, a representative TA. TRIDENT-B1 is composed of four polypeptide chains (see Figure 3A; VL1 / VH1 (site A) is identical to VL3 / VH3 (site C) and binds to CD137, and VL2 / VH2 (site B) binds to HER2). TRIDENT-B1 contains the binding domains of CD137 MAB-6 (1.1) and hHER2 MAB-1 (1.3).

[0390] The first polypeptide chain of TRIDENT-B2 is identical to the first polypeptide chain of DART-B1 (SEQ ID NO: 143).

[0391] The second polypeptide chain of TRIDENT-B2 is identical to the second polypeptide chain of DART-B1 (SEQ ID NO: 144).

[0392] Alternative TRIDENT-B2 first and second polypeptide chains (e.g., α- and β-coil) may contain heterodimer-promoting (E-coil and K-coil) domains containing cysteine ​​residues. E VAA CE K‐ E VAAL E K‐ E VAAL E K‐ E VAAL E K (SEQ ID NO: 39) and K VAA CK E‐ K VAAL K E‐ K VAAL K E‐ K VAAL K It is also specifically contemplated to employ TRIDENT-B2 variants having the sequences SEQ ID NO:E (SEQ ID NO:40). In such alternative TRIDENT-B2 variants, the first polypeptide chain optionally consists of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:39-SEQ ID NO:21-SEQ ID NO:146, and the second polypeptide chain optionally consists of: SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40.

[0393] The third polypeptide chain of TRIDENT-B2 contains, in an N-terminal to C-terminal direction, an N-terminal VH domain (VH) of a monoclonal antibody capable of binding to CD137. CD137 )(CD1 37 MAB-6 VH1, SEQ ID NO: 46), a human IgG1 CH1 domain (SEQ ID NO: 3), a human IgG1 hinge region (SEQ ID NO: 7), and "hole-bearing" CH2 and CH3 domains containing L234A / L235A / M252Y / S254T / T256E / H435R substitutions (SEQ ID NO: 149).

[0394] Thus, the third polypeptide chain of TRIDENT-B2 is composed of: SEQ ID NO:46-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149.

[0395] The amino acid sequence of the third polypeptide chain of TRIDENT-B2 is (SEQ ID NO: 155): QVQLQESGPG LVKPSETLSL TCTVSGGSIS SYYWSWIRQP PGKGLEWIGR IYTSGSTNYN PSLKSRVTMS VDTSKNQFSL KLSSVTAADT AVYYCARDGW YDEDYNYYGM DVWGQGTMVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLYIT REPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPS REEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHN RYTQKSLSLS PGK is.

[0396] The fourth polypeptide chain of TRIDENT-B2 contains, in an N-terminal to C-terminal direction, an N-terminal VL domain (VL) of a monoclonal antibody capable of binding to CD137. CD137 )(CD1 37 MAB-6 VL1 (SEQ ID NO: 50), human IgG CLκ domain (SEQ ID NO: 1), and C-terminus.

[0397] Thus, the fourth polypeptide chain of TRIDENT-B2 is composed of: SEQ ID NO:50-SEQ ID NO:1.

[0398] The amino acid sequence of the fourth polypeptide chain of TRIDENT-B2 is (SEQ ID NO: 156): EDFAVYYCQQ DYDLPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC is.

[0399] C. Alternative CD137×TA binding molecules In light of the present disclosure, it will be appreciated that additional CD137xTA binding molecules having the general structure of any of the above exemplary molecules and including binding sites for alternative TAs can be constructed by substituting the VL and VH domains of alternative tumor antigen antibodies for the VL and VH domains of anti-PD-L1 or anti-HER2. Similarly, alternative CD137xTA binding molecules incorporating alternative linkers and / or Heterodimer-Promoting Domains and / or antibody constant regions (e.g., CL, CH2-CH3 domains) as provided herein can be similarly constructed.

[0400] D. Control molecule To more meaningfully demonstrate the properties of the CD137xTA binding molecules of the present invention, control Fc-bearing diabodies and control antibodies whose VL and VH domains can be used to generate other control and reference binding molecules are described herein.

[0401] Palivizumab (see, e.g., Protein Data Bank (PDB) Identification No. 2HWZ) is a humanized monoclonal antibody (IgG) directed against an epitope in the A antigenic site of the RSV F protein, and is a suitable control antibody whose VL and VH domains can be used to generate control diabodies and other control binding molecules. Alternative anti-RSV glycoprotein F antibodies include motavizumab (see, e.g., PDB Identification No. 3IXT), and motavizumab, which is a nucleotide sequence derived from the CDR1 of the light chain of the RSV glycoprotein F. Examples of variants of palivizumab include those engineered to remove methylamino acid residues. Variants of palivizumab were used to generate negative control molecules described below.

[0402] The amino acid sequence of the VH domain of the palivizumab variant is (SEQ ID NO: 137) (CDR H Residues are underlined): QVTLRESGPA LVKPTQTLTL TCTFSGFSLS TSGMSVG WIR QPPGKALEWL AD IWWDDKKD YNPSLKS RLT ISKDTSKNQV VLKVTNMDPA DTATYYCAR S MITNWYFDV W GAGTTVTVSS is.

[0403] The amino acid sequence of the VL domain of the variant of Palivizumab is (SEQ ID NO: 138) (CDR L Residues are underlined): DIQMTQSPST LSASVGDRVT ITC RASQSVG YMH WYQQKPG KAPKLLIY DT SKLAS GVPSR FSGSGSGTEF TLTISSLQPD DFATYYC FQG SGYPFT FGGG TKLEIK is.

[0404] Several molecules containing the epitope-binding site of the above-mentioned anti-CD137 antibodies are used herein for comparison purposes, including urelumab (also known as BMS-663513, see U.S. Patent No. 8,137,667) and utomilumab (also known as PF-05082566, see U.S. Patent No. 8,337,850), as well as murine and humanized hCD137 MAB-3 (see WO 2018 / 156740). The complete heavy chains of urelumab (WHO Drug Information, 2011, Recommended INN: List 66, 25(3):334) and utomilumab (WHO Drug Information, 2017, Recommended INN: List 77, 31(1):140-141) are shown. The amino acids of the VH and VL domains of humanized hCD137 MAB-3 (1B.3), used as a comparison herein, are provided in WO 2018 / 156740 (see paragraphs

[0254] and

[0261] ).

[0405] E. Overview of CD137×TA binding molecules and control molecules Table 5 summarizes the attributes of the domains DART‐A to DART‐A9, TRIDENT‐A, and TRIDENT‐A4 to A6.

[0406] [Table 6] JPEG2025186381000016.jpg170155

[0407] Table 6 shows the properties of additional DART and TRIDENT molecules prepared as comparators and negative controls.

[0408] [Table 7]

[0409] III. Manufacturing method The binding molecules of the present invention can be recombinantly produced and expressed using any method known in the art. The molecules can be produced recombinantly by obtaining a nucleic acid encoding the binding molecule and using this nucleic acid to generate a vector useful for recombinant expression of the molecule in a host cell (e.g., a CHO cell). Another method that can be used is to express the molecule in a plant (e.g., tobacco) or in transgenic milk.

[0410] A vector containing a polynucleotide of interest (e.g., a polypeptide of a binding molecule of the invention) A polynucleotide encoding a nucleic acid or vector can be introduced into a host cell by any of a number of suitable means, including: electroporation; particle bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). Techniques for introducing nucleic acids or vectors into host cells are well established in the art, and any suitable technique may be employed.

[0411] Any host cell capable of overexpressing heterologous DNA can be used to express the binding molecule of interest (e.g., antibody, diabody, trivalent binding molecule). Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, NSO, HEK-293, HeLa, and CHO cells. Methods for culturing host cells are known in the art.

[0412] The binding molecules are typically isolated and / or purified from host cells, culture medium, etc. Techniques for the purification of recombinant binding molecules comprising antibody domains (e.g., Fc domains) are known in the art and include, for example, the use of HPLC, FPLC, or affinity chromatography (e.g., with Protein A or Protein G). After purification, the binding molecules of the invention can be formulated into pharmaceutical compositions, optionally with pharmaceutically acceptable excipients or other substances described below.

[0413] IV. Pharmaceutical Compositions Compositions of the invention include bulk drug compositions (e.g., impure or non-sterile compositions) that can be used to manufacture pharmaceutical compositions, and pharmaceutical compositions that can be used to prepare unit dosage forms (i.e., compositions suitable for administration to a subject or patient). These compositions comprise the CD137xTA binding molecules of the invention, or a combination of such agents with a pharmaceutically acceptable carrier. As provided herein, compositions of the invention comprise a prophylactically or therapeutically effective amount of a CD137xTA bispecific Fc-bearing diabody of the invention and a pharmaceutically acceptable carrier.

[0414] The present invention also encompasses pharmaceutical compositions comprising the CD137xTA binding molecules of the present invention, one or more additional molecules effective in stimulating an immune response (e.g., immune checkpoint inhibitors), and / or one or more additional molecules that specifically bind to a tumor antigen (e.g., tumor-specific monoclonal antibodies or diabodies) specific for at least one particular TA as described above, and a pharmaceutically acceptable carrier.

[0415] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal regulatory agency or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle used in administering a therapeutic agent. These pharmaceutical carriers can be sterile liquids. When the pharmaceutical composition is administered intravenously, aqueous carriers such as saline, aqueous dextrose, and glycerol solutions are preferred.

[0416] Generally, the ingredients of the compositions of the invention are supplied separately or mixed together in unit dosage form as a lyophilized powder or water-free concentrate, or in liquid form in an airtight container, such as an ampule or sachet, indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0417] The present invention also provides a pharmaceutical pack comprising one or more containers containing a CD137xTA binding molecule of the present invention, alone or in combination with other agents, such as a pharmaceutically acceptable carrier. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally, such one or more containers can be associated with a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting approval by the agency of the manufacture, use, or sale for human administration.

[0418] A kit can include any of the CD137xTA binding molecules of the present invention. The kit can further include, in one or more containers, one or more other prophylactic and / or therapeutic agents useful for treating cancer; and / or the kit can further include one or more cytotoxic antibodies that bind to one or more tumor antigens (TAs). In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

[0419] V. Method of Administration The compositions of the invention can be provided for the treatment, prevention, and amelioration of one or more symptoms associated with cancer or other diseases or disorders by administering to a subject an effective amount of a molecule of the invention or a pharmaceutical composition comprising a molecule of the invention. In a preferred aspect, the composition is substantially purified (i.e., substantially free of substances that limit the effectiveness of the composition or produce undesirable side effects). In a specific embodiment, the subject is an animal. In another specific embodiment, the subject is a mammal, such as a non-primate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey, such as a cynomolgus monkey, human, etc.). In another embodiment, the subject is a human.

[0420] Methods of administering the molecules of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous); epidural; and mucosal (e.g., intranasal and oral routes). In a specific embodiment, the CD137xTA binding molecules of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous layers (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local. Additionally, pulmonary administration can be employed, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent.

[0421] The present invention also provides that the CD137xTA binding molecules of the invention are packaged in an airtight container, such as an ampoule or sachet, indicating the quantity of the molecule. In one embodiment, the CD137xTA binding molecules are supplied as a dry, sterile, lyophilized powder or water-free concentrate in an airtight container, which can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. Lyophilized CD137xTA binding molecules of the invention should be stored in their original container at 2-8°C, and the molecules should be administered within 12 hours, 6 hours, 5 hours, 3 hours, or 1 hour after reconstitution.

[0422] In an alternative embodiment, the CD137xTA binding molecules of the invention are supplied in liquid form in an airtight container indicating the amount and concentration of the molecule, fusion protein, or conjugated molecule. In a specific embodiment, the CD137xTA binding molecules of the invention are supplied in liquid form in an airtight container and do not require reconstitution.

[0423] The amount of the composition of the invention that will be effective in the treatment, prevention, or amelioration of one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0424] As used herein, an "effective amount" of a pharmaceutical composition is, in one embodiment, an amount sufficient to obtain beneficial or desired results, including, but not limited to, reducing symptoms caused by the disease; attenuating symptoms of an infectious disease (e.g., cancer cell proliferation, tumor presence, tumor metastasis, etc.) thereby improving the quality of life of a person afflicted with the disease; reducing the dosage of other medications required to treat the disease; enhancing the effectiveness of other medications, such as by targeting and / or internalization; slowing the progression of the disease; and / or extending the survival time of an individual.

[0425] Such an effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly reduce the proliferation of the viral presence (or its effects) and reduce and / or slow the progression of a disease (e.g., cancer). In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more additional agents (e.g., chemotherapeutic agents, or other agents considered standard of care for a particular condition), and can be considered to be given in an effective amount when a single agent can achieve or achieves a desired result in combination with one or more other agents. While individual needs will vary, determining optimal ranges of effective amounts of each component is within the ability of one of ordinary skill in the art.

[0426] For CD137xTA binding molecules encompassed by the present invention, the dosage administered to a patient may be determined based on the recipient subject's weight (kg) or may be based on a fixed dose.

[0427] The dosage and frequency of administration of the CD137xTA binding molecules of the invention can be reduced or modified by enhancing uptake and tissue penetration of the CD137xTA binding molecules by modifications such as lipidation.

[0428] The dosage of the CD137xTA binding molecules of the invention administered to a patient may be calculated for use as a single agent therapy. Alternatively, the CD137xTA binding molecules are used in combination with other therapeutic compositions, and therefore the dosage administered to a patient is lower than when the molecules are used as a single agent therapy.

[0429] Treatment of a subject with a therapeutically or prophylactically effective amount of a CD137xTA binding molecule of the invention can include a single treatment or a series of multiple treatments. In some examples, a subject is treated with a molecule of the invention once a week, once every two weeks (i.e., once per week), or once every three weeks for approximately 1 to 52 weeks. The pharmaceutical compositions of the invention can be administered once daily, twice daily, or three times daily. Alternatively, the pharmaceutical compositions can be administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once a year. It will also be understood that the effective dosage of the molecules used for treatment may increase or decrease over the course of a particular treatment.

[0430] VI. Uses of the Compositions of the Invention The CD137xTA binding molecules of the present invention have the ability to bind to T cells (APCs) (e.g., by binding to CD137 expressed on the surface of said T cells) and to bind to TA-expressing tumor cells (e.g., by binding to TA expressed on the surface of said tumor cells). Thus, the CD137xTA binding molecules of the present invention have the ability to co-localize T cells to TA-expressing tumor cells, and thus to bind to T cells associated with or expressing TA. The molecules can be used to treat any disease or condition characterized by the TA. Thus, pharmaceutical compositions comprising such molecules can be employed in the diagnosis or treatment of cancers that express TA, including, but not limited to, bladder cancer, bone cancer, brain and spinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), stomach cancer, testicular cancer, thymic cancer, and uterine cancer, particularly cancers that highly express TA.

[0431] The CD137xTA binding molecules of the present invention can further be used in the manufacture of a medicament for the treatment of the above-mentioned conditions.

[0432] In certain embodiments, the CD137xTA binding molecules of the invention are used in combination with one or more other prophylactic and / or therapeutic agents useful in the treatment of cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biotherapeutic or hormonal therapeutic agent. In other embodiments, the biotherapeutic agent is a cytotoxic antibody-based molecule, including, but not limited to, an antibody, an antigen-binding fragment of an antibody (e.g., scFv, Fab, F(ab)2, TandAb, etc.), or a multispecific binding molecule (e.g., diabody, bispecific antibody, trivalent binding molecule, etc.) that binds to one or more tumor antigens (TA).

[0433] The CD137xTA binding molecules of the present invention can enhance the activity of tumor targeting agents. Therefore, the CD137xTA binding molecules of the present invention can be further used in combination with other tumor targeting agents, including, but not limited to, antibodies, antigen-binding fragments of antibodies (e.g., scFv, Fab, F(ab)2, TandAb, etc.), and multispecific binding molecules (e.g., diabodies, bispecific antibodies, trivalent binding molecules, etc.) capable of binding to the desired TA. It is specifically contemplated that the tumor targeting agent may bind to the same or a different TA as the CD137xTA binding molecule used in such combination. In certain embodiments, the tumor targeting agent is a multispecific molecule that binds to a TA and an epitope expressed on T cells, including, for example, CD3 and / or CD8, to mediate T cell targeted killing. Exemplary tumor targeting agents include, but are not limited to, molecules that bind to a TA and CD3 ("TAxCD3"). Representative TAxCD3 binding molecules (e.g., bispecific antibodies, DART® molecules, BiTe® molecules, TandAbs, etc., and trivalent molecules) that can be used in such combinations, as well as methods for making them, are known in the art (e.g., WO 2013 / 026835; WO 2013 / 158856; WO 2014 / 047231; WO 2014 / 110601). No.; WO 2014 / 131711; WO 2015 / 026894; WO 2015 / 026892; WO 2015 / 184203; WO 2015 / 184207; WO 2016 / 036937; WO 2016 / 182751; WO 2017091656; WO 2017 / 142928; WO 2017 / 118675).

[0434] The CD137xTA binding molecules of the present invention can be used in combination with tumor targeting agents (e.g., TAxCD3 binding molecules) to achieve upregulation of the inhibitory immune regulator, Programmed Death-1 (PD-1), also known as CD279. PD-1 mediates its inhibition of the immune system by binding to PD-L1 and PD-L2 (also known as B7-H1 and B7-DC) (Flies, DB et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity," J. Immunother. 30(3):251-260; U.S. Patent No. 6,803,192; U.S. Patent No. 7,794,710). Therefore, the addition of an agent that blocks the inhibitory activity of PD-1 (a "PD-1 / PD-L1 checkpoint inhibitor") down-regulates PD-1 expression and inhibits the TAxCD3 bond. The activity of CD137xTA and tumor targeting agents, such as combined molecules, can be further enhanced. The present invention particularly encompasses PD-1 / PD-L1 checkpoint inhibitors that contain an epitope binding site of an antibody that binds to PD-1.

[0435] Thus, the CD137×TA binding molecules of the present invention can be further used in combination with other tumor targeting agents, and even in combination with PD-1 / PD-L1 checkpoint inhibitors, including, but not limited to, antibodies capable of binding to PD-1 and / or PD-L1, antigen-binding fragments of antibodies (e.g., scFv, Fab, F(ab)2, TandAb, etc.), and multispecific binding molecules (e.g., diabodies, bispecific antibodies, trivalent binding molecules, etc.). Exemplary PD-1 / PD-L1 checkpoint inhibitors that can be used in such combinations, and methods for producing them, are known in the art. PD-1 binding molecules that can be used in the methods of the invention include: nivolumab (CAS Registry Number 946414-94-4, also known as 5C4, BMS-936558, ONO-4538, MDX-1106, marketed by Bristol-Myers Squibb as OPDIVO®); pembrolizumab (formerly known as lambrolizumab, CAS Registry Number 1374853-91-4, also known as MK-3475, SCH-900475, marketed by Merck as KEYTRUDA®); cemiplimab (CAS Registry Number 1801342-60-8, also known as REGN-2810, SAR-439684, marketed as LIBTAYO®). The complete heavy and light chain amino acid sequences of nivolumab (WHO Drug Information, 2013, Recommended INN: List 69, 27(1):68-69), pembrolizumab (WHO Drug Information, 2014, Recommended INN: List 75, 28(3):407), and cemiplimab (WHO Drug Information 2018, Proposed INN: List 119) are known in the art. Additional anti-PD-1 antibodies (e.g., hPD-1 mAb 7(1.2)) with unique binding characteristics that can be used in the methods and compositions of the invention have recently been identified (see WO 2017 / 019846).

[0436] When the above-described combinations are employed, one or more of the above molecules may be administered to a subject "concurrently" (e.g., a CD137×TA binding molecule may be administered at the same time as a TA×CD3 binding molecule and / or a PD-1 / PD-L1 checkpoint inhibitor) and / or one or more of the above molecules may be administered "sequentially" (e.g., a CD137×TA binding molecule may be administered followed by a TA×CD3 binding molecule and / or a PD-1 / PD-L1 checkpoint inhibitor, or vice versa).

[0437] VII. EMBODIMENTS OF THE INVENTION Having now generally described the present invention, the same will be more readily understood by reference to the following numbered embodiments ("E"), which are provided by way of example and are not intended to limit the invention unless otherwise specified.

[0438] E1. A CD137 binding molecule comprising a first binding site that immunospecifically binds to an epitope of CD137, said first binding site comprising a CDR L 1. CDR L 2 and CDR L 3 a first light chain variable domain comprising: H 1. CDR H 2 and CDR H The first heavy chain contains 3 a variable domain; (A) CDRs of the first light chain variable domain L 1. CDR L 2 and CDR L 3 is CD1 37 light chain CDRs of MAB-6 VL1 (SEQ ID NO: 50); (B) CDRs of the first heavy chain variable domain H 1. CDR H 2 and CDR H 3 is CD1 37 A CD137 binding molecule which is the heavy chain CDRs of MAB-6 VH1 (SEQ ID NO: 46).

[0439] E2. The first heavy chain variable domain is selected from the group consisting of hCD137 MAB-6 VH1 (SEQ ID NO: 1). 46). A CD137-binding molecule as described in E1, comprising the amino acid sequence:

[0440] E3. The first light chain variable domain comprises: (A) hCD137 MAB-6 VLx (SEQ ID NO: 54); (B) hCD137 MAB-6 VL1 (SEQ ID NO: 50); (C) hCD137 MAB-6 VL2 (SEQ ID NO: 55); or (D) hCD137 MAB-6 VL3 (SEQ ID NO: 56) The CD137-binding molecule of E1 or E2, comprising the amino acid sequence:

[0441] E4.(A) the first heavy chain variable domain comprises the amino acid sequence of: hCD137 MAB-6 VH1 (SEQ ID NO: 46); (B) A CD137 binding molecule described in any one of E1 to E3, wherein the first light chain variable domain comprises the amino acid sequence of: hCD137 MAB-6 VL1 (SEQ ID NO: 50).

[0442] E5. (A) The first heavy chain variable domain is: hCD137 MAB-6 VH1 (sequence containing the amino acid sequence of sequence number 46); (B) A CD137 binding molecule described in any one of E1 to E3, wherein the first light chain variable domain comprises the amino acid sequence of: hCD137 MAB-6 VL3 (SEQ ID NO: 56).

[0443] E6. The molecule is a bispecific molecule comprising a second binding site that immunospecifically binds to a tumor antigen (TA), and the second binding site comprises a CDR L 1. CDR L 2 and CDR L 3 and a second light chain variable domain comprising: H 1. CDR H 2 and CDR HThe second heavy containing 3 The CD137-binding molecule according to any one of E1 to E5, comprising a chain variable domain.

[0444] E7. The CD137-binding molecule of E6, wherein said TA is selected from the antigens presented in Tables 1-2.

[0445] E8. The TA is PD-L1 and: (A) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3, hPD - L1 is the light chain CDRs of MAB-2 VLx (SEQ ID NO: 63); (B) CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, hPD -L1 A CD137 binding molecule as described in E6, which is the heavy chain CDRs of MAB-2 VHx (SEQ ID NO: 59).

[0446] E9.(A)(1) The CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is the light chain CDRs of hPD-L1 MAB-2 VL1 (SEQ ID NO: 63); or (2) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of PD-L1 MAB-2 VLx (SEQ ID NO: 58); or (3) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of PD-L1 MAB-2 VL2 (SEQ ID NO: 72); (B)(1) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDRH 3 is, the heavy chain CDRs of hPD-L1 MAB-2 VH1 (SEQ ID NO: 59); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VHx (SEQ ID NO: 57); (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 67); (4) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH3 (SEQ ID NO: 68); (5) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 69); (6) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 70); or (7) CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h The CD137 binding molecule described in E8, which is the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 71).

[0447] E10. The second heavy chain variable domain comprises: (A) hPD-L1 MAB-2 VHx (SEQ ID NO: 59); (B) hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (C) hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (D) hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (E) hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (F) hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (G) hPD-L1 MAB-2 VH6 (SEQ ID NO: 71) 2. The CD137-binding molecule of claim E9, comprising the amino acid sequence:

[0448] E11. The second light chain variable domain comprises: (A) hPD-L1 MAB-2 VLx (SEQ ID NO: 63); (B) hPD-L1 MAB-2 VL1 (SEQ ID NO: 58); or (B) hPD-L1 MAB-2 VL2 (SEQ ID NO: 72) The CD137-binding molecule of E9 or E10, comprising the amino acid sequence:

[0449] E12. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VHx (SEQ ID NO: 59); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VLx (SEQ ID NO: 63).

[0450] E13. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VLx (SEQ ID NO: 63).

[0451] E14. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VLx (SEQ ID NO: 63).

[0452] E15. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VLx (SEQ ID NO: 63).

[0453] E16. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (B) the second light chain variable domain is hPD-L1 MAB-2 VLx (SEQ ID NO: 63). The CD137-binding molecule of E10 or E11, comprising the amino acid sequence of:

[0454] E17. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VLx (SEQ ID NO: 63).

[0455] E18. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH6 (SEQ ID NO: 71); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VLx (SEQ ID NO: 63).

[0456] E19. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VL1 (SEQ ID NO: 58).

[0457] E20. (A) the second heavy chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (B) The CD137-binding molecule of E10 or E11, wherein the second light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VL2 (SEQ ID NO: 72).

[0458] E21. The above TA is 5T4 and: (A)(1) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of 5T4 MAB-1 VL (SEQ ID NO: 93); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, 5 the heavy chain CDRs of T4 MAB-1 VH (SEQ ID NO: 92); or (B)(1) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of 5T4 MAB-2 VL (SEQ ID NO: 95); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, 5 A CD137 binding molecule as described in E6, which is the heavy chain CDRs of T4 MAB-2 VH (SEQ ID NO: 96).

[0459] E22. The CD137xTA binding molecule of E21, wherein said second heavy chain variable domain comprises the amino acid sequence of 5T4 MAB-1 VH (SEQ ID NO: 92).

[0460] E23. The CD137xTA binding molecule of E21 or E22, wherein said second light chain variable domain comprises the amino acid sequence of 5T4 MAB-1 VL (SEQ ID NO: 93).

[0461] E24. The above TA is HER2 and: (A) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3, but hHE R2-MAB-1 VLx (SEQ ID NO: 79) light chain CDRs; (B) CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3, but hHE A CD137 binding molecule described in E6, which is the heavy chain CDRs of R2-MAB-1 VHx (SEQ ID NO: 78).

[0462] E25.(A)(1) CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is the light chain CDR of hHER2-MAB-1 VL1 (SEQ ID NO: 83); (2) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of HER2-MAB-1 VL2 (SEQ ID NO: 84); or (3) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of HER2-MAB-1 VL3 (SEQ ID NO: 85); (B)(1) the CDR of the second heavy chain variable domain H 1. CDRH 2 and CDR H 3 is, the heavy chain CDRs of hHER2-MAB-1 VH1 (SEQ ID NO: 80); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of HER2-MAB-1 VH2 (SEQ ID NO: 81); or (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h The CD137 binding molecule of E24, which is the heavy chain CDRs of HER2-MAB-1 VH3 (sequence number 82).

[0463] E26. The second heavy chain variable domain comprises: (A) hHER2-MAB-1 VHx (SEQ ID NO: 78); (B) hHER2-MAB-1 VH1 (SEQ ID NO: 80); (C) hHER2-MAB-1 VH2 (SEQ ID NO: 81); or (D) hHER2-MAB-1 VH3 (SEQ ID NO: 82) The CD137-binding molecule of E25, comprising the amino acid sequence:

[0464] E27. The second light chain variable domain comprises: (A) hHER2-MAB-1 VLx (SEQ ID NO: 79); (B) hHER2-MAB-1 VL1 (SEQ ID NO: 83); (C) hHER2-MAB-1 VL2 (SEQ ID NO: 84); or (D) hHER2-MAB-1 VL3 (SEQ ID NO: 85) The CD137-binding molecule of E25 or E26, comprising the amino acid sequence:

[0465] E28. (A)(1) the second heavy chain variable domain comprises the amino acid sequence of hHER2-MAB-1 VHx (SEQ ID NO: 78); (2) the second light chain variable domain comprises the amino acid sequence of hHER2-MAB-1 VLx (SEQ ID NO: 79); or (B)(1) the second heavy chain variable domain comprises the amino acid sequence of hHER2-MAB-1 VH1 (SEQ ID NO: 80); (2) The CD137-binding molecule described in any one of E24 to E26, wherein the second light chain variable domain comprises the amino acid sequence of hHER2-MAB-1 VL3 (SEQ ID NO: 85).

[0466] E29. The CD137 binding molecule of any one of E1 to E29, which is an antibody, a bispecific antibody, a bispecific bivalent Fc-bearing diabody, or a bispecific tetravalent Fc-bearing diabody, or a bispecific trivalent molecule.

[0467] E30. The CD137 binding molecule of any one of E1-E29, wherein said molecule is bispecific and bivalent and comprises a first, second, and third polypeptide chain, wherein said polypeptide chains form a covalent complex.

[0468] E31. Any of E1-E29, wherein the molecule is bispecific and tetravalent and comprises a first, second, third, and fourth polypeptide chain, wherein the polypeptide chains form a covalent complex. A CD137-binding molecule according to any one of claims 1 to 4.

[0469] E32. The CD137 binding molecule of any one of E1-E29, wherein said molecule is bispecific and trivalent and comprises a first, second, third, and fourth polypeptide chain, wherein said polypeptide chains form a covalent complex.

[0470] E33. The TA is PD-L1, and: (A) the first polypeptide chain and the third polypeptide chain are, in an N-terminal to C-terminal direction: (i) SEQ ID NO:63 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:30 - SEQ ID NO:43; (ii) SEQ ID NO:63 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:30 - SEQ ID NO:43; (iii) SEQ ID NO:58 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:30 - SEQ ID NO:43; (iv) SEQ ID NO:58 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:30 - SEQ ID NO:43; (v) SEQ ID NO:72 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:30 - SEQ ID NO:43; or (vi) SEQ ID NO: 72 - SEQ ID NO: 16 - SEQ ID NO: 46 - SEQ ID NO: 18 - SEQ ID NO: 37 - SEQ ID NO: 30 - SEQ ID NO: 43 Including, (B) the second polypeptide chain and the fourth polypeptide chain are arranged, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:59-SEQ ID NO:18-SEQ ID NO:40; (ii) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:59-SEQ ID NO:18-SEQ ID NO:38; (iii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:57-SEQ ID NO:18-SEQ ID NO:40; (iv) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:57-SEQ ID NO:18-SEQ ID NO:38; (v) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:67-SEQ ID NO:18-SEQ ID NO:40; (vi) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:67-SEQ ID NO:18-SEQ ID NO:38; (vii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:40; (viii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:38; (ix) SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:40; (x) SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:38; (xi) SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:40; (xii) SEQ ID NO:56-SEQ ID NO:16-SEQ ID NO:68-SEQ ID NO:18-SEQ ID NO:38; (xiii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:69-SEQ ID NO:18-SEQ ID NO:40; (xiv) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:69-SEQ ID NO:18-SEQ ID NO:38; (xv) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:70 - SEQ ID NO:18 - SEQ ID NO:40 ; (xvi) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:70-SEQ ID NO:18-SEQ ID NO:38; (xvii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:71-SEQ ID NO:18-SEQ ID NO:40; (xviii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:71-SEQ ID NO:18-SEQ ID NO:38; (xix) SEQ ID NO:56 - SEQ ID NO:16 - SEQ ID NO:69 - SEQ ID NO:18 - SEQ ID NO:40; or (xx) SEQ ID NO: 56 - SEQ ID NO: 16 - SEQ ID NO: 69 - SEQ ID NO: 18 - SEQ ID NO: 38 The CD137-binding molecule of E31, comprising:

[0471] E34. The above TA is PD-L1 and: (A) the first polypeptide chain and the third polypeptide chain comprise the amino acid sequence of SEQ ID NO:116, SEQ ID NO:118, or SEQ ID NO:120; (B) A CD137 binding molecule described in E31 or E33, wherein the second polypeptide chain and the fourth polypeptide chain comprise the amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO: 139.

[0472] E35. The molecule above is: (A) SEQ ID NO: 116 and SEQ ID NO: 117; (B) SEQ ID NO: 118 and SEQ ID NO: 119; (C) SEQ ID NO: 120 and SEQ ID NO: 119; (D) SEQ ID NO: 118 and SEQ ID NO: 121; (E) SEQ ID NO: 120 and SEQ ID NO: 121; (F) SEQ ID NO: 120 and SEQ ID NO: 122; (G) SEQ ID NO: 120 and SEQ ID NO: 123; (H) SEQ ID NO: 120 and SEQ ID NO: 124; (I) SEQ ID NO: 120 and SEQ ID NO: 125; (J) SEQ ID NO: 120 and SEQ ID NO: 126; or (K) SEQ ID NO: 120 and SEQ ID NO: 139 The CD137-binding molecule according to E34, comprising:

[0473] E36. The above TA is PD-L1 and: (A) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; (ii) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146; (iii) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; (iv) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146; (v) SEQ ID NO:55 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; (vi) SEQ ID NO:55 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146; (vi) SEQ ID NO:56 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; or (vii) SEQ ID NO:56 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146 Including, (B) the second polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38; (ii) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (iii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38; (iv) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (v) SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38; (vi) SEQ ID NO:55-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (vi) SEQ ID NO:56 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:38; or (vii) SEQ ID NO:56 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:40 Including, (C) the third polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:59-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149; (ii) SEQ ID NO:57-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149; (iii) SEQ ID NO:67-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149; (iv) SEQ ID NO:68-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149; (v) SEQ ID NO:69-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149; (vi) SEQ ID NO:70 - SEQ ID NO:3 - SEQ ID NO:7 - SEQ ID NO:149; or (vii) SEQ ID NO: 71 - SEQ ID NO: 3 - SEQ ID NO: 7 - SEQ ID NO: 149 Including, (D) the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO: 63-SEQ ID NO: 1; or (ii) SEQ ID NO: 72 to SEQ ID NO: 1 The CD137-binding molecule of E32, comprising:

[0474] E37. The above TA is PD-L1 and: (A) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:59 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; or (ii) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:59 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146 Including, (B) the second polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:63 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:38; or (ii) SEQ ID NO: 63-SEQ ID NO: 16-SEQ ID NO: 46-SEQ ID NO: 18-SEQ ID NO: 40; (C) the third polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO: 46 - SEQ ID NO: 3 - SEQ ID NO: 7 - SEQ ID NO: 149 Including, (D) the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO: 54-SEQ ID NO: 1; or (ii) SEQ ID NO: 50 - SEQ ID NO: 1 The CD137-binding molecule of E32, comprising:

[0475] E38. The above TA is PD-L1 and: (A) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127, SEQ ID NO: 133, or SEQ ID NO: 135; (B) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128, SEQ ID NO: 134, or SEQ ID NO: 136; (C) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, or SEQ ID NO: 131; (D) A CD137-binding molecule described in E32 or E36, wherein the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 132.

[0476] E39. The molecule above is: (A) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130; (B) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 131, and SEQ ID NO: 132; (C) SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 131, and SEQ ID NO: 132; or (D) SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 131, and SEQ ID NO: 132 The CD137-binding molecule according to E38, comprising:

[0477] E40. The TA is HER2 and: (A) the first polypeptide chain and the third polypeptide chain are, in an N-terminal to C-terminal direction: (i) SEQ ID NO:79 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:30 - SEQ ID NO:43; (ii) SEQ ID NO:79 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:30 - SEQ ID NO:43; (iii) SEQ ID NO:85 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:30 - SEQ ID NO:43; or (iv) SEQ ID NO:85 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:30 - SEQ ID NO:43 Including, (B) the second polypeptide chain and the fourth polypeptide chain are arranged, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:78-SEQ ID NO:18-SEQ ID NO:40; (ii) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:78-SEQ ID NO:18-SEQ ID NO:38; (iii) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:80-SEQ ID NO:18-SEQ ID NO:40; or (iv) A CD137-binding molecule described in E31, comprising SEQ ID NO: 50-SEQ ID NO: 16-SEQ ID NO: 80-SEQ ID NO: 18-SEQ ID NO: 38.

[0478] E41. The TA is HER2 and: (A) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; (ii) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146; (iii) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; or (iv) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146 Including, (B) the second polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38; (ii) SEQ ID NO:54-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (iii) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:38; or (iv) SEQ ID NO:50-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (C) the third polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:78-SEQ ID NO:3-SEQ ID NO:7-SEQ ID NO:149; or (ii) SEQ ID NO: 80 - SEQ ID NO: 3 - SEQ ID NO: 7 - SEQ ID NO: 149 Including, (D) the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO: 79-SEQ ID NO: 1; or (ii) SEQ ID NO: 85 - SEQ ID NO: 1 The CD137-binding molecule of E32, comprising:

[0479] E42. The TA is HER2 and: (A) the first polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:78 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; (ii) SEQ ID NO:54 - SEQ ID NO:16 - SEQ ID NO:78 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146; (iii) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:80 - SEQ ID NO:18 - SEQ ID NO:37 - SEQ ID NO:21 - SEQ ID NO:146; or (iv) SEQ ID NO:50 - SEQ ID NO:16 - SEQ ID NO:46 - SEQ ID NO:18 - SEQ ID NO:39 - SEQ ID NO:21 - SEQ ID NO:146 Including, (B) the second polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO:79-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38; (ii) SEQ ID NO:79-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (iii) SEQ ID NO:85-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:38; or (iv) SEQ ID NO:85-SEQ ID NO:16-SEQ ID NO:46-SEQ ID NO:18-SEQ ID NO:40; (C) the third polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO: 46 - SEQ ID NO: 3 - SEQ ID NO: 7 - SEQ ID NO: 149 Including, (D) the fourth polypeptide chain comprises, in an N-terminal to C-terminal direction: (i) SEQ ID NO: 54-SEQ ID NO: 1; or (ii) SEQ ID NO: 50 - SEQ ID NO: 1 The CD137-binding molecule of E32, comprising:

[0480] E43. The molecule above is: (A) SEQ ID NO: 151 and SEQ ID NO: 152; (B) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 153, and SEQ ID NO: 154; or (C) SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 155, and SEQ ID NO: 165 The CD137-binding molecule according to any one of E40 to E42, comprising:

[0481] E44. A pharmaceutical composition comprising the CD137-binding molecule of any one of E1 to E43 and a physiologically acceptable carrier.

[0482] E45. Use of a CD137 binding molecule according to any one of E6 to E43 or a pharmaceutical composition according to E44 in the treatment of a disease or condition associated with or characterized by expression of said TA.

[0483] E46.CDR L 1. CDR L 2 and CDR L a light chain variable domain comprising CDRs H 1. CDR H 2 and CDR H and a heavy chain variable domain comprising: (A) CDRs of the light chain variable domain L 1. CDR L 2 and CDR L 3, hPD-L 1 the light chain CDRs of MAB-2 VL2 (SEQ ID NO: 72); (B)(1) CDRs of the heavy chain variable domain H 1. CDR H 2 and CDR H 3, hPD -L1 are the heavy chain CDRs of MAB-2 VH2 (SEQ ID NO: 67); (2) CDR of the heavy chain variable domain H 1. CDR H 2 and CDR H 3, hPD‐ L1 is the heavy chain CDR of MAB-2 VH3 (SEQ ID NO: 68); (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH4 (SEQ ID NO: 69); (4) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of PD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (5) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h A PD-L1 binding molecule which is the heavy chain CDRs of PD-L1 MAB-2 VH6 (SEQ ID NO: 71).

[0484] E47. The heavy chain variable domain: (A) hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (B) hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (C) hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (D) hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (E) hPD-L1 MAB-2 VH6 (SEQ ID NO: 71) PD-L1-binding molecule of E46, comprising the amino acid sequence:

[0485] E48. The PD-L1-binding molecule of E46 or E47, wherein said light chain variable domain comprises the amino acid sequence of hPD-L1 MAB-2 VL2 (SEQ ID NO: 72).

[0486] E49. The PD-L1-binding molecule of any one of E46 to E48, wherein said molecule is an antibody or antigen-binding fragment thereof.

[0487] E50. The PD-L1-binding molecule of any one of E46 to E48, wherein the molecule is a multispecific binding molecule.

[0488] E51. The PD-L1-binding molecule of E50, wherein said molecule is a bispecific diabody, bispecific antibody, or trivalent binding molecule.

[0489] E52. A pharmaceutical composition comprising the PD-L1-binding molecule of any one of E46 to E51 and a physiologically acceptable carrier.

[0490] E53. Use of a PD-L1-binding molecule according to any one of E46 to E51 or a pharmaceutical composition according to E52 in the treatment of a disease or condition associated with a suppressed immune system or characterized by expression of PD-L1.

[0491] E54. The use according to E53, wherein said disease or condition associated with a suppressed immune system or characterized by expression of PD-L1 is cancer.

[0492] E55. The use according to E45 or E54, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, cerebrospinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), stomach cancer, testicular cancer, thymic cancer, and uterine cancer.

[0493] E56. A method of enhancing the activity of a tumor-targeting agent, comprising administering the tumor-targeting agent in combination with a CD137-binding molecule described in any one of E1-E43, a PD-L1-binding molecule described in any one of E46-E51, or a pharmaceutical composition described in E44 or E52.

[0494] E57. A method of treating a disease or condition associated with a suppressed immune system or characterized by the development of TA, comprising the step of administering to a subject in need thereof a CD137-binding molecule described in any one of E1-E43, a PD-L1-binding molecule described in any one of E46-E53, or a pharmaceutical composition described in E44 or E53.

[0495] E58. The method of E57, wherein said condition associated with a suppressed immune system or characterized by the expression of TA is cancer.

[0496] E59. The method of E57 or E58, further comprising administering a tumor targeting agent.

[0497] E60. The method of E56 or E59, wherein said tumor targeting agent is an antibody, an epitope-binding fragment of an antibody, or an agent that mediates T cell targeted killing of a target cell.

[0498] E61. The method of any one of E57 to E60, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, cerebrospinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), stomach cancer, testicular cancer, thymic cancer, and uterine cancer.

[0499] E62. A nucleic acid encoding a CD137-binding molecule according to any one of E1 to E43 or a PD-L1-binding molecule according to any one of E46 to E51.

[0500] E63. An expression vector comprising a nucleic acid according to E62.

[0501] E64. A cell comprising a nucleic acid according to E62 or an expression vector according to E63.

[0502] E65. The cell of E64, wherein said cell is a mammalian cell. [Example]

[0503] Having now generally described the present invention, the same will be more readily understood by reference to the following examples, which illustrate various methods relating to the compositions in the diagnostic or therapeutic methods of the present invention. These examples are intended to illustrate, but not to limit, the present invention.

[0504] Example 1 method The ability of test articles (e.g., antibodies, diabodies, or trivalent molecules) to mediate target-dependent signaling of the NF-κB pathway was assessed using a CD137-expressing reporter cell line (Jurkat-NF-κB-Luc) in a CD137 reporter assay performed essentially as follows: For bispecific molecules, target cells (type and number indicated in the figures and below) in 100 μL of assay medium (RPMI-1640, 10% FBS) were plated into sterile assay microplates and incubated overnight at 37°C. On day 2, CD137-overexpressing Jurkat-NF-κB-Luc reporter cells (4 × 10 in 50 μL of assay medium) were plated into sterile assay microplates and incubated overnight at 37°C. 4 ~7.5×10 4 Cells) and 50 μL of serially diluted test article were added in rapid succession to each well containing target cells and to control wells without target cells. Plates were incubated at 37°C for 4-5 hours. BioGlo substrate (Promega) was then added to each well (50 μL), the plates were incubated at room temperature ("RT") for an additional 5-10 minutes, and signaling was measured by detecting luminescence using, for example, a Perkin Elmer Envision device with a readout in relative light units (RLU). For monospecific anti-CD137 antibodies, target cells were not used; instead, a 4-fold excess of goat anti-mouse or goat anti-human antibody was added to crosslink the antibody.

[0505] An ELISA assay to evaluate test articles for CD137 binding was performed essentially as follows: flat-bottom maxisorb 96-well plates were filled with soluble human or cynomolgus CD137 (His tag (shCD137)) at 0.5 or 1 μg / mL, respectively. Plates were coated with the extracellular domain of human or cynomolgus monkey CD137 fused to either His or scyCD137 His, or to a human Fc region (shCD137 hFc or scyCD137 hFc). Plates were washed, blocked with PBS buffer containing 0.5% bovine serum albumin and 0.1% Tween 20, and incubated with test articles (e.g., cell supernatants or purified mAbs). For hybridoma supernatants, anti-CD137 antibodies were used at 1.0 μg / mL and six 3-fold serial dilutions. The amount of test article binding to immobilized CD137 (human or cynomolgus monkey) was assessed using a goat anti-mouse IgG-HRP secondary antibody. All samples were analyzed on a plate reader (Victor 2 Wallac, Perkin Elmer), and EC50 values ​​were calculated from dose-response curves by nonlinear regression analysis.

[0506] T cell cytokine release assays (using suboptimally stimulated primary T cells in the absence of target cells) were performed essentially as follows: 50 μL of serially diluted test article (antibody (+ / - cross-linked with anti-human Fc(Fab)'2)) and 2 × 10 beads 6 50 μL of pre-washed Dynabeads αCD3 (REF 11151D; Invitrogen by Thermo Fisher Scientific, or similar) at 10 6 100 μL / well of cells / mL (Dynabeads Untouched) Human pan T cells (purified from donor PBMCs using a Human T Cell Kit (Invitrogen Cat# 11344D) or similar, according to the manufacturer's protocol) were added to each well of the assay plate. The final volume of each well on the plate was 200 μL. For control wells without test article or αCD3 beads, assay medium was added to a total volume of 200 μL, and the plate was incubated in a tissue culture incubator for 72 hours. The supernatant from each well was then removed. The cells were collected, and the released cytokines, IL-2, IL-10, TNF-α, and IFN-γ, were assayed using Cytokine ELISA Kits (e.g., R&D System Human IL-2 DuoSet ELISA (Cat: DY202), Human IFN-gamma DuoSet ELISA (Cat: DY285), and Human IFN-γ DuoSet ELISA (Cat: DY285)). TNF-alpha was measured using DuoSet ELISA (Cat: DY210), or similar commercially available reagents, according to the manufacturer's instructions. Microsoft Excel and SoftMax Pro were used for data analysis to extrapolate cytokine levels, which were plotted using Prism.

[0507] FACS analysis to evaluate test articles for binding to cell surface CD137 was performed essentially as follows: 100 μL of CD137-expressing CHO cells (CHO / CD137) (1.0 × 10 5 ~1×10 6 100 μL of serially diluted test article or control (1:1000 cells / well) was added to each well of one or more microtiter assay plates, mixed, and incubated at RT for approximately 30 minutes. Cells were washed with FACS buffer, and then secondary antibody (goat anti-human APC, PE, or FITC) was added to each well (1:1000), after which the components were mixed and the wells were incubated at RT for approximately 30 minutes. Cells were washed and resuspended in 250 μL of FACS buffer and analyzed for cellular sequence events by flow cytometry (BD LSR Fortessa or FACSCanto II). Data analysis was performed by FloJo v10.

[0508] Competition between anti-CD137 antibodies and CD137 ligands for binding to CD137 was determined using a real-time, label-free biolayer interferometry assay on an Octet biosensor (Pall ForteBio) essentially as follows: anti-CD137 antibodies were immobilized on an anti-human Fc biosensor and then allowed to associate with the extracellular domain of human CD137 fused to a mouse Fc domain (shCD137 mFc; 10 μg / mL) for 30 seconds. The sensor was then immersed in recombinant human CD137 ligand (R&D Systems; 5 μg / mL) for 30 seconds to monitor whether the ligand bound to the preformed CD137 / antibody complex or blocked its binding.

[0509] The binding kinetics of anti-CD137 antibodies (with human Fc regions) were investigated using BIACORE™ SPR analysis. Anti-CD137 antibodies were captured on a Fab'2 goat anti-human Fc surface. Association and dissociation of shCD137 His or scyCD137 His (12.5 nM, 50 nM, 200 nM) were monitored, and the association and dissociation rate constants were calculated and the KD determined by fitting the sensograms using a 1:1 binding model.

[0510] FACS analysis to evaluate test articles for binding to cell surface PD-L1 was performed essentially as follows: 100 μL of PD-L1-expressing CHO cells (CHO / PD-L1) (1.0 × 10 5 ~1.0×10 6 Cells / well) and 100 μL of serially diluted test article were added to each well of one or more microtiter assay plates, mixed, and incubated at RT for approximately 30 minutes. Cells were washed with FACS buffer, and then secondary antibody (goat anti-human FITC, PE, or APC) was added to each well, after which the components were mixed and the wells were incubated at RT for approximately 30 minutes. Cells were washed and resuspended in 250 μL of FACS buffer and analyzed for cellular sequence events by flow cytometry (BD LSR Fortessa or FACSCanto II). Data analysis was performed by FloJo v10.

[0511] The ability of test articles to antagonize the PD-1 / PD-L1 axis (i.e., block PD-1 / PD-L1 interaction and prevent downregulation of T cell responses) was assessed in a Jurkat-luc-NFAT / CHO / PD-L1 luciferase PD-L1 reporter assay performed essentially as follows: CHO / PD-L1 cells were cultured in 100 μL of culture medium. Plates were seeded at 40,000 cells / well in (DMEM / F12 + 10% FBS + 200 μg / mL hygromycin B + 250 μg / mL G418) and incubated overnight. The following day, the medium was removed, and NFAT-luc2 / PD-1 Jurkat cells (Promega) at 125,000 cells / well in 50 μL of assay buffer (RPMI + 2% FBS) and 50 μL of serially diluted test article were added to each well and incubated at 37°C for 6 hours. 80 μL of BioGlo substrate (Promega) was then added to each well, and the plate was incubated at RT for an additional 5–10 minutes. PD-1 / PD-L1 blockade was measured by detecting luminescence (e.g., using a Perkin Elmer Envision device) with relative light units (RLU) as the readout.

[0512] T cell cytokine release assays (using suboptimally stimulated primary T cells in the presence of target cells) were performed essentially as follows: human pan T cells (purified from donor PBMCs (see above)) were resuspended in assay medium and placed in a cell culture incubator overnight. TA-positive target cells (e.g., CHO / PD-L1 cells, JIMT-1 cells, N87 cells) and control TA-negative cells (e.g., CHO cells) were obtained from the cultures. After washing, target cells (numbers indicated in the figures and below) were pre-seeded into flat-bottom clear 96-well plates and placed in a cell culture incubator overnight. The next day, the settled human pan T cells were measured for density and viability by trypan blue exclusion using a Beckman Coulter Vi-Cell counter, resulting in a concentration of 2 × 10 6 The density of cells / mL The next day, the supernatant was discarded, and 50 μL of serially diluted test article (antibody, diabody, trivalent molecule, etc.) and 2.0 × 10 beads were added. 6 50 μL of pre-washed D ynabeads αCD3 (REF 11151D; Invitrogen by Thermo Fisher Scientific) and 2.0 × 10 6 5 cells / mL 0 μL / well of human pan T cells and 50 μL of assay medium were added to each well of the assay plate. The final volume of each well on the plate was 200 μL. For control wells that did not contain test article or αCD3 beads, assay medium was added to a total volume of 200 μL, and the plate was incubated in a tissue culture incubator for 72 hours. Supernatants were then collected from each well, and the released cytokines of IFN-γ and IL-2 were assayed using a Cytokine ELISA Kit (e.g., R&D System). Human IL-2 DuoSet ELISA (Cat: DY202), Human IFN-gamma DuoSet ELISA (Cat: DY285), or similar commercially available reagents were measured according to the manufacturer's instructions. Excel and SoftMax Pro were used for data analysis to extrapolate cytokine levels, which were plotted using Prism.

[0513] Quantification of CD137×TA binding molecules in cynomolgus monkey serum was performed essentially as follows: Assay plates were coated overnight with 2.0 μg / mL of His-tagged soluble human CD137 fusion protein (huCD137) (containing the extracellular portion of human CD137 fused to a histidine-containing peptide). After blocking nonspecific sites with 0.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST), the plates were incubated with CD137×TA binding molecule standard calibrators, quality controls, and test samples. Immobilized huCD137-His captures CD137×TA binding molecules present in the standard calibrators, quality controls, and test samples. Captured CD137×TA binding molecules were detected by adding 0.05 μg / mL of human IgG(Fc)-HRP. The activity of bound HRP was quantified by luminescence light generation using Thermo Scientific SuperSignal ELISA Pico chemiluminescent substrate. Luminescence light intensity, expressed as relative light units (RLU), was measured on a Victor X4 plate reader. A standard curve was constructed by fitting the RLU signals from the AEX3370 standards to a five-parameter logistic model. The concentration of CD137xTA binding molecules in serum samples was interpolated from the RLU signal of the sample and the equation describing the standard curve. The lower limit of quantification (LLOQ) for this assay was 6.1 ng / mL.

[0514] T cell and NK cell proliferation assays were performed essentially as follows: Panel T cell and NK cell marker antibodies, including CD3, CD4, CD8, CD56, and CD159a, were added to well-mixed, anticoagulated whole blood samples from cynomolgus monkey studies, mixed thoroughly with a pipette, and incubated at room temperature in the dark for 25–35 minutes. 1x BD FACS Lysing Solution was then added to each well, mixed with a pipette, and each plate was then incubated at room temperature in the dark for an additional 10–20 minutes. Each plate was centrifuged at 400 × g for 5 minutes, and the supernatant was discarded. For a wash step, FACS buffer was added to each well and mixed. Each well was then centrifuged at 400 × g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in BD Cytofix / Cytoperm solution and incubated at 2–8°C for 20–40 minutes. At the end of the incubation, each plate was washed as described above, and the cell pellet was resuspended with Ki67 antibody or isotype control and incubated for 30-60 minutes at 2-8°C. T cell and NK cell proliferation were monitored by CD3 + CD8 + Ki67 + and CD56 + CD159a + Quantification was performed by monitoring the Ki67+ population.

[0515] Example 2 Isolation and characterization of a human non-blocking anti-CD137 mAb To identify CD137-binding domains with improved properties, particularly when incorporated into different CD137xTA binding molecules, a panel of monoclonal antibodies with fully human variable domains specific for human CD137 was generated by immunizing mice with a His-tagged soluble human CD137 fusion protein (huCD137) (containing the extracellular portion of human CD137 fused to a histidine-containing peptide) using the TRIANNI MOUSE® platform. Supernatants from the resulting hybridomas were evaluated for CD137 binding, their ability to mediate dose-dependent T cell signaling in a CD137 reporter assay, and their ability to induce cytokine (e.g., IFN-γ, TNF-α) release from T cells. The VH and VL domains of several hybridomas were cloned and expressed in CHO cells as human IgG1 (L234A / L235A) antibodies and evaluated for binding affinity, ligand-blocking activity, binding to CD137 on the cell surface, and again in CD137 reporter and cytokine release assays. These antibodies were assayed against an irrelevant negative control antibody and / or the chimeric anti-CD137 antibody chCD137 described above. MAB-3 (see WO 2018 / 156740 and above) was included in these evaluations. The methods used in these evaluations are provided above. One antibody, designated CD137 MAB-6(1.1), was selected for further study. The amino acid sequences of the VH and VL domains of CD137 MAB-6(1.1) are provided above, and representative results from these evaluations are summarized in Tables 7A-D.

[0516] [Table 8]

[0517] [Table 9]

[0518] [Table 10]

[0519] [Table 11]

[0520] Epitope binning was performed by cross-competition studies. The results of the binding competition studies indicate that CD137 MAB-6 binds to a distinct epitope from comparator antibodies containing the variable domain of utomilumab, comparator antibodies containing the variable domain of urelumab, and all anti-CD137 antibodies described in WO 2018 / 156740, including chCD137 MAB-3. In summary, these studies demonstrate that CD137 MAB-6 binds to a unique, non-blocking epitope and exhibits higher binding affinity than the aforementioned chCD137 MAB-3, as determined by ELISA, FACS, and BIACORE™ assays. CD137 MAB-6 also exhibits higher activity in T-cell cytokine release assays.

[0521] Example 3 Characterization of CD137×TA binding molecules CD137×TA binding molecules capable of binding to CD137 and the representative TA, PD-L1, were generated incorporating the VH and VL domains of CD137 MAB-6(1.1) and hPD-1 MAB-2(1.1). In particular, a tetravalent bispecific diabody, designated "DART-A," containing two identical bispecific diabody-binding domains and possessing the antibody-like Y structure shown in Figure 1B, and a trivalent binding molecule, designated "TRIDENT-A," containing one monospecific diabody-type binding domain and a non-diabody-type binding domain and possessing the structure shown in Figure 3A, were generated. The domain attributes of these molecules, as well as certain bispecific control and comparator molecules with identical structures, are described above (see, e.g., Tables 5-6).

[0522] DART-A, TRIDENT-A, the comparator molecule TRIDENT-2 (containing the binding domain of hCD137 MAB-3 (1B.3)), and a negative control (hIgG1, an irrelevant antibody as an isotype control) were evaluated for their ability to bind to cell surface CD137 by FACS performed essentially as described above, with test articles used at 10 μg / mL and five-fold serial dilutions. Representative assay results, shown in Figure 4, demonstrate that all CD137×PD-L1 bispecific molecules were able to efficiently bind to CD137 expressed on the cell surface. The comparator molecules appear to exhibit relatively good binding in this assay.

[0523] DART-A, TRIDENT-A, hPD-1 MAB-2(1.1), and negative control hIgG1 were assessed for their ability to bind to the surface of PD-L1-expressing CHO cells (CHO / PD-L1) by FACS analysis and for their ability to antagonize the PD-1 / PD-L1 axis (i.e., block PD-1 / PD-L1 interaction and prevent downregulation of T cell responses) in a Jurkat-luc-NFAT / CHO / PD-L1 luciferase reporter assay. Both assays were performed essentially as described above. Test articles were used at 10 μg / mL and 5-fold serial dilutions for FACS analysis and at 50 μg / mL and 5-fold serial dilutions for the PD-L1 reporter assay. Representative assay results, shown in Figures 5A-5B, demonstrate that DART-A and TRIDENT-A could efficiently bind to PD-L1 expressed on the cell surface (Figure 5A) and block the PD-1 / PD-L1 interaction (Figure 5B). Note that the binding curves of molecules with two PD-L1 binding sites (DART-A and hPD-1 MAB-2(1.1)) reached saturation relatively quickly, indicating that some of these molecules exhibit bivalent binding (i.e., bind to two PD-L1 molecules on the surface). Bivalent binding is more likely to occur in the presence of high concentrations of target ligands expressed on CHO / PD-L1 cells. We also observed that molecules with two PD-L1 binding sites exhibited higher PD-L1 blocking activity compared to trivalent molecules.

[0524] DART-A, TRIDENT-A, comparison molecules: DART-2 and TRIDENT-2 (each containing the binding domain of hCD137 MAB-3 (1B.3)), DA The functional activity of RT-3 (containing the binding domain of utomirumab), a replica of the agonistic anti-CD137 mAb urelumab (r-urelumab), and negative controls: DART-1 (RSV x PD-L1 binding molecule) and hIgG1, was assessed in a CD137 reporter assay performed essentially as described above in the presence and absence of PD-L1-expressing JIMT-1 cells (10,000 cells / well) using test articles at 1 μg / mL and five-fold serial dilutions. Representative assay results, shown in Figure 6, demonstrate that both tetravalent and trivalent CD137 x PD-L1 bispecific molecules (DART-A and TRIDENT-A, respectively) containing the binding domain CD137 MAB-6 (1.1) mediated target-dependent signaling. In contrast, only the trivalent molecule (TRIDENT-2) containing the binding domain of hCD137 MAB-3 (1B.3) exhibited activity in the presence of target cells; the corresponding tetravalent molecule, DART-2, did not. Furthermore, of all tetravalent molecules tested, DART-A exhibited the highest activity. None of the CD137 x TA bispecific molecules exhibited activity in the absence of target cells. As expected, the agonist r-urelumab exhibited activity in both the presence and absence of PD-L1-expressing target cells, and the negative control exhibited no activity.

[0525] The functional activities of DART-A, TRIDENT-A, comparator molecules: DART-2, TRIDENT-2, DART-3, and negative controls: DART-1 and hIgG1 were assessed in a primary T cell cytokine release assay in the presence of PD-L1-expressing JIMT-1 cells (10,000 cells / well) performed essentially as described above, with test articles used at 1 μg / mL and five-fold serial dilutions. Representative results for representative cytokines INF-γ and IL-2 are shown in Figures 7A and 7B, respectively. As observed in the CD137 reporter assay described above, both tetravalent and trivalent CD137xPD-L1 bispecific molecules (DART-A and TRIDENT-A, respectively) containing the binding domain CD137 MAB-6 (1.1) mediated cytokine release; in contrast, only the trivalent molecule (TRIDENT-2) containing the binding domain of hCD137 MAB-3 (1B.3) was active; the tetravalent molecule DART-2 did not show any activity. Again, of all tetravalent molecules tested, DART-A showed the highest activity, and the negative control showed no activity.

[0526] These studies demonstrate that the fully human binding domain CD137 MAB-6(1.1) is active in both tetravalent and trivalent CD137xTA bispecific molecules and lacks agonist activity in the absence of PD-L1-expressing target cells. Although TRIDENT-2 exhibited relatively high binding to CHO / CD137 cells, the activity of TRIDENT-A and TRIDENT-2 was comparable, whereas DART-2 showed no activity in either functional assay. Indeed, CD137 MAB-6(1.1) has higher activity in the tetravalent antibody-like structure shown in Figure 1B than either hCD137 MAB-3(1B.3) or molecules containing the binding domain of utomirumab.

[0527] Example 4 Pharmacokinetics of CD137×TA molecules The pharmacokinetics of the trivalent CD137 x TA bispecific molecules TRIDENT-A (containing the binding domain of CD137 MAB-6 (1.1)) and TRIDENT-2 (containing the binding domain of hCD137 MAB-3 (1B.3)) were evaluated in cynomolgus monkeys. Briefly, two female cynomolgus monkeys were injected with a single dose of each test article at 1 mg / kg or 10 mg / kg (four groups). The animals were observed for 22 days; no necropsies were performed. Animals were monitored for food consumption, body weight, and whole blood hematology, and clinical chemistry procedures were performed throughout the study. Transient increases in liver enzymes (ALT, AST, and bilirubin) were observed. The test articles were well tolerated, and no adverse events were observed.

[0528] Serum concentrations of the molecules were monitored over time essentially as described above. The Cmax, AUC, t1 / 2β, and CL values ​​are presented in Table 8 and demonstrate that the trivalent CD137xTA bispecific molecule containing the binding domain of CD137 MAB-6 (1.1) exhibits a serum half-life approximately two-fold longer than that containin...

Claims

1. 1. A CD137 binding molecule comprising a first binding site that immunospecifically binds to an epitope of CD137, wherein the first binding site is a CDR L 1. CDR L 2 and CDR L Including 3 a first light chain variable domain comprising: H 1. CDR H 2 and CDR H 3. and a variable domain; (A) the CDRs of the first light chain variable domain L 1. CDR L 2 and CDR L 3 is CD1 37 are the light chain CDRs of MAB-6 VL1 (SEQ ID NO: 50); (B) the CDRs of the first heavy chain variable domain H 1. CDR H 2 and CDR H 3 is CD1 37 A CD137 binding molecule which is the heavy chain CDRs of MAB-6 VH1 (SEQ ID NO: 46).

2. The CD137 binding molecule of claim 1, wherein the first heavy chain variable domain comprises the amino acid sequence of hCD137 MAB-6 VH1 (sequence number 46).

3. The first light chain variable domain comprises: (A) hCD137 MAB-6 VL1 (SEQ ID NO: 54); (B) hCD137 MAB-6 VL1 (SEQ ID NO: 50); (B) hCD137 MAB-6 VL2 (SEQ ID NO: 55); or (C) hCD137 MAB-6 VL3 (SEQ ID NO: 56) The CD137 binding molecule of claim 1 or 2, comprising the amino acid sequence:

4. (A) the first heavy chain variable domain comprises the amino acid sequence of: hCD137 MAB-6 VH1 (SEQ ID NO:46); (B) The CD137 binding molecule of any one of claims 1 to 3, wherein the first light chain variable domain comprises the amino acid sequence of hCD137 MAB-6 VL3 (sequence number 56).

5. The molecule is a bispecific molecule that comprises a second binding site that immunospecifically binds to a tumor antigen (TA), and the second binding site is L 1. CDR L 2 and CDR L Contains 3 a second light chain variable domain and a CDR H 1. CDR H 2 and CDR H a second heavy chain variable domain comprising: The CD137-binding molecule of any one of claims 1 to 4, comprising a domain.

6. The CD137-binding molecule of claim 5, wherein the TA is selected from the antigens presented in Tables 1-2.

7. The TA is PD-L1, and: (A) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is hPD -L1 are the light chain CDRs of MAB-2 VLx (SEQ ID NO: 63); (B) the CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is hPD The CD137 binding molecule of claim 5, which is the heavy chain CDR of -L1 MAB-2 VHx (sequence number 59).

8. (A)(1) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of hPD-L1 MAB-2 VL1 (SEQ ID NO: 58); or (2) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of PD-L1 MAB-2 VL2 (SEQ ID NO: 72); (B)(1) the CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is, the heavy chain CDRs of hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 67); (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH3 (SEQ ID NO: 68); (4) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 69); (5) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of PD-L1 MAB-2 VH2 (SEQ ID NO: 70); or (6) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h The CD137-binding molecule of claim 7, which is the heavy chain CDR of PD-L1 MAB-2 VH2 (SEQ ID NO: 71).

9. The second heavy chain variable domain comprises: (A) hPD-L1 MAB-2 VH1 (SEQ ID NO: 57); (B) hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (C) hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (D) hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (E) hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (F) hPD-L1 MAB-2 VH6 (SEQ ID NO: 71) The CD137 binding molecule of claim 8, comprising the amino acid sequence:

10. The second light chain variable domain comprises: (A) hPD-L1 MAB-2 VL1 (SEQ ID NO: 58); or (B) hPD-L1 MAB-2 VL2 (SEQ ID NO: 72) The CD137 binding molecule of claim 8 or 9, comprising the amino acid sequence:

11. The TA is 5T4: (A)(1) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of 5T4 MAB-1 VL (SEQ ID NO: 93); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is 5 the heavy chain CDRs of T4 MAB-1 VH (SEQ ID NO: 92); or (B)(1) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, the light chain CDRs of 5T4 MAB-2 VL (SEQ ID NO: 95); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is 5 The CD137 binding molecule of claim 5, which is the heavy chain CDR of T4 MAB-2 VH (sequence number 96).

12. The CD137xTA binding molecule of claim 11, wherein the second heavy chain variable domain comprises the amino acid sequence of 5T4 MAB-1 VH (sequence number 92).

13. The CD137xTA binding molecule of claim 11 or 12, wherein the second light chain variable domain comprises the amino acid sequence of 5T4 MAB-1 VL (SEQ ID NO: 93).

14. The TA is HER2: (A) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is hHE R2-MAB-1 VLx (SEQ ID NO: 79) light chain CDRs; (B) the CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is hHE The CD137 binding molecule of claim 5, which is the heavy chain CDR of R2-MAB-1 VHx (sequence number 78).

15. (A)(1) the CDRs of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is, are the light chain CDRs of hHER2-MAB-1 VL1 (SEQ ID NO: 83); (2) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of HER2-MAB-1 VL2 (SEQ ID NO: 84); or (3) the CDR of the second light chain variable domain L 1. CDR L 2 and CDR L 3 is h the light chain CDRs of HER2-MAB-1 VL3 (SEQ ID NO: 85); (B)(1) the CDRs of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is, are the heavy chain CDRs of hHER2-MAB-1 VH1 (SEQ ID NO: 80); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of HER2-MAB-1 VH2 (SEQ ID NO: 81); or (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h The CD137 binding molecule of claim 14, which is the heavy chain CDR of HER2-MAB-1 VH3 (sequence number 82).

16. The second heavy chain variable domain comprises: (A) hHER2-MAB-1 VH1 (SEQ ID NO: 80); (B) hHER2-MAB-1 VH2 (SEQ ID NO: 81); or (C) hHER2-MAB-1 VH3 (SEQ ID NO: 82) The CD137 binding molecule of claim 15, comprising the amino acid sequence:

17. The second light chain variable domain comprises: (A) hHER2-MAB-1 VL1 (SEQ ID NO: 83); (B) hHER2-MAB-1 VL2 (SEQ ID NO: 84); or (C) hHER2-MAB-1 VL3 (SEQ ID NO: 85) 17. The CD137 binding molecule of claim 15 or 16, comprising the amino acid sequence:

18. The CD137-binding molecule of any one of claims 1 to 17, wherein the molecule is an antibody, or a bispecific tetravalent Fc-bearing diabody, or a bispecific trivalent molecule.

19. A CD137 binding molecule described in any one of claims 1 to 17, wherein the molecule is bispecific and tetravalent and comprises a first, second, third, and fourth polypeptide chain, and the polypeptide chains form a covalent complex.

20. A CD137 binding molecule described in any one of claims 1 to 17, wherein the molecule is bispecific and trivalent and comprises a first, second, third, and fourth polypeptide chain, wherein the polypeptide chains form a covalent complex.

21. The TA is PD-L1: (A) the first polypeptide chain and the third polypeptide chain comprise the amino acid sequence of SEQ ID NO:116, SEQ ID NO:118, or SEQ ID NO:120; (B) The CD137 binding molecule of claim 19, wherein the second polypeptide chain and the fourth polypeptide chain comprise the amino acid sequence of SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO:

139.

22. The molecule is: (A) SEQ ID NO: 116 and SEQ ID NO: 117; (B) SEQ ID NO: 118 and SEQ ID NO: 119; (C) SEQ ID NO: 120 and SEQ ID NO: 119; (D) SEQ ID NO: 118 and SEQ ID NO: 121; (E) SEQ ID NO: 120 and SEQ ID NO: 121; (F) SEQ ID NO: 120 and SEQ ID NO: 122; (G) SEQ ID NO: 120 and SEQ ID NO: 123; (H) SEQ ID NO: 120 and SEQ ID NO: 124; (I) SEQ ID NO: 120 and SEQ ID NO: 125; (J) SEQ ID NO: 120 and SEQ ID NO: 126; or (K) SEQ ID NO: 120 and SEQ ID NO: 139 The CD137 binding molecule of claim 21, comprising:

23. The TA is PD-L1: (A) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:127, SEQ ID NO:133, or SEQ ID NO:135; (B) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:128, SEQ ID NO:134, or SEQ ID NO:136; (C) the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129, or SEQ ID NO: 131; (D) The CD137 binding molecule of claim 20, wherein the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO:

132.

24. The molecule is: (A) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, and SEQ ID NO: 130; (B) SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 131, and SEQ ID NO: 132; (C) SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 131, and SEQ ID NO: 132; or (D) SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 131, and SEQ ID NO:

132. The CD137 binding molecule of claim 23, comprising:

25. A pharmaceutical composition comprising the CD137-binding molecule of any one of claims 1 to 24 and a physiologically acceptable carrier.

26. Use of a CD137 binding molecule described in any one of claims 6 to 24 or a pharmaceutical composition described in claim 25 in the treatment of a disease or condition associated with or characterized by the expression of said TA.

27. CDR L 1. CDR L 2 and CDR L a light chain variable domain comprising: H 1. CDR H 2 and CDR H and a heavy chain variable domain comprising: (A) the CDRs of the light chain variable domain L 1. CDR L 2 and CDR L 3 is hPD-L 1 MAB-2 VL2 (SEQ ID NO: 72); (B) (1) the CDRs of the heavy chain variable domain H 1. CDR H 2 and CDR H 3 is hPD -L1 are the heavy chain CDRs of MAB-2 VH2 (SEQ ID NO: 67); (2) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH3 (SEQ ID NO: 68); (3) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h are the heavy chain CDRs of PD-L1 MAB-2 VH4 (SEQ ID NO: 69); (4) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h the heavy chain CDRs of PD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (5) the CDR of the second heavy chain variable domain H 1. CDR H 2 and CDR H 3 is h A PD-L1 binding molecule which is the heavy chain CDRs of PD-L1 MAB-2 VH6 (SEQ ID NO: 71).

28. The heavy chain variable domain comprises: (A) hPD-L1 MAB-2 VH2 (SEQ ID NO: 67); (B) hPD-L1 MAB-2 VH3 (SEQ ID NO: 68); (C) hPD-L1 MAB-2 VH4 (SEQ ID NO: 69); (D) hPD-L1 MAB-2 VH5 (SEQ ID NO: 70); or (E) hPD-L1 MAB-2 VH6 (SEQ ID NO: 71) 28. The PD-Ll-binding molecule of claim 27, comprising the amino acid sequence:

29. 29. The PD-Ll-binding molecule of claim 27 or 28, wherein the Light Chain Variable Domain comprises the amino acid sequence of hPD-Ll MAB-2 VL2 (SEQ ID NO: 72).

30. 30. The PD-L1-binding molecule of any one of claims 27 to 29, wherein the molecule is an antibody or an antigen-binding fragment thereof.

31. 31. A pharmaceutical composition comprising the PD-L1-binding molecule of any one of claims 27 to 30 and a physiologically acceptable carrier.

32. 32. Use of the PD-L1-binding molecule of any one of claims 27 to 30 or the pharmaceutical composition of claim 31 in the treatment of a disease or condition associated with a suppressed immune system or characterized by expression of PD-L1.

33. 33. The use of claim 32, wherein the disease or condition associated with a suppressed immune system or characterized by expression of PD-L1 is cancer.

34. 34. The use of claim 26 or 33, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, cerebrospinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), stomach cancer, testicular cancer, thymic cancer, and uterine cancer.

35. 32. A method of enhancing the activity of a tumor-targeting agent, comprising administering the tumor-targeting agent in combination with a CD137-binding molecule of any one of claims 1-24, a PD-L1-binding molecule of any one of claims 27-30, or a pharmaceutical composition of claim 25 or 31.

36. 31. A method of treating a disease or condition associated with a suppressed immune system or characterized by the development of a TA, comprising administering to a subject in need thereof a CD137-binding molecule of any one of claims 1-24, a PD-L1-binding molecule of any one of claims 27-30, or a pharmaceutical composition of claim 25 or 31.

37. 37. The method of claim 36, wherein the condition associated with a suppressed immune system or characterized by the expression of TA is cancer.

38. 38. The method of claim 36 or 37, further comprising administering a tumor targeting agent.

39. 39. The method of claim 35 or 38, wherein the tumor targeting agent is an antibody, an epitope-binding fragment of an antibody, or an agent that mediates T cell-targeted killing of target cells.

40. The cancers include: bladder cancer, bone cancer, brain and spinal cancer, breast cancer, cervical cancer, colorectal cancer, gallbladder or bile duct cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian cancer, liver cancer, pharyngeal cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, squamous cell carcinoma of the head and neck (SCCHN), gastric cancer, 39. The method of any one of claims 36 to 38, wherein the cancer is selected from the group consisting of uterine cancer, testicular cancer, thymic cancer, and uterine cancer.

41. A nucleic acid encoding the CD137-binding molecule of any one of claims 1 to 24, or the PD-L1-binding molecule of any one of claims 27 to 30.

42. 42. An expression vector comprising the nucleic acid of claim 41.

43. 43. A cell comprising the nucleic acid of claim 41 or the expression vector of claim 42.

44. 44. The cell of claim 43, wherein the cell is a mammalian cell.

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