Compositions and methods for universal tumor cell killing

Abstract

Provided herein is a method for killing tumor cells by administering to a subject a multispecific antigen-binding molecule having a first antigen-binding region specific to a first target antigen and a second antigen-binding region specific to CD28 protein. In some embodiments, the tumor cells do not express the target antigen. In some embodiments, the tumor cells are not expected to express the target antigen.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 329,762, filed April 11, 2022, and U.S. Provisional Application No. 63 / 347,330, filed May 31, 2022, the entire contents of each of which are incorporated by reference herein. [Background technology]

[0002] Cancer is one of the leading causes of death in North America. Despite decades of research, many cancers remain ineffective or develop resistance to traditional chemotherapy and other therapeutic agents. In recent years, advances in immunotherapy have provided some promising treatments, but even these tend to prove effective only in certain patients and cancer types. Thus, there is a need for new cancer treatments. Summary of the Invention

[0003] The methods and compositions provided herein are based, in part, on the unexpected discovery that CD28 bispecific antibodies targeting a tumor-associated antigen, an antigen that targets cells within the tumor microenvironment, or an immune antigen (e.g., an antigen expressed on the surface of immune cells within a tumor or within the tumor microenvironment) can induce killing of cancer or tumor cells that lack expression of such antigen.

[0004] In certain aspects, provided herein are methods and compositions for administering to a subject a multispecific (e.g., bispecific) antigen binding molecule having a first antigen binding region specific for a target antigen and a second antigen binding region specific for a CD28 protein, thereby resulting in the killing of tumor cells within a tumor in the subject, where the tumor cells do not express or are not expected to express the target antigen. In some embodiments, at least a subset of tumor cells within the tumor do not express the target antigen.

[0005] In some aspects, provided herein are methods and compositions for inducing killing of tumor cells and / or inducing T cell activation against tumor cells in a tumor in a subject, the methods comprising administering to the subject a multispecific antigen binding molecule having a first antigen binding region specific for a target antigen and a second antigen binding region specific for a CD28 protein. In some embodiments, at least a subset of tumor cells in the tumor do not express the target antigen.

[0006] In some aspects, methods and compositions are also provided herein for treating cancer in a subject having a tumor, the methods comprising administering to the subject a multispecific antigen binding molecule having a first antigen binding region specific for a target antigen and a second antigen binding region specific for a CD28 protein. In some embodiments, at least a subset of tumor cells within the tumor do not express the target antigen.

[0007] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigen is an antigen associated with the tumor microenvironment (e.g., the microenvironment of a tumor in a subject). For example, in some embodiments, the target antigen is an antigen on an immune cell, on a tumor cell stroma, or on an extracellular matrix within the tumor microenvironment. Examples of extracellular matrix antigens include nectin (e.g., nectin-3 or nectin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) protein antigens.

[0008] In some embodiments, the methods provided herein may further include determining that at least a subset of tumor cells within the tumor do not express the target antigen, hi some embodiments, the tumor cells do not express the target antigen if expression of the target antigen is below the level of detection or below the signal to noise ratio.

[0009] In some aspects, provided herein are methods of treating cancer in a subject, comprising: i) determining that the subject has a tumor comprising tumor cells that do not express a target antigen; and ii) administering to the subject a multispecific antigen binding molecule comprising a first antigen binding region specific for the target antigen and a second antigen binding region specific for a CD28 protein.

[0010] In some aspects, provided herein is a method of selecting a subject for cancer treatment, comprising: i) determining that the subject has a tumor comprising tumor cells that do not express a target antigen; and ii) administering to the subject a multispecific antigen binding molecule having a first antigen binding region specific for the target antigen and a second antigen binding region specific for a CD28 protein, optionally wherein the tumor cells do not express the target antigen if expression of the target antigen is below detection level or below signal to noise ratio, thereby selecting the subject for cancer treatment.

[0011] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the tumor may be a heterogeneous tumor further comprising tumor cells expressing a TAA. In some embodiments, the tumor microenvironment of the tumor comprises cells expressing a TAA. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the tumor cells in the tumor do not express a TAA. In some embodiments, at least 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, or 90%-100% of the tumor cells in the tumor do not express a TAA.

[0012] In some embodiments, the target antigen is an antigen associated with the tumor microenvironment of the tumor, e.g., an antigen associated with the tumor stroma, an antigen associated with the extracellular matrix of the tumor, an antigen associated with blood vessels within the tumor microenvironment, or an antigen associated with cancer-associated fibroblasts.

[0013] In some embodiments, the target antigen is an antigen associated with the tumor stroma selected from PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin isoform EDB.

[0014] In some embodiments, the target antigen is an antigen associated with the extracellular matrix of a tumor selected from nectin (e.g., nectin-3 or nectin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) proteins.

[0015] In some embodiments, the tumor microenvironment of a tumor comprises cells that express the target antigen.

[0016] In some embodiments, the target antigen is an antigen expressed on the surface of cancer-associated fibroblasts (e.g., alpha-smooth muscle actin (alpha-SMA), fibroblast activation protein (FAP), S100A4, platelet-derived growth factor receptor (PDGFRα / β), vimentin, PDPN, CD70, CD10, GPR77, CD10, CD74, CD146, CAV1, Saa3-, or CD49e).

[0017] In some embodiments, the target antigen is an antigen expressed on the surface of blood vessels within the tumor microenvironment, such as DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, or VEGFR2.

[0018] In some embodiments, the target antigen is an immune antigen. In some embodiments, the immune antigen is an antigen expressed on the surface of an immune cell. The immune cell can be a macrophage, neutrophil, eosinophil, basophil, mast cell, monocyte, dendritic cell, natural killer cell, T cell, or B cell. In some embodiments, the immune cell infiltrates a tumor or the tumor microenvironment of the tumor. The immune antigen can be selected from any of the immune antigens listed in Table 4.

[0019] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of tumor cells do not express the target antigen. In some embodiments, at least 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, or 90%-100% of the tumor cells in the tumor do not express the target antigen.

[0020] In some embodiments, the tumor comprises immune cells (e.g., B cells and / or cells expressing CD20). In some embodiments, tumor cells that do not express the target antigen also do not express CD20. The target antigen may be selected from CD38, EGFR, CD22, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7. The target antigen may be CD22.

[0021] In some embodiments, the multispecific antigen-binding molecule can be a bispecific antibody (e.g., any of the bispecific antibodies listed in Table 3) or a bispecific antibody fragment, such as a bispecific T-engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb). In some embodiments, the multispecific antigen-binding molecule is administered with a second multispecific antigen-binding molecule having a first antigen-binding region specific for a second target antigen (e.g., a second tumor-associated antigen (TAA2)) and a second antigen-binding region specific for a CD3 protein. In some embodiments, the second multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T-engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb).

[0022] The second target antigen may be selected from any of the antigens listed in Table 2. The second target antigen may be CD20 protein. In some embodiments, the second multispecific antigen binding molecule is selected from any of the multispecific antigen binding molecules in Table 5. The multispecific antigen binding molecule may exhibit a costimulatory effect when administered with the second multispecific antigen binding molecule. In some embodiments, the costimulatory effect is one or more of the following: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in PBMCs, and enhancing T cell-mediated cytotoxicity. In some embodiments, the tumor cell is a tumor cell of a B cell cancer. B cell cancers include, but are not limited to, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (lymphoma of the eye).

[0023] In some embodiments, the tumor is a solid tumor. The tumor may be an adenocarcinoma, an adrenal tumor, anal tumor, bile duct tumor, bladder tumor, bone tumor, blood-borne tumor, brain / CNS tumor, breast tumor, cervical tumor, colorectal tumor, endometrial tumor, esophageal tumor, Ewing's tumor, eye tumor, gallbladder tumor, gastrointestinal tumor, kidney tumor, laryngeal tumor or hypopharyngeal tumor, liver tumor, lung tumor, mesothelioma, multiple myeloma, muscle tumor, nasopharyngeal tumor, neuroblastoma, oral tumor, osteosarcoma, ovarian tumor, pancreatic tumor, penile tumor, pituitary tumor, primary tumor, prostate tumor, retinoblastoma, rhabdomyosarcoma, salivary gland tumor, soft tissue sarcoma, melanoma, metastatic tumor, basal cell carcinoma, Merkel cell tumor, testicular tumor, thymus tumor, thyroid tumor, uterine tumor, vaginal tumor, vulvar tumor, or Wilms' tumor.

[0024] The multispecific antigen-binding molecule may be administered systemically, intravenously, subcutaneously, or intramuscularly. The multispecific antigen-binding molecule may be administered to the subject in a pharma- ceutically acceptable formulation. In some embodiments, the method further comprises administering an additional anti-cancer agent. The additional anti-cancer agent may be a chemotherapeutic agent, an immune checkpoint inhibitor, a CAR-T cell, or a tumor vaccine. The immune checkpoint inhibitor may be an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, or an anti-LAG3 antibody.

[0025] In some embodiments, the subject is afflicted with a treatment-resistant cancer.

[0026] In some aspects, provided herein is a method of treating cancer in a subject having a tumor, inducing or effecting the killing of tumor cells in the tumor in the subject, and / or inducing T cell activation against tumor cells in the tumor in the subject, the method comprising administering to the subject a first multispecific antigen binding molecule having an antigen binding region specific for a first target antigen and an antigen binding region specific for CD28 protein, and a second multispecific antigen binding molecule having an antigen binding region specific for a second target antigen and an antigen binding region specific for CD3 protein, wherein the first target antigen is not the same antigen as the second target antigen. In some embodiments, the first and / or second target antigen is a tumor associated antigen (TAA).

[0027] In some embodiments, the method further includes determining that the tumor comprises a subset of tumor cells that do not express the first target antigen.

[0028] In some aspects, provided herein is a method of treating cancer in a subject having a tumor, inducing or effecting the killing of tumor cells in the tumor in the subject, and / or inducing T cell activation against tumor cells in the tumor in the subject, the method comprising: determining whether the tumor contains tumor cells that do not express a first target antigen; administering to the subject a first multispecific antigen binding molecule having an antigen binding region specific for the first target antigen and an antigen binding region specific for CD28 protein, and a second multispecific antigen binding molecule having an antigen binding region specific for a second target antigen and an antigen binding region specific for CD3 protein, wherein the first target antigen is not the same antigen as the second target antigen. In some embodiments, the first and / or second target antigen is a tumor associated antigen (TAA).

[0029] In some embodiments, at least a subset of tumor cells in the tumor do not express the first target antigen. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of tumor cells in the tumor do not express the first target antigen. In some embodiments, at least a subset of tumor cells in the tumor do not express the second target antigen. In some embodiments, the tumor is a heterogeneous tumor comprising cells that express the first target antigen and cells that express the second target antigen. In some embodiments, tumor cells in the tumor do not express both the first target antigen and the second target antigen.

[0030] In some embodiments, the first target antigen is an antigen associated with the tumor microenvironment of the tumor, e.g., an antigen associated with the tumor stroma, an antigen associated with the extracellular matrix of the tumor, an antigen associated with blood vessels within the tumor microenvironment, or an antigen associated with cancer-associated fibroblasts.

[0031] In some embodiments, the first target antigen is an antigen associated with the tumor stroma selected from PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin isoform EDB.

[0032] In some embodiments, the first target antigen is an antigen associated with the extracellular matrix of a tumor selected from nectin (e.g., nectin-3 or nectin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) proteins.

[0033] In some embodiments, the tumor microenvironment of a tumor comprises cells that express the target antigen.

[0034] In some embodiments, the first target antigen is an antigen expressed on the surface of cancer-associated fibroblasts (e.g., alpha-smooth muscle actin (alpha-SMA), fibroblast activation protein (FAP), S100A4, platelet-derived growth factor receptor (PDGFRα / β), vimentin, PDPN, CD70, CD10, GPR77, CD10, CD74, CD146, CAV1, Saa3-, or CD49e).

[0035] In some embodiments, the first target antigen is an antigen expressed on the surface of blood vessels within the tumor microenvironment, such as DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, and VEGFR2.

[0036] In some embodiments, the first target antigen is an immune antigen. In some embodiments, the immune antigen is an antigen expressed on the surface of an immune cell. The immune cell can be a macrophage, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a dendritic cell, a natural killer cell, a T cell, or a B cell. In some embodiments, the immune cell infiltrates a tumor or a tumor microenvironment of the tumor. The immune antigen can be selected from any of the immune antigens listed in Table 4.

[0037] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 101%, 102%, 103%, 104%, 105%, 106%, 10 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the tumor cells do not express the first or second target antigen. In some embodiments, at least 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, or 90%-100% of the tumor cells in the tumor do not express the first and / or second target antigen.

[0038] The first target antigen may be selected from any of the antigens listed in Table 2. The first target antigen may be CD22. The first target antigen may be a non-immune antigen. The non-immune antigen may be CD38, EGFR, MUC16, PSMA, CA9, FOLR1, HER2, or SLAMF7. In some embodiments, the first multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T-engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb). In some embodiments, the first multispecific antigen-binding molecule is selected from a bispecific antibody listed in Table 3. In some embodiments, the second target antigen is selected from any of the antigens listed in Table 2. The second target antigen may be CD20. The second target antigen may be an immune antigen. In some embodiments, the immunizing antigen is CD22, CD20, CD72, CD19, CD21, CD24, or CD79. The second multispecific antigen-binding molecule can be a bispecific antibody or a bispecific antibody fragment. In some embodiments, the second multispecific antigen-binding molecule is selected from the bispecific antibodies listed in Table 5. In some embodiments, the second multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T-engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb).

[0039] In some embodiments, the first multispecific antigen binding molecule exhibits a costimulatory effect when administered together with the second multispecific antigen binding molecule, which may be one or more of the following: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in PBMCs, and enhancing T cell-mediated cytotoxicity.

[0040] The tumor may originate from a B-cell cancer, such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (lymphoma of the eye).

[0041] In some embodiments, the tumor is a solid tumor. The tumor may be an adenocarcinoma, an adrenal tumor, anal tumor, bile duct tumor, bladder tumor, bone tumor, blood-borne tumor, brain / CNS tumor, breast tumor, cervical tumor, colorectal tumor, endometrial tumor, esophageal tumor, Ewing's tumor, eye tumor, gallbladder tumor, gastrointestinal tumor, kidney tumor, laryngeal tumor or hypopharyngeal tumor, liver tumor, lung tumor, mesothelioma, multiple myeloma, muscle tumor, nasopharyngeal tumor, neuroblastoma, oral tumor, osteosarcoma, ovarian tumor, pancreatic tumor, penile tumor, pituitary tumor, primary tumor, prostate tumor, retinoblastoma, rhabdomyosarcoma, salivary gland tumor, soft tissue sarcoma, melanoma, metastatic tumor, basal cell carcinoma, Merkel cell tumor, testicular tumor, thymus tumor, thyroid tumor, uterine tumor, vaginal tumor, vulvar tumor, or Wilms' tumor.

[0042] In some embodiments, the first multispecific antigen-binding molecule is administered systemically, intravenously, subcutaneously, or intramuscularly. The second multispecific antigen-binding molecule may be administered systemically, intravenously, subcutaneously, or intramuscularly. The first multispecific antigen-binding molecule and / or the second multispecific antigen-binding molecule may be administered to the subject in a pharma- ceutically acceptable formulation.

[0043] In some embodiments, the method further comprises administering an additional anti-cancer agent. In some embodiments, the additional anti-cancer agent is a chemotherapeutic agent, an immune checkpoint inhibitor, a CAR-T cell, or a tumor vaccine. In some embodiments, the additional anti-cancer agent is an immune checkpoint inhibitor. The immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, or an anti-LAG3 antibody.

[0044] In some embodiments, the subject may be suffering from a treatment-resistant cancer. [Brief description of the drawings]

[0045] [Figure 1A] 1 shows that CD28 is expressed on intratumoral CD8 T cells from r / r NHL patients both pre- and post-odronextamab treatment.Representative images of multiplex IHC staining (CD3, CD8, CD28, and DAPI) of DLBCL patient baseline samples from an odronextamab Phase I study are shown. [Figure 1B] Shows that CD28 is expressed on intratumoral CD8 T cells from r / r NHL patients both pre- and post-odronextamab treatment. Shows the density of CD4+CD28+ and CD8+CD28+ cells in baseline DLBCL and FL samples. n=64. [Figure 1C] Figure 2 shows that CD28 is expressed on intratumoral CD8 T cells from r / r NHL patients both before and after odronextamab treatment. Representative images of DLBCL patient samples at baseline (top left) and 5 weeks after initiation of odronextamab treatment (top right) are shown. Density of CD8+Cd28+ cells and CD8+CD28+ cells in corresponding DLBCL and FL samples at baseline and 5 weeks of treatment. [Figure 2A] Figure 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMC in combination with increasing doses of odronextamab and a fixed concentration of REGN5837 (ranging from 7.72x10-" ​​to 1.00x10-7M). Figure 2 shows that tumor cell killing was assessed by percent dead cells. [Figure 2B]We show that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMC in combination with increasing doses of odronextamab and a constant concentration of REGN5837 (ranging from 7.72x10-” to 1.00x10-7M). We show that activation of CD4 T cells was indicated by CD25 upregulation. [Figure 2C] Figure 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMC in combination with increasing doses of odronextamab and a constant concentration of REGN5837 (ranging from 7.72x10-" ​​to 1.00x10-7M). Proliferation was assessed by percent dividing CD4 T cells. [Figure 2D] We show that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMC in combination with increasing doses of odronextamab and a constant concentration of REGN5837 (ranging from 7.72x10-” to 1.00x10-7M). We show that activation of CD8 T cells was indicated by CD25 upregulation. [Figure 2E] Figure 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMC in combination with increasing doses of odronextamab and a constant concentration of REGN5837 (ranging from 7.72x10-" ​​to 1.00x10-7M). Proliferation was assessed by percent dividing CD8 T cells. [Figure 2F]Figure 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMCs in combination with increasing doses of odronextamab and a fixed concentration of REGN5837 (ranging from 7.72x10-" ​​to 1.00x10-7M). Supernatants were assessed for cytokine release of IL-2, IL-4, IL-6, IL-10, TNF-a, IFN-y, and IL-17A. Arrows indicate fold change in EC50 or fold change in maximum cytokine concentration between the highest concentration of REGN5837 (1.00x10-7M) and no REGN5837. [Figure 2G] Figure 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. Enhancement of T cell killing of CD22-negative cells by REGN5837 (R5837) was measured by incubating purified human T cells with different ratios of mixed CD22+ and CD22- WSU-DLCL2 tumor cells with fixed concentrations of REGN5837 (ranging from 4.63x10-10 to 1.67x10-8M) and 5pM odronextamab. Killing of CD22+ targets is shown in red and killing of CD22- targets in blue. [Figure 2H] Figure 2 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell activation, cytotoxicity, and effector function in vitro. Figure 2 shows activation of T cells in WSU-DLCL2 CD22+ and CD22- mixed cultures as measured by upregulation of CD25. [Figure 3A] 1 shows that REGN5837 enhances the antitumor efficacy of odronextamab in the context of prophylactic treatment in the WSU-DLCL2 tumor model and expands intratumoral CD8 T cells. 2 shows the treatment scheme for WSU-DLCL2 tumors implanted in NSG animals. [Figure 3B]1 shows that REGN5837 enhances the antitumor efficacy of odronextamab in the context of prophylactic treatment in the WSU-DLCL2 tumor model and expands intratumoral CD8+ T cells. Individual tumor volumes are shown plotted. Percentages indicate the number of tumor-free mice. [Figure 3C] Figure 1 shows that REGN5837 enhances the antitumor efficacy of odronextamab in the context of prophylactic treatment in the WSU-DLCL2 tumor model and expands intratumoral CD8 T cells. Mean tumor growth (left) and survival (right) are shown. Statistical significance for mean tumor growth was calculated using two-way ANOVA and Tukey's multiple comparisons. Statistical significance for survival was calculated using the Kaplan-Meier method with the log-rank test. *P<0.05, **P<0.01, ***P<0.001. [Figure 3D] We show that REGN5837 enhances the antitumor efficacy of odronextamab in the context of prophylactic treatment in the WSU-DLCL2 tumor model and expands intratumoral CD8 T cells. To examine the immune phenotype of intratumoral T cell responses, WSU-DLCL2 tumor-bearing animals were sacrificed 26 days after implantation. UMAP plots of all live cells from the tumor were overlaid with color-coded immune cell subsets identified by FlowSOM (Figure 3D, left). Density UMAP plots (Figure 3D, right) revealed the bias of certain populations in response to combination therapy. [Figure 3E] 1 shows that REGN5837 enhances the antitumor efficacy of odronextamab in the context of prophylactic treatment in the WSU-DLCL2 tumor model and expands intratumoral CD8+ T cells. The density of WSU-DLCL2 cells (left) and the density of intratumoral CD8+ T cells (right) are shown. [Figure 3F]Figure 2 shows that REGN5837 enhances the antitumor efficacy of odronextamab and expands intratumoral CD8+ T cells in the context of prophylactic treatment in the WSU-DLCL2 tumor model. Pie charts (left) showing the proportion of activated memory subsets of intratumoral CD8+ T cells in response to treatment. Density of effector memory and central memory intratumoral CD8+ T cells (right). Statistics were calculated using one-way ANOVA with Tukey's test. ***P<0.001, ****P<0.0001. [Figure 4A] 1 shows that REGN5837-mediated costimulation enhances the antitumor efficacy of odronextamab against B cell malignancies in the context of therapeutic treatment. 2 shows a treatment scheme for therapeutic treatment of WSU-DLCL2 tumors implanted in NSG animals. [Figure 4B] 1 shows that REGN5837-mediated costimulation enhances the antitumor efficacy of odronextamab against B-cell malignancies in the context of therapeutic treatment. Individual tumor volumes are shown plotted. Percentages indicate the number of tumor-free mice. [Figure 4C] Figure 1 shows that REGN5837-mediated costimulation enhances the antitumor efficacy of odronextamab against B-cell malignancies in the context of therapeutic treatment. Mean tumor growth (left) and survival (right) are shown. Statistical significance for mean tumor growth was calculated using two-way ANOVA and Tukey's multiple comparisons. Statistical significance for survival was calculated using the Kaplan-Meier method with the log-rank test. *P<0.05, **P<0.01. [Figure 4D] 1 shows that REGN5837-mediated costimulation enhances the antitumor efficacy of odronextamab against B cell malignancies in the context of therapeutic treatment.1 shows a treatment scheme for therapeutic treatment of NALM6-luc tumors implanted in NSG animals engrafted with PBMCs. [Figure 4E] 14 shows that REGN5837-mediated costimulation enhances the anti-tumor efficacy of odronextamab against B-cell malignancies in the context of therapeutic treatment.15 BLI showing tumor burden in individual mice is shown. [Figure 4F]Figure 1 shows that REGN5837-mediated costimulation enhances the antitumor efficacy of odronextamab against B-cell malignancies in the context of therapeutic treatment. Mean values ​​of NALM6-luc tumor growth are shown. Significance was calculated using two-way ANOVA and Tukey's multiple comparisons. *P<0.05, ***P<0.001. [Figure 5A] 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. 2 shows a treatment scheme for WSU-DLCL2 tumors implanted in human immune reconstituted animals. [Figure 5B] 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. Individual tumor volumes are plotted. [Figure 5C] Figure 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. Mean tumor growth (left) and survival (right) are shown. Statistical significance for mean tumor growth was calculated using two-way ANOVA and Tukey's multiple comparisons. Statistical significance for survival was calculated using the Kaplan-Meier method with the log-rank test. *P<0.05, **P<0.01. [Figure 5D] 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. Time course of peripheral CD8 T cell counts (left) and peripheral B cell counts (right) in response to treatment is shown. [Figure 5E] 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. 2 shows the time course of serum cytokines induced in response to treatment. [Figure 5F-1]We show that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune system reconstituted animals bearing WSU-DLCL2 tumors. To examine the immune phenotype of intratumoral T cell responses, WSU-DLCL2 tumor-bearing human immune system animals were sacrificed 30 days after implantation. UMAP plots of all live cells from blood, spleen, and tumor were overlaid with color-coded immune cell subsets identified by FlowSOM (Figure 5F, left). Density UMAP plots (Figure 5F, right) revealed the bias of certain populations in response to the combination therapy. [Figure 5F-2] (Continued)Ibid. [Figure 5F-3] (Continued)Ibid. [Figure 5G] 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. The density of intratumoral CD4 T cells (top right), CD8 T cells (top left), and WSU-DLCL2 cells (bottom) are shown. [Figure 5H] Combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune-reconstituted animals bearing WSU-DLCL2 tumors. UMAP plot of all intratumoral T cells overlaid with color-coded metaclusters identified by FlowSOM (left). Density UMAP plot (right) reveals bias in certain metaclusters. [Figure 5I] Figure 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors.Figure 2 shows heat maps of T cell activation, memory, and dysfunction markers used by FlowSOM to identify T cell metaclusters. [Figure 5J]Figure 1 shows that the combination of REGN5837 with odronextamab enhances peripheral and intratumoral T cell responses in human immune reconstituted animals bearing WSU-DLCL2 tumors. The frequencies of selected T cell clusters that are enriched or decreased in response to combination therapy are shown. Statistics were calculated using one-way ANOVA with Tukey's test. *P<0.05, **P<0.01, ****P<0.0001. [Figure 6A] Figure 6A shows synergistic activation of CD8 T cells in peripheral blood of cynomolgus monkeys when REGN5837 is combined with odronextamab. Cynomolgus monkeys received a single dose of REGN5837 at either 1 or 10 mg / kg (shown in brackets) combined with increasing doses of odronextamab. Blood was collected at the indicated hours after dosing. Figure 6A shows B cell counts 5 hours after dosing (left) and over the course of the experiment (right). [Figure 6B] Figure 6B shows synergistic activation of CD8+ T cells in peripheral blood of cynomolgus monkeys when REGN5837 is combined with odronextamab. Cynomolgus monkeys received a single dose of REGN5837 at either 1 or 10 mg / kg (shown in brackets) combined with increasing doses of odronextamab. Blood was collected at the indicated hours after administration. Figure 6B shows peripheral CD8+ T cell counts 5 hours after administration (left) and during the experimental period (right). [Figure 6C] Figure 6C shows synergistic activation of CD8+ T cells in peripheral blood of cynomolgus monkeys when REGN5837 is combined with odronextamab. Cynomolgus monkeys received a single dose of REGN5837 at either 1 or 10 mg / kg (shown in brackets) combined with increasing doses of odronextamab. Blood was collected at the indicated hours after dosing. Figure 6C shows ICOS upregulation on peripheral CD8+ T cells 5 hours after dosing (left) and proliferation 4 days after dosing (right). [Figure 6D]FIG. 6D shows synergistic activation of CD8 T cells in peripheral blood of cynomolgus monkeys when REGN5837 is combined with odronextamab. Cynomolgus monkeys received a single dose of REGN5837 at either 1 or 10 mg / kg (indicated in brackets) combined with increasing doses of odronextamab. Blood was collected at the indicated hours after dosing. FIG. 6D shows serum cytokines induced 5 hours after dosing. Statistics were calculated using one-way ANOVA with Tukey's test. Black stars indicate significance compared to placebo. n=3 animals / group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 7A] Figure 7 shows that expression of CD22 expression is variable in DLBCL patient resection samples. Figure 7A shows chromogenic immunohistochemical staining for CD20 and CD22 of untreated DLBCL patient resection samples. Representative images of CD22 high (left), moderate (middle), and low (right) expression. [Figure 7B] Figure 7B shows that expression of CD22 expression is variable in DLBCL patient resection samples. Representative images of DLBCL patient samples showing high (top) and low (bottom) % expression of CD22 by multiple IHC staining of PAX5, CD20, and CD22. [Figure 7C] Figure 7C shows that expression of CD22 expression is variable in DLBCL patient resection samples. Figure 7C shows the percentage of B cell marker positive cells from untreated DLBCL patient samples. [Figure 8] Figure 1 shows that CD28, CTLA4, CD80, and CD86 are detectable in DLBCL patient samples by chromogenic IHC. A shows representative images of chromogenic staining for CD28, CTLA4, CD80, and CD86 in untreated DLBCL patient resection samples (Tristar). B shows the density of CD28', CTLA4', CD86', and CD80' cells from 25 DLBCL patient samples. [Figure 9A]Figure 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell cytotoxicity and proliferation. Figure 2 shows that REGN5837, a non-targeting control antibody, or a CD28 superagonist (REGN2329) were tethered to assay plates using a wet coating method. Human PBMCs were incubated with the antibody-coated assay plates and cytokine release was measured 50-54 hours later. Data shown are from 4 individual donors. Cytokines for which a significant release response was observed compared to the non-binding control group are indicated (Tukey's post-hoc test). [Figure 9B] 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell cytotoxicity and proliferation. The number of CD22 (left) and CD20 (right) epitopes per cell of the WSU-DLCL2 cell line reported as antibody binding capacity is shown. [Figure 9C] Figure 2 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell cytotoxicity and proliferation. Figure 2 shows a summary of REGN5837-mediated human CD4 and CD8 T cell proliferation in the presence or absence of odronextamab when cultured with NALM-6 cells or Raji CD80 / CD86 DKO cells. NC: not calculated. [Figure 9D] 1 shows that REGN5837 bispecific antibody enhances odronextamab-mediated T cell cytotoxicity and proliferation. CD22 (left) and CD20 (right) expression in WSU-DLCL2 CD22KO (red) and WSU-DLCL2 CD22WT (green) lines by flow cytometry. [Figure 10A] 1 shows that treatment with odronextamab monotherapy suppresses tumor growth but does not result in complete tumor rejection in a WSU-DLBCL (DLBCL) tumor model. 2 shows the treatment scheme for WSU-DLCL2 tumors implanted in NSG animals. [Figure 10B]Figure 10B shows that treatment with odronextamab monotherapy suppresses tumor growth but does not result in complete tumor rejection in the WSU-DLBCL (DLBCL) tumor model. Figure 10B shows individual tumor growth curves in response to increasing doses of odronextamab. [Figure 10C] Figure 10C shows that treatment with odronextamab monotherapy suppresses tumor growth but does not result in complete tumor rejection in the WSU-DLBCL (DLBCL) tumor model. [Figure 10D] Figure 1 shows that treatment with odronextamab monotherapy suppresses tumor growth but does not result in complete tumor rejection in the WSU-DLBCL (DLBCL) tumor model. Survival rates are shown. Statistical significance of survival rates between treatment with isotype and increasing R1979 was calculated using the Kaplan-Meier method with the log-rank test. ***P<0.001, ****P<0.0001. [Figure 11A] Figure 11A shows that the combination of REGN5837 and odronextamab promotes intratumoral T cell proliferation and activation and killing of WSU-DLBCL2 tumor cells in vivo. Figure 11A shows the treatment scheme for immunophenotyping of WSU-DLCL2 tumors in NSG mice 26 days after implantation. [Figure 11B] Figure 11B shows that the combination of REGN5837 and odronextamab promotes intratumoral T cell proliferation and activation and killing of WSU-DLBCL2 tumor cells in vivo. Figure 11B shows the tumor burden 26 days after implantation plotted for each treatment group. [Figure 11C] Figure 11C shows that the combination of REGN5837 and odronextamab promotes intratumoral T cell proliferation and activation and killing of WSU-DLBCL2 tumor cells in vivo. Figure 11C shows the percentage of the indicated cell populations from total viable cells, showing proliferation of CD4 and CD8 T cells and a bias from WSU-DLCL2 cells. [Figure 11D]Figure 11D shows that the combination of REGN5837 and odronextamab promotes intratumoral T cell proliferation and activation and killing of WSU-DLBCL2 tumor cells in vivo. Figure 11D shows the density of intratumoral CD4 T cells plotted. [Figure 11E] Figure 11 shows that the combination of REGN5837 and odronextamab promotes intratumoral T cell proliferation and activation and killing of WSU-DLBCL2 tumor cells in vivo. Figure 11E shows the percentage of memory subsets plotted for intratumoral CD4 T cells. Statistics were calculated using one-way ANOVA with Tukey's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 12A] Figure 12 shows that REGN5837 increases IL-2 production and enhances T cell proliferation in the presence of signal 1 mediated by the allogeneic response with odronextamab. The ability of REGN5837 to mediate IL-2 release and T cell proliferation in the presence of human B cell leukemia cell lines was measured using enriched human primary T cells and allogeneic NALM-6 cells. Figure 12A shows that the assay was performed in the presence of odronextamab (500 pM) or in the absence of odronextamab, which provides signal 1 only by the allogeneic response and increasing doses of REGN5837 or a non-binding bispecific CD28 control antibody. Plates were incubated for 72 hours, at which point culture supernatants were harvested for IL-2 analysis. To assess proliferation, tritium was added and cells were incubated for an additional 16 hours. [Figure 12B] Figure 12B shows that REGN5837 increases IL-2 production and enhances T cell proliferation in the presence of signal 1 mediated alloreactivity by odronextamab. The ability of REGN5837 to mediate IL-2 release and T cell proliferation in the presence of human B cell leukemia cell lines was measured using enriched human primary T cells and allogeneic NALM-6 cells. Figure 12B shows a summary of the concentration-dependent increase in IL-2 release and T cell proliferation mediated by REGN5837 in the presence and absence of odronextamab. [Figure 13A] 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune reconstituted animals bearing DLBCL tumors. 2 shows the time course of peripheral CD4 T cell counts. [Figure 13B] 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune reconstituted animals bearing DLBCL tumors. 2 shows the time course of serum IL-10 induced in response to treatment. [Figure 13C] Shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune system reconstituted animals bearing DLBCL tumors. Mean WSU-DLCL2 tumor growth before sacrifice for immunophenotyping is shown. Statistical significance between combination therapy and isotype (black stars) or combination therapy and REGN5837 monotherapy (red stars) was calculated using two-way ANOVA and Tukey's multiple comparisons. [Figure 13D] 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune-reconstituted animals bearing DLBCL tumors. Percentages of total intratumoral viable cells are shown for WSU-DLCL2 cells (left) and T cells (right). [Figure 13E] 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune reconstituted animals bearing DLBCL tumors. Percentages of memory and activated subsets of intratumoral CD8 T cells are shown. [Figure 13F] 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune reconstituted animals bearing DLBCL tumors. Percentages of memory and activated subsets of intratumoral CD4 T cells are shown. [Figure 13G] 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune reconstituted animals bearing DLBCL tumors. 2 shows the intratumoral density of memory CD8 T cells. [Figure 13H] Figure 1 shows that the combination of odronextamab and REGN5837 expands peripheral and intratumoral T cells in human immune reconstituted animals bearing DLBCL tumors. Intratumoral density of memory CD4 T cells is shown. Statistics were calculated using one-way ANOVA with Tukey's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14A] Figure 14A shows that odronextamab efficiently depletes splenic and blood B cells while promoting effector memory T cell induction in human immune reconstituted animals bearing DLBCL tumors. Figure 14A shows the percentage of splenic B cells (left) and T cells (right) 30 days after implantation of WSU-DLCL2 tumors. [Figure 14B] Figure 14B shows that odronextamab efficiently depletes splenic and blood B cells while promoting effector memory T cell induction in human immune reconstituted animals bearing DLBCL tumors. Figure 14B shows the percentage of blood B cells (left) and T cells (right) 30 days after implantation of WSU-DLCL2 tumors. [Figure 14C] Figure 14C shows that odronextamab efficiently depletes splenic and blood B cells while promoting effector memory T cell induction in human immune reconstituted animals bearing DLBCL tumors. Figure 14C shows enumeration of B and T cell subsets in the spleen. [Figure 14D] Figure 14D shows that odronextamab efficiently depletes splenic and blood B cells while promoting effector memory T cell induction in human immune reconstituted animals bearing DLBCL tumors. Figure 14D shows a density UMAP plot of total viable cells in blood (right). [Figure 14E] Figure 14C shows that odronextamab efficiently depletes splenic and blood B cells while promoting effector memory T cell induction in human immune reconstituted animals bearing DLBCL tumors. Figure 14E shows the enumeration of blood B and T cell subsets. [Figure 15A]Figure 15A shows the synergistic activation of CD4 T cells in peripheral blood of cynomolgus monkeys when REGN5837 is combined with odronextamab. Figure 15A shows peripheral CD4 T cell counts 4 days after dosing (left) and over the course of the experiment. [Figure 15B] Figure 15B shows synergistic activation of CD4+ T cells in peripheral blood of cynomolgus monkeys when REGN5837 is combined with odronextamab. Figure 15B shows ICOS upregulation on peripheral CD4+ T cells 5 hours after dosing (left) and proliferation 4 days after dosing (right). Statistics were calculated using one-way ANOVA with Tukey's test. Black stars indicate significance compared to placebo. n=3 animals / group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 16] 1 shows that CD22 expression in DLBCL patient samples is more variable than CD20 expression. [Figure 17] We show that in mixed cultures of CD22WT and CD22KO tumor cells, the combination of CD22xCD28 and CD20xCD3 can result in enhanced T cell killing of bystander CD22KO target cells. [Figure 18] 1 shows exemplary expression profiles of EGFR target antigens in several cancers. [Figure 19] 1 shows exemplary expression profiles of PMSA target antigens in several cancers. [Figure 20] 1 shows exemplary expression profiles of CA9 target antigens in several cancers. [Figure 21] 1 shows exemplary expression profiles of HER2 target antigens in several cancers. [Figure 22] 1 shows exemplary expression profiles of SLAMF7 target antigens in several cancers. [Figure 23] 1 shows exemplary expression profiles of MUC16 target antigens in several cancers. [Figure 24] 1 shows exemplary expression profiles of FOLR1 target antigens in several cancers. [Diagram 25] 1 shows exemplary expression profiles of CD38 target antigens in several cancers. [Figure 26] 1 shows exemplary expression profiles of CD22 target antigens in several cancers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] As shown herein, CD22 on T cells + Incubation of target cells expressing CD3A with an anti-CD28 / anti-CD22 bispecific antibody enhanced tumor cell lysis mediated by a bispecific CD3A antibody. The disclosure herein shows that combination therapy with an anti-CD28 / anti-CD22 bispecific antibody enhances the lysis of tumor cells mediated by CD22A. + and CD22 - The method is based in part on the discovery that the use of a bispecific CD3 antibody enhanced tumor cell lysis mediated by both CD22 and CD3+ antibodies in a target population. + and CD22 - CD4 and CD8 T cell activation was also enhanced in both target populations. Thus, Applicants demonstrate that an anti-CD28 bispecific antibody targeting a tumor-associated antigen can enhance the killing of tumor cells lacking that tumor-associated antigen.

[0047] Provided herein are methods and compositions for administering to a subject a multispecific antigen-binding molecule having a first antigen-binding region specific for a target antigen and a second antigen-binding region specific for a CD28 protein, thereby resulting in the killing of tumor cells within a tumor in the subject, wherein the tumor cells do not express or are not expected to express the target antigen. In some embodiments, at least a subset of tumor cells within the tumor do not express the target antigen.

[0048] In some aspects, provided herein are methods and compositions for inducing killing of tumor cells and / or inducing T cell activation against tumor cells in a tumor in a subject, the methods comprising administering to the subject a multispecific antigen binding molecule having a first antigen binding region specific for a target antigen and a second antigen binding region specific for a CD28 protein. In some embodiments, at least a subset of tumor cells in the tumor do not express the target antigen.

[0049] Also provided herein are methods and compositions for treating cancer in a subject having a tumor, the methods comprising administering to the subject a multispecific antigen-binding molecule having a first antigen-binding region specific for a target antigen and a second antigen-binding region specific for a CD28 protein. In some embodiments, at least a subset of tumor cells within the tumor do not express the target antigen.

[0050] As used herein, when referring to tumor cells within a tumor that "do not express the target antigen," this phrase encompasses the experimental discovery or confirmation that at least a subset of tumor cells do not express the target antigen, as well as methods that involve predicting that tumor cells do not express the target antigen (i.e., with or without further experimental confirmation).

[0051] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigen is an antigen associated with the tumor microenvironment (e.g., the microenvironment of a tumor in a subject). For example, in some embodiments, the target antigen is an antigen on an immune cell, on a tumor cell stroma, or on an extracellular matrix within the tumor microenvironment. Examples of extracellular matrix antigens include nectin (e.g., nectin-3 or nectin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM).

[0052] definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0053] As used herein, the term "administering" or "administration" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration. Such pharmaceutical agents may contain, for example, a bispecific antibody or bispecific antibody fragment provided herein.

[0054] As used herein, the term "antibody" can refer to both intact antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as V H Each light chain comprises a light chain variable region (abbreviated herein as V L V H Area and V L The regions can be further subdivided into hypervariable regions called "complementarity determining regions" (CDRs), separated by more conserved regions called "framework regions" (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The term "antibody" includes, for example, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), single chain antibodies, and antigen-binding antibody fragments.

[0055] As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antibody refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), e.g., CDR3 peptides) or FR3-CDR3-FR4 constrained peptides). Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the scope of the expression "antigen-binding fragment" as used herein.

[0056] Furthermore, the term "antibody" (unless otherwise specified or otherwise clear from the context) includes, but is not limited to, intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies, e.g., IgNAR or fragments thereof), camelid antibodies, camelized antibodies, masked antibodies (e.g., Probody®), affibodies, anti-idiotypic (anti-Id) antibodies (e.g., anti-anti-Id antibodies), Small Modular The antibodies may include any construct or format known in the art that utilizes the structural and / or functional characteristics of antibodies, including ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAbs®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibobodies®, TrimerX®, MocroProteins, Fynomers®, Centyrin®, and KALBITOR®, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0057] The antibody used in the present invention may be a bispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by hybrid hybridomas or triomas are disclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S. Patent No. 5,959,084. Bispecific antibody fragments are described in U.S. Patent No. 5,798,229.

[0058] Bispecific agents can also be produced by creating heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identifying clones that produce and coassemble both antibodies. Bispecific agents can also be produced by chemical conjugation or genetic joining of full-length immunoglobulin chains or portions thereof, such as Fab and Fv sequences.

[0059] An antibody may be "humanized", which is intended to encompass antibodies made by non-human cells with variable and constant regions that have been modified to more closely resemble antibodies made by human cells, e.g., by modifying a non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. A humanized antibody of the present disclosure may, for example, include amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "humanized antibody" also encompasses antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0060] As used herein, "cancer-associated fibroblasts" (CAFs) encompass fibroblasts found in and around tumor tissue that are activated from normal tissue-resident fibroblasts or transdifferentiated from non-fibroblast lineages, e.g., epithelial cells and adipocytes, by stimuli in the tumor microenvironment. Exemplary cancer-associated fibroblast antigens include α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), S100A4, platelet-derived growth factor receptor (PDGFRα / β), vimentin, PDPN, CD70, CD10, GPR77, CD10, CD74, CD146, CAV1, Saa3-, and CD49e. Further details regarding antigens and biomarkers can be found in Han, C., Liu, T. & Yin, R. Biomarkers for cancer-associated fibroblasts. Biomark Res 8, 64 (2020).

[0061] "Cancer" broadly refers to the uncontrolled, abnormal proliferation of the host's own cells, resulting in invasion of surrounding tissues and potentially tissues distal to the initial site of abnormal cell proliferation within the host. Major types include carcinomas, which are cancers of epithelial tissues (e.g., skin, squamous cells); sarcomas, which are cancers of connective tissues (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias, which are cancers of hematopoietic tissues (e.g., bone marrow tissue); lymphomas and myelomas, which are cancers of immune cells; and central nervous system cancers, including cancers originating from brain and spinal cord tissue. "Cancer(s)" and "neoplasm(s)" are used interchangeably herein. As used herein, "cancer" refers to all types of cancers or neoplasms or malignant tumors, including leukemias, carcinomas, and sarcomas, whether new or recurrent. Specific examples of cancers are carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed tumors. Non-limiting examples of cancers are new or recurrent cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, and medulloblastoma. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a metastasis.

[0062] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell, such as an antibody-based specificity for a desired antigen (e.g., a tumor antigen), together with a T cell receptor activating intracellular domain to create a chimeric protein that exhibits specific anti-target cell immune activity. Generally, CARs consist of an extracellular single-chain antigen binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0063] As used herein, the phrase "co-administration" or "co-administered" refers to any administration form of two or more different therapeutic agents, such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective simultaneously in the subject, which may include the synergistic effect of the two agents). For example, the different therapeutic agents can be administered in the same formulation, or in separate formulations, either simultaneously or sequentially. In certain embodiments, the different therapeutic agents can be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or within 1 week of each other. Thus, subjects undergoing such treatment can benefit from the combined effect of the different therapeutic agents.

[0064] A "costimulatory domain" or "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. The costimulatory domain can be a human costimulatory domain. Exemplary costimulatory molecules include CD28, 4-1BB, CD27, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0065] "Costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation activation, differentiation, etc. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3.

[0066] A "costimulatory signal" refers to a signal that, in combination with a primary signal, causes T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0067] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. An epitope may be a conformational epitope or a linear epitope. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is one formed by adjacent amino acid residues of a polypeptide chain. In certain circumstances, an epitope may include a portion of a saccharide, phosphoryl group, or sulfonyl group on an antigen.

[0068] As used herein, "immune cells" include any white blood cells that develop from stem cells in bone marrow. Examples include macrophages, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, natural killer cells, T cells, or B cells. Immune cells can be present in tumors or tumor microenvironments.

[0069] As used herein, the phrase "pharmacologically acceptable" refers to those agents, compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0070] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in the transfer or transport of a drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; and (10) gluten. (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol, (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) aqueous pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, and (22) other non-toxic compatible substances used in pharmaceutical formulations.

[0071] As used herein, a therapeutic agent that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0072] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is at least about 95% nucleotide sequence identity of the nucleotide bases, more preferably at least about 96%, 97%, 98%, or 99%, as measured by any well-known sequence identity algorithm, e.g., FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0073] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, more preferably at least 98% or 99% sequence identity, when optimally aligned, for example, by the GAP or BESTFIT programs using default gap weights. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol.Biol.24:307-331, incorporated herein by reference. Examples of amino acid groups with side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0074] The term "specifically binds" or "specific binding" as used herein when referring to a polypeptide refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biological products. Thus, under given conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an antigen) if the particular ligand or antibody does not bind in significant amounts to other proteins present in the sample or to other proteins that the ligand or antibody may come into contact with in an organism. Generally, a first molecule that "specifically binds" to a second molecule has a binding affinity of about 10 to the second molecule. 5 M -1 (For example, 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1 For example, in the case of the ability of a PIG-specific CAR to bind a peptide presented on MHC (e.g., class I MHC or class II MHC), the CAR typically specifically binds to that peptide / MHC with an affinity of at least about 10-4 M or less, KD, and binds to a given antigen / binding partner with an affinity (represented by KD) that is at least 10-fold, at least 100-fold, or at least 1000-fold lower than the affinity of binding to a nonspecific and unrelated peptide / MHC complex (e.g., one that contains BSA or casein peptides).

[0075] As used herein, the term "subject" refers to a human or non-human animal that is selected for treatment or therapy.In certain embodiments provided herein, the subject is a human subject.In some embodiments provided herein, the subject is a subject that requires the methods provided herein, for example, a subject that has cancer.

[0076] "Tumor microenvironment" refers to the cellular environment in which a tumor resides, including, for example, the stroma, the interstitial fluid surrounding the tumor, surrounding blood vessels, immune cells, other cells, fibroblasts, signaling molecules, and the extracellular matrix.

[0077] As used herein, the term "treatment" refers to a clinical intervention designed to change the natural course of the individual being treated during the course of a clinical condition. The desired effects of treatment include reducing the rate of progression, improving or alleviating the pathological condition, and remission or improving the prognosis of a particular disease, disorder, or condition. For example, an individual is successfully "treated" when one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0078] As used herein, the phrases "therapeutically effective amount" and "effective amount" mean an amount of an agent effective to produce a desired therapeutic effect in at least a subpopulation of cells of a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0079] therapeutic antibodies General In certain aspects, the methods and compositions provided herein relate to the use of therapeutic antibodies (e.g., bispecific antibodies disclosed herein, e.g., bispecific antibodies having an antigen-binding region specific for a first target antigen and an antigen-binding region specific for a CD28 protein, optionally administered with a second bispecific anti-CD3 antibody and / or an immune checkpoint inhibitor).

[0080] As mentioned above, the term "antibody" as used herein encompasses both complete antibody molecules and antigen-binding fragments of complete antibody molecules. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), e.g., CDR3 peptides) or FR3-CDR3-FR4 constrained peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the scope of the expression "antigen-binding fragment" as used herein.

[0081] Antigen-binding fragments of antibodies usually contain at least one variable domain. The variable domain may be of any size or amino acid composition and usually contains at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be located in any suitable configuration relative to each other. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragments of antibodies may contain monomeric VH or VL domains.

[0082] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody disclosed herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies disclosed herein may include homodimers or heterodimers (or other multimers) of any of the configurations of variable and constant domains listed above in non-covalent association (e.g., by disulfide bond(s)) with each other and / or with one or more monomeric VH or VL domains.

[0083] As with complete antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies usually contain at least two different variable domains, where each variable domain can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the antigen-binding fragments of antibodies disclosed herein using routine techniques available in the art.

[0084] In certain embodiments provided herein, at least one variable domain of a multispecific antibody can specifically bind to a T cell costimulatory domain, e.g., CD28. In certain embodiments provided herein, at least one variable domain of a multispecific antibody disclosed herein can specifically bind to CD3.

[0085] In some embodiments, the antibodies provided herein may function by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies disclosed herein in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound on target cells, thereby resulting in the lysis of the target cells. CDC and ADCC are well known in the art and can be measured using available assays (see, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, an antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0086] In certain embodiments provided herein, the multispecific (e.g., bispecific or trispecific) antibodies provided herein are human antibodies. As used herein, the term "human antibody" is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies disclosed herein may contain amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) not encoded by human germline immunoglobulin sequences, for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0087] In some embodiments, the antibodies provided herein may be recombinant human antibodies. As used herein, the term "recombinant human antibodies" is intended to encompass all human antibodies that are produced, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from the combination of recombinant human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means, including the insertion of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0088] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked by interchain disulfide bonds, forming an approximately 75-80 kDa molecule (half antibody) composed of covalently linked light and heavy chains. These forms have been very difficult to separate even after affinity purification.

[0089] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level normally observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present disclosure encompasses antibodies with one or more mutations in the hinge, CH2, or CH3 regions, which may be desirable, for example, to improve the yield of the desired antibody form in manufacturing.

[0090] Sequence variants The monospecific or multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can easily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and / or CDR residues in the VH and / or VL domains are backmutated to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, in which certain individual residues in the framework and / or CDR regions are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, enhanced binding (e.g., as assessed by measuring cell-binding titers or FACS binding) or binding affinity (e.g., KD), improved or enhanced biological antagonist or agonist properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the scope of the present disclosure.

[0091] In some embodiments, the multispecific (e.g., bispecific or trispecific) antibodies provided herein comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, in certain embodiments, the anti-CD40 antagonist antibodies or multispecific (e.g., bispecific or trispecific) CD3 antibodies provided herein have HCVR, LCVR, and / or CDR amino acid sequences that have, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0092] Fc variants According to certain embodiments provided herein, antibodies and multispecific antigen-binding molecules are provided that include an Fc domain that includes one or more mutations that enhance or attenuate binding of the antibody to the FcRn receptor, for example, at acidic pH compared to neutral pH. For example, the disclosure includes antibodies that include a mutation in the CH2 or CH3 region of the Fc domain, where the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH ranging from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0093] For example, the present disclosure includes multispecific antigen binding molecules (e.g., anti-CD28 / anti-TAA bispecific antibodies or anti-CD3 / anti-TAA bispecific antibodies) comprising an Fc domain that includes one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations, as well as other mutations in the antibody variable domains disclosed herein, are contemplated herein.

[0094] bioequivalence Provided herein are antigen-binding molecules that have amino acid sequences that differ from the amino acid sequences of the exemplary molecules disclosed herein, but retain the ability to bind to the same antigen or antigens. Such variant molecules may contain one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but exhibit essentially the same biological activity as the biological activity of the bispecific antigen-binding molecules described.

[0095] The present disclosure includes antigen-binding molecules that are biologically equivalent to any of the exemplary antigen-binding molecules described herein. Two antigen-binding proteins or antibodies are considered biologically equivalent if, for example, they are pharmacopoeial equivalents or pharmacopoeial alternatives whose rate and extent of absorption do not show significant differences when administered in either single or repeated doses at the same molar dose under similar experimental conditions. Some antigen-binding proteins are considered equivalent or pharmacopoeial alternatives when they are equivalent in degree of absorption but not in absorption rate, and can still be considered biologically equivalent because such differences in absorption rate are intentional and reflected in the label, e.g., are not important for achieving effective body drug concentration in chronic use, and are not medically significant with respect to the particular formulation being studied.

[0096] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0097] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be switched between the reference and biologic products one or more times without expected increased risk of adverse effects, e.g., clinically relevant changes in immunogenicity, or decreased efficacy, compared to continued treatment without switching.

[0098] In one embodiment, two antigen binding proteins are bioequivalent if they both act in a condition or conditions of use by a common mechanism or mechanism of action, to the extent such mechanism is known.

[0099] Bioequivalence may be demonstrated by in vivo and in vitro methods. Methods for measuring bioequivalence include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites in blood, plasma, serum, or other biological fluid is measured as a function of time; (b) in vitro studies that correlate with and reasonably predict in vivo human bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time; and (d) well-controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0100] Biologically equivalent variants of the exemplary bispecific antigen-binding molecules described herein can be constructed, for example, by making various substitutions of residues or sequences, or by removing terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be removed or replaced with other amino acids to prevent the formation of unnecessary or improper intramolecular disulfide bridges during renaturation. In other contexts, biologically equivalent antigen-binding proteins can include variants of the exemplary bispecific antigen-binding molecules described herein that contain amino acid changes that alter the glycosylation characteristics of the molecule, for example, mutations that eliminate or remove glycosylation.

[0101] antibody binding As used herein, the term "binding" refers to an interaction or association between at least two entities or molecular structures, e.g., an antibody-antigen interaction, typically in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment or Fc-containing protein to any given antigen, e.g., a cell surface protein or fragment thereof.

[0102] For example, binding affinity, when measured, for example, by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument, using an antigen as the ligand and an antibody, Ig, antibody-binding fragment, or Fc-containing protein as the analyte (or antiligand), typically corresponds to a KD value of about 10-7 M or less, e.g., about 10-8 M or less, e.g., about 10-9 M or less. Cell-based binding strategies, such as binding assays by fluorescence-activated cell sorting (FACS), are also routinely used, and FACS data correlate well with other methods such as radioligand competitive binding and SPR (Benedict, CA, J Immunol Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J Immunol Methods. 2005, 302(1-2):68-77).

[0103] Thus, the antibodies or antigen-binding proteins provided herein bind to a given antigen or cell surface molecule (receptor) with an affinity corresponding to a KD value that is at least 10-fold lower than the binding affinity to a non-specific antigen (e.g., BSA, casein). According to the present disclosure, antibody affinities corresponding to KD values ​​that are 10-fold lower or lower than the non-specific antigen may be considered undetectable binding, although such antibodies may be combined with a second antigen-binding arm to create a bispecific antibody as disclosed herein.

[0104] The term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. There is an inverse correlation between KD and binding affinity, such that the smaller the KD value, the higher, i.e., the stronger the affinity. Thus, the term "higher affinity" or "stronger affinity" refers to a higher ability to form an interaction, and thus a smaller KD value, and conversely, the term "lower affinity" or "weaker affinity" refers to a lower ability to form an interaction, and thus a larger KD value. In some cases, the higher binding affinity (or KD) of a particular molecule (e.g., an antibody) to an interaction partner molecule (e.g., an antigen X) compared to the binding affinity of that molecule (e.g., an antibody) to another interaction partner molecule (e.g., an antigen Y) can be expressed as a binding ratio determined by dividing the higher KD value (lower or weaker affinity) by the smaller KD (higher or stronger affinity), and is expressed, for example, as a 5-fold or 10-fold higher binding affinity in some cases.

[0105] The term "kd" (sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody-binding fragment. The value is also referred to as the koff value.

[0106] The term "ka" (M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0107] The term "kA" (M-1 or 1 / M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing ka by kd.

[0108] The term "EC50" or "EC50" refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a certain exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximum effect is observed. In certain embodiments, the EC50 value is equal to the concentration of an antibody disclosed herein that exhibits half-maximal binding to cells expressing CD28 or tumor-associated antigens (e.g., antigens disclosed in Table 2), for example, as measured by FACS binding assay. Thus, with increased EC50 value or half-maximal effective concentration value, reduced or weaker binding is observed.

[0109] In one embodiment, a decrease in binding of a multispecific CD28 antibody may be defined as an increase in the EC50 antibody concentration that allows binding to half-maximal amounts of target cells.

[0110] In another embodiment, the EC50 value represents the concentration of a multispecific CD28 antibody disclosed herein that induces half-maximal depletion of target cells by T cell cytotoxic activity. Thus, at reduced EC50 or half-maximal effective concentration values, enhanced cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed.

[0111] Preparation of antigen-binding domains and construction of bispecific molecules An antigen-binding domain specific to a particular antigen can be prepared by any antibody production technique known in the art. Once obtained, two different antigen-binding domains specific to two different antigens (e.g., CD28 and a human tumor antigen) can be appropriately positioned relative to each other to create a bispecific antigen-binding molecule disclosed herein using routine methods. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecules disclosed herein can be derived from chimeric, humanized, or fully human antibodies. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecules disclosed herein can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody production technique), a high affinity chimeric antibody against a particular antigen (e.g., CD28 or a human tumor-associated antigen) with human variable regions and mouse constant regions is first isolated. Antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. To generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules disclosed herein, the mouse constant regions are replaced with the desired human constant regions.

[0112] Genetically engineered animals can be used to generate human bispecific antigen-binding molecules. For example, genetically modified mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, and the mice express only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically modified mice can be used to generate fully human bispecific antigen-binding molecules that contain two different heavy chains associated with the same light chain that contains a variable domain derived from one of two different human light chain variable region gene segments (see US2011 / 0195454). Fully human refers to an antibody or antigen-binding fragment or immunoglobulin domain that contains an amino acid sequence encoded by DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain. In some examples, the fully human sequence is derived from a protein endogenous to humans. In other examples, the fully human protein or fully human protein sequence includes a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any theory, chimeric proteins or sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, e.g., compared to any wild-type human immunoglobulin region or domain.

[0113] Multispecific CD28 antigen binding molecule In certain embodiments, the methods and compositions provided herein relate to CD28 antigen binding molecules (i.e., antigen binding molecules that comprise at least one antigen binding region that binds to CD28). In certain embodiments, the multispecific CD28 antigen binding molecules provided herein further comprise an antigen binding domain that binds to a target antigen (e.g., an antigen expressed on a cancer cell, e.g., a tumor-associated antigen (TAA)).

[0114] In certain embodiments, provided herein is a second antigen binding molecule that is a multispecific CD3 antigen binding molecule provided herein, which further comprises an antigen binding domain that binds to a target antigen (e.g., an antigen expressed on a cancer cell, e.g., a tumor-associated antigen (TAA)).

[0115] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigen is an antigen associated with the tumor microenvironment (e.g., the microenvironment of a tumor in a subject). For example, in some embodiments, the target antigen is an antigen on an immune cell, on a tumor cell stroma, or on an extracellular matrix within the tumor microenvironment. Examples of extracellular matrix antigens include nectin (e.g., nectin-3 or nectin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM).

[0116] As used herein, the term "multispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex that comprises at least a first antigen-binding region and a second antigen-binding region. In some embodiments, each antigen-binding domain in a multispecific antigen-binding molecule may comprise at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In certain embodiments, the first antigen-binding domain specifically binds to a first antigen (e.g., CD28), and the second antigen-binding domain specifically binds to a different second antigen (e.g., a tumor-associated antigen).

[0117] In some embodiments, the multispecific CD28 antigen binding molecule is a multispecific CD28 antibody, for example, a bispecific CD28 antibody. The CD28 multispecific antibody provided herein can be, for example, bispecific or trispecific. A multispecific antibody can be specific for different epitopes of one target polypeptide, or can contain antigen binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J.Immunol.147:60-69; Kufer et al., 2004, Trends Biotechnol.22:238-244. The bispecific CD28 antibody provided herein can be linked to or co-expressed with another functional molecule, for example, another peptide or protein. For example, to create a bispecific or multispecific antibody having second or additional binding specificities, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, e.g., another antibody or antibody fragment.

[0118] As used herein, the term "CD28" refers to cluster of differentiation 28, a protein expressed on T cells that provides a costimulatory signal required for T cell activation and survival. T cell stimulation by CD28 in addition to the T cell receptor (TCR) can generate a strong signal for the production of various interleukins. Human CD28 comprises the amino acid sequence as follows: Table 1: Human protein sequence of CD28 >NP_001230006.1;GeneID=940;Isoform 2 precursor MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSWKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >NP_001230007.1;GeneID=940;Isoform 3 precursor MLRLLLALNLFPSIQVTGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >NP_006130.1;GeneID=940; Isoform 1 precursor MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >XP_011510496.1;GeneID=940; Isoform X1 MPCGLSALIMCPKGMVAVVVAVDDGDSQALAGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >XP_011510497.1;GeneID=940; Isoform X2 MPCGLSALIMCPKGMVAVVVAVDDGDSQALAGNKILVKQSPMLVAYDNAVNLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS >XP_011510499.1;GeneID=940; Isoform X3 MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0119] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly stated to be from a non-human species. Thus, the term "CD28" refers to human CD28, unless expressly stated to be from a non-human species, e.g., "mouse CD28," "monkey CD28," etc.

[0120] As used herein, the phrase "cell surface expressed CD28" refers to one or more CD28 proteins (or proteins) expressed on the surface of a cell in vitro or in vivo such that at least a portion of the CD28 protein is exposed to the extracellular side of the cell membrane and is accessible to contact with the antigen-binding portion of an antibody. Cell surface expressed CD28 includes CD28 protein in the context of a functional T cell receptor on the cell membrane. Cell surface expressed CD28 may include or consist of CD28 protein expressed on the surface of a cell that normally expresses CD28 protein. Alternatively, cell surface expressed CD28 may include or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD28 on its surface but has been artificially engineered to express CD28 on its surface.

[0121] In some embodiments, the disclosure includes bispecific antibodies in which one immunoglobulin arm binds CD28 and the other immunoglobulin arm is specific for a target antigen (e.g., a tumor antigen or "TAA"). In some embodiments, the disclosure includes trispecific antibodies in which a first immunoglobulin arm binds CD28, a second immunoglobulin arm is specific for a tumor antigen, and a third immunoglobulin arm binds an additional T cell antigen (e.g., CD3) or an additional tumor antigen.

[0122] In some embodiments, the multispecific CD28 antibody may comprise any of the antibodies disclosed in US2020 / 0239576. In some embodiments, the CD28 binding arm may comprise any of the HCVR / LCVR or CDR amino acid sequences disclosed in US2020 / 0239576. In certain embodiments, the CD28 binding arm binds to human CD28 and induces human T cell activation. In certain embodiments, the CD28 binding arm binds weakly to human CD28 and induces human T cell activation. In other embodiments, the CD28 binding arm binds weakly to human CD28 and induces the killing of cells expressing tumor-associated antigens in the context of a bispecific or multispecific antibody.

[0123] In certain embodiments, a multispecific antibody or antigen-binding fragment used in the present disclosure comprises an antigen-binding arm that binds to ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2 to induce T cell activation.

[0124] In certain embodiments, the multispecific CD28 antigen binding molecule comprises an antigen binding domain specific for a tumor-associated antigen.

[0125] In certain embodiments, the tumor-associated antigen is an immune tumor antigen. In certain embodiments, the tumor-associated antigen is a non-immune tumor antigen.

[0126] The tumor-associated antigen may be any of the antigens selected from Table 2 below. [Table 1-1] [Table 1-2]

[0127] In certain embodiments, the tumor associated antigen is AIM-2, ALDH1A1, alpha-actinin 4, alpha fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCMA, BCR-ABL fusion protein b3a2, β-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD20, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin A, cyclin B ... linD1, cyclin A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAS7, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Rα2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, lengusin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAG E-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, MUC16, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP,PAX5, PBF, pml-RARα fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, Secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, S Selected from TEAP1, STEAP2, survivin, SYT-SSX1 or SYT-SSX2 fusion proteins, TAG-1, TAG-2, telomerase, TGF-βRII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, and XAGE-1b / GAGED2a.

[0128] In some embodiments, tumor associated antigens may include ADAM17, BCMA, CA-IX, CD19, CD20, CD21, CD22, CD24, CD30, CD33, CD38, CD52, CD56, CD70, CD72, CD74, CD79b, CD123, CD138, CDH3, CEA, EphA2, EpCAM, ERBB2, ENPP3, EGFR, EGFR-vIII, FLT3, FOLRLl, GD-2, Glypican-3, gpA33, GPNMB, GPRC5D, HER2, HER3, LMP1, LMP2A, MUC16, mesothelin, PSMA, PSCA, RON, ROR1, ROR2, STEAP1, STEAP2, SSTR2, SSTR5, 5T4, and Trop-2. In some embodiments, the tumor antigen may be CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, or BCMA.

[0129] In some embodiments, the tumor-associated antigen may be a non-immune related antigen selected from CD38, EGFR, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7.

[0130] In some embodiments, the tumor-associated antigen can be an immune-related antigen selected from CD22, CD20, CD72, CD19, CD21, CD24, and CD79.

[0131] CD22 (Siglec2) is a receptor expressed on the plasma membrane of B cells. CD22 mediates B cell / B cell interactions. It may also be involved in the localization of B cells in lymphoid tissues. CD22 binds sialylated glycoproteins, one of which is CD45. CD22 also preferentially binds α-2,6-linked sialic acid. It also plays a role in positive regulation by interacting with Src family tyrosine kinases and may act as an inhibitory receptor by recruiting cytoplasmic phosphatases via their SH2 domains that block signaling by dephosphorylating signaling molecules.

[0132] CD20 is a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. CD20 is considered a B cell tumor-associated antigen because it is expressed by more than 95% of B cell non-Hodgkin's lymphomas (NHL) and other B cell malignancies, and is absent from precursor B cells, dendritic cells, and plasma cells. Human CD20 protein has the amino acid sequence shown in SEQ ID NO:5 of U.S. Patent Application No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0133] MUC16 refers to mucin 16. MUC16 is a single transmembrane domain, highly glycosylated integral membrane protein that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is set forth in SEQ ID NO: 1899 of US Patent Application No. US2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety.

[0134] BCMA refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is shown in SEQ ID NO: 115 of US Patent Application No. US2020 / 0024356, the contents of which are incorporated herein by reference in their entirety. It can also be found in GenBank Accession No. NP_001183.2.

[0135] PSMA refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is a non-deciduomeric integral membrane glycoprotein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. The amino acid sequence of human PSMA is set forth in SEQ ID NO: 7 of US Patent Application No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0136] In some embodiments, the CD28 multispecific antibody may be a bispecific CD28xCD19 antibody, a bispecific CD28xCD22 antibody, a bispecific CD28xCD20 antibody, a bispecific CD28xCD72 antibody, a bispecific CD28xCD20 antibody, a bispecific CD28xCD19 antibody, a bispecific CD28xCD21 antibody, a bispecific CD28xCD24 antibody, or a bispecific CD28xCD79 antibody.

[0137] In some embodiments, the multispecific CD28 antibody may be a bispecific CD28xCD38 antibody, a bispecific CD28xEGFR antibody, a bispecific CD28xMUC16 antibody, a bispecific CD28xPSMA antibody, a bispecific CD28xCA9 antibody, a bispecific CD28xCD20 antibody, a bispecific CD28xFOLR1 antibody, a bispecific CD28xHER2 antibody, and a bispecific CD28xSLAM7 antibody.

[0138] In certain embodiments, the multispecific antigen-binding molecule is a multispecific antibody or an antigen-binding fragment thereof. Each antigen-binding domain of the multispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be denoted with the prefix "A1" and the CDRs of the second antigen-binding domain may be denoted with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3. In the context of a trispecific antigen-binding molecule (e.g., a trispecific antibody) comprising a first, second, and third antigen-binding domain, the CDRs of the first antigen-binding domain may be denoted with the prefix "A1", the CDRs of the second antigen-binding domain may be denoted with the prefix "A2", and the CDRs of the third antigen-binding domain may be denoted with the prefix "A3". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3, and the CDRs of the third antigen-binding domain may be referred to herein as A3-HCDR1, A3-HCDR2, and A3-HCDR3.

[0139] The bispecific antigen-binding molecule described above or herein can be a bispecific antibody. In some examples, the bispecific antibody comprises a human IgG heavy chain constant region. In some examples, the human IgG heavy chain constant region is of isotype IgG1. In some examples, the human IgG heavy chain constant region is of isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0140] In some embodiments, the multispecific CD28 antibody is any of the multispecific antibodies in Table 3. [Table 2]

[0141] In some embodiments, the target antigen is an antigen associated with the tumor microenvironment of the tumor. As used herein, antigens associated with the tumor microenvironment include any antigen on cells in the stroma, interstitial fluid surrounding the tumor, blood vessels surrounding the tumor, and extracellular matrix. Also included are antigens associated with immune cells, other cells, fibroblasts, or signaling molecules in the tumor microenvironment.

[0142] In some embodiments, the target antigen is an antigen associated with the tumor stroma selected from PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin isoform EDB.

[0143] In some embodiments, the target antigen is an antigen associated with the extracellular matrix of a tumor selected from nectin (e.g., nectin-3 or nectin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) proteins.

[0144] In some embodiments, the target antigen is an antigen expressed on the surface of cancer-associated fibroblasts (e.g., alpha-smooth muscle actin (alpha-SMA), fibroblast activation protein (FAP), S100A4, platelet-derived growth factor receptor (PDGFRα / β), vimentin, PDPN, CD70, CD10, GPR77, CD10, CD74, CD146, CAV1, Saa3-, or CD49e).

[0145] In some embodiments, the target antigen is an antigen expressed on the surface of blood vessels within the tumor microenvironment, such as DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, and VEGFR2.

[0146] In some embodiments, the target antigen is an immune antigen. In some embodiments, the immune antigen is an antigen expressed on the surface of an immune cell. The immune cell can be a macrophage, neutrophil, eosinophil, basophil, mast cell, monocyte, dendritic cell, natural killer cell, T cell, or B cell. In some embodiments, the immune cell infiltrates a tumor or the tumor microenvironment of the tumor. The immune antigen can be selected from any of the immune antigens listed in Table 4. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0147] The first antigen-binding domain and the second antigen-binding domain can be linked to each other directly or indirectly to form the bispecific antigen-binding molecule disclosed herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be linked to a separate multimerization domain. The association of one multimerization domain with another multimerization domain promotes the association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with another multimerization domain of the same or similar structure or composition. For example, the multimerization domain can be a polypeptide that includes an immunoglobulin CH3 domain. A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin (including a CH2-CH3 domain), such as an IgG Fc domain selected from isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0148] A bispecific antigen-binding molecule disclosed herein typically comprises two multimerization domains, e.g., two Fc domains, each part of an individual, separate antibody heavy chain. The first and second multimerization domains can be, for example, of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains can be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0149] In certain embodiments, the multimerization domain is an Fc fragment containing at least one cysteine ​​residue or an amino acid sequence of 1 to about 200 amino acids in length. In other embodiments, the multimerization domain is a cysteine ​​residue, or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that include or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0150] Any bispecific antibody format or technology can be used to create the bispecific antigen-binding molecule disclosed herein.For example, to create a bispecific antigen-binding molecule, an antibody or its fragment with a first antigen-binding specificity can be functionally linked (for example, by chemical coupling, gene fusion, non-covalent association, or another method) to one or more other molecular entities, such as another antibody or antibody fragment with a second antigen-binding specificity. Certain exemplary bispecific formats that can be used in the methods provided herein include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knob-in-hole, common light chain (e.g., common light chain with knob-in-hole), CrossMab, CrossFab, SEED body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6,1-11 and references cited therein).

[0151] In the context of the bispecific antigen-binding molecules provided herein, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the present disclosure includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain that exhibits an altered (e.g., enhanced or weakened) binding interaction between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule contains a modification in the CH2 or CH3 region, where the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P).

[0152] In certain embodiments, provided herein is a bispecific antigen-binding molecule comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds to Protein A and the second Ig CH3 domain contains a mutation that reduces or eliminates Protein A binding, e.g., an H95R modification (according to IMGT exon numbering; H435R in EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F in EU). See, e.g., U.S. Pat. No. 8,586,713. Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in EU); N44S, K52N, and V82I for IgG2 antibodies (IMGT; N384S, K392N, and V422I in EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU).

[0153] In certain embodiments, the Fc domain may be a chimera combining Fc sequences from two or more immunoglobulin isotypes. For example, the chimeric Fc domain may comprise a portion or all of the CH2 sequence from the CH2 region of human IgG1, human IgG2, or human IgG4, and a portion or all of the CH3 sequence from human IgG1, human IgG2, or human IgG4. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence from a human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, from N-terminus to C-terminus, [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules described herein comprises, from N-terminus to C-terminus, [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules disclosed herein are described in U.S. Patent Application Publication No. 2014 / 0243504, published August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains having these overall structural arrangements and variants thereof may exhibit altered Fc receptor binding, thereby affecting Fc effector function.

[0154] The multispecific (e.g., bispecific or trispecific) CD28 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the light chain and light chain variable domains compared to the corresponding germline sequence from which the antibody was derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can easily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and / or CDR residues in the VH and / or VL domains are backmutated to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, in which certain individual residues in the framework and / or CDR regions are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, enhanced binding (e.g., as assessed by measuring cell-binding titers or FACS binding) or binding affinity (e.g., KD), improved or enhanced biological antagonist or agonist properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the scope of the present disclosure.

[0155] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of tumor cells do not express the target antigen. In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 109%, 101%, 102%, 103%, 104%, 105%, 106%, Less than 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the tumor cells do not express the target antigen. In some embodiments, a range of percentages of tumor cells within a tumor do not express the target antigen, where the upper and lower percentage limits are disclosed herein.

[0156] Measurement of tumor antigen expression or levels can be accomplished by any method known in the art, including direct detection of antigen surface expression by flow cytometry. Thus, as used herein, the phrase "cells in a tumor that do not express a target antigen" can refer to surface expression of antigen proteins. For example, tumor antigens can be identified by direct survey of the tumor immune peptidome, i.e., all endogenous peptides presented on the cell surface by MHC molecules. In this approach, after extraction from tumor cells, peptides are eluted from complexes with MHC molecules and then subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS). MS spectra can be compared to a customized database created by combining sequencing data from the patient's tumor with reference protein sequences. In some embodiments, tumor antigen quantification with a platform called SureQuant-IsoMHC, which utilizes a series of pMHC isotope substitutes and internal standard triggered targeted mass spectrometry to generate a built-in multi-point calibration curve for determining endogenous pMHC concentration. For further details, see Stopfer LE, Gajadhar AS, Patel B, Gallien S, Frederick DT, Boland GM, Sullivan RJ, White FM. Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants. Proc Natl Acad Sci USA. 2021 Sep 14; 118 (37), which is incorporated by reference in its entirety. In addition, T cell receptor (TCR) mimicking antibodies can be used to estimate antigen copy number. Alternatively, measuring expression of a target antigen (e.g., any target antigen disclosed herein) can be achieved by measuring nucleic acid expression (e.g., RNA, e.g., mRNA expression) in tumor cells. Nucleic acid expression can be achieved by nucleic acid amplification and related techniques.

[0157] Multispecific CD3 antibody In some embodiments, the multispecific CD28 antibodies disclosed herein may be administered together with a CD3 antibody (eg, a multispecific CD3 antibody).

[0158] In some embodiments, the multispecific CD3 antibody is any of the multispecific CD3 antibodies listed in Table 5. [Table 4]

[0159] In some embodiments, the disclosure includes antibodies having the HCVR, LCVR, and / or CDR amino acid sequences of an antibody described herein, an anti-CD3 antibody disclosed in WO2014 / 047231 or WO2017 / 053856, a bispecific anti-CD20x anti-CD3 antibody disclosed in WO2014 / 047231, a bispecific anti-PSMAx anti-CD3 antibody disclosed in WO2017 / 023761, a bispecific anti-MUC16x anti-CD3 antibody disclosed in WO2018 / 067331 or WO2018 / 058003, a bispecific anti-STEAP2x anti-CD3 antibody disclosed in WO2018 / 058001, or a bispecific anti-BCMAx anti-CD3 antibody disclosed in WO2020 / 018820 (each of which is incorporated herein by reference).

[0160] Additional exemplary multispecific CD3 antibodies that can be used in the compositions and methods disclosed herein include, but are not limited to, those described in, for example, U.S. Pat. No. 10,787,521 B2, U.S. Patent Application Publication Nos. 2018 / 0222987 A1, and US2019 / 0241657 A1, and International Patent Application Publication Nos. WO2016 / 036937 A1, WO2017 / 210443 A1, WO2019 / 050521 A1, The bispecific CD3xCD123 antibodies disclosed in International Patent Application Publication No. WO2019 / 210147A1, WO2019 / 232528A1, and WO2020 / 092404A1; the bispecific CD3xSTEAP2 antibodies disclosed in International Patent Application Publication No. WO2018 / 058001A1; WO2014 / 047231A1, WO2015 / 143079A1, WO2016 / 081490A1, WO2017 / 112775A1, Nos. WO2017 / 210485A1, WO2018 / 114748A1, WO2018 / 093821A8, WO2018 / 223004A1, WO2018 / 188612A1, WO2019 / 155008A1, WO2019 / 228406A1, WO2020 / 088608A1, WO2020 / 156405A1, and U.S. Patent Application Publication Nos. US2020 / 0199231A1 and US2020 / 0172627A1. bispecific CD3xCD20 antibodies disclosed; bispecific CD3xSSTR2 antibodies disclosed in International Patent Application Publication No. WO2018 / 005706A1; bispecific CD3xCD38 antibodies disclosed in International Patent Application Publication Nos. WO2015 / 149077A1 and WO2020 / 018556A1, and US Patent Application Publication Nos. US2018 / 0305465A1 and US2020 / 0102403A1; bispecific CD3xSTEAP1 antibodies disclosed in Olivier Nolan-Stevaux (2020) Abstract of Proceedings of the Annual Meeting of the American Association for Cancer Research 2020;bispecific CD3x5T4 antibodies as disclosed in International Patent Application Publication No. WO2013 / 041687A1, US Patent Application Publication No. US2017 / 0342160A1, US20200277397A1; bispecific CD3xENPP3 antibodies as described in International Patent Application Publication No. WO2020 / 180726A1; bispecific CD3xMUC16 antibodies as disclosed in International Patent Application Publication No. WO2018 / 067331A9 and WO2019 / 246356A1; 06A1, WO2014 / 140248A1, WO2016 / 166629A1, WO2017 / 031104A1, WO2017 / 134134A1, WO2017 / 095267A1, WO2019 / 220369A3, WO2019 / 075359A1, WO2019 / 226761A1, WO2020 / 025596A1, WO2020 / 191346A1, WO2020018820A1, U.S. Patent Application Publication No. US2013 / 0273055 A1, the bispecific CD3xBCMA antibody disclosed in US2019 / 0263920A1; International Patent Application Publication Nos. WO2012 / 055961A1, WO2016 / 048938A1, WO2017 / 087603A1, WO2017 / 096368A1, WO2018 / 188612A1, WO2019 / 237081A1, WO2020 / 048525A1, WO2020 / 135335A1, U.S. Patent Application Publication Nos. US2016 / 0326249A1, U.S. Patent Application Publication Nos. US2016 / 0326249A1, US2019 / 0263920A1; bispecific CD3xCD19 antibodies disclosed in International Patent Application Publication Nos. S2020 / 0283523A1, US2019 / 0284279A1, US Patent Nos. US9,315,567B2, US7,575,923B2, and US7,635,472B2; bispecific CD3xGPRC5D antibodies disclosed in International Patent Application Publication Nos. WO2018 / 017786A3 and WO2019 / 220369A3; bispecific CD3xPSMA antibodies disclosed in US Patent Application Publication No. US2017 / 0320947A1;The trispecific CD3xCD28xCD38 antibody disclosed in U.S. Patent Application Publication No. US2020 / 0140552A1; or other multispecific CD3 antibodies disclosed in International Patent Application Publication Nos. WO2016 / 086189A2, WO2020 / 088608A1, WO2019191120A1, and WO2016 / 105450A3, the contents of each of which are incorporated herein by reference in their entirety;

[0161] In some embodiments, the multispecific (e.g., bispecific or trispecific) antigen-binding molecules described above that specifically bind to CD3 and tumor antigens may include anti-CD3 antigen-binding molecules that bind to CD3 with weak binding affinity, such as exhibiting a KD of greater than about 40 nM as measured by in vitro binding affinity assays. The bispecific antigen-binding molecules described above may include anti-CD3 antigen-binding molecules that bind to CD3 and exhibit an EC50 of greater than about 100 nM as measured by FACS titration assays. The bispecific antigen-binding molecules described above may include anti-CD3 antigen-binding molecules that do not exhibit measurable or observable binding to CD3 as measured by in vitro binding affinity assays or FACS titration assays, but retain the ability to activate human PBMC cells and / or induce cytotoxic activity against cell lines expressing tumor antigens.

[0162] Treatment method Provided herein are methods and compositions for administering to a subject a multispecific antigen-binding molecule having a first antigen-binding region specific for a target antigen and a second antigen-binding region specific for a CD28 protein, thereby resulting in the killing of tumor cells within a tumor in the subject, wherein the tumor cells do not express or are not expected to express the target antigen. In some embodiments, at least a subset of tumor cells within the tumor do not express the target antigen.

[0163] In some aspects, provided herein are methods and compositions for inducing killing of tumor cells and / or inducing T cell activation against tumor cells in a tumor in a subject, the methods comprising administering to the subject a multispecific antigen binding molecule having a first antigen binding region specific for a target antigen and a second antigen binding region specific for a CD28 protein. In some embodiments, at least a subset of tumor cells in the tumor do not express the target antigen.

[0164] Also provided herein are methods and compositions for treating cancer in a subject having a tumor, the methods comprising administering to the subject a multispecific antigen-binding molecule having a first antigen-binding region specific for a target antigen and a second antigen-binding region specific for a CD28 protein. In some embodiments, at least a subset of tumor cells within the tumor do not express the target antigen.

[0165] In some aspects, provided herein is a method of selecting a subject for cancer treatment, comprising: i) determining that the subject has a tumor comprising tumor cells that do not express a target antigen; and ii) administering to the subject a multispecific antigen binding molecule having a first antigen binding region specific for the target antigen and a second antigen binding region specific for a CD28 protein, optionally wherein the tumor cells do not express the target antigen if expression of the target antigen is below detection level or below signal to noise ratio, thereby selecting the subject for cancer treatment.

[0166] In some embodiments, the multispecific antigen-binding molecule is a bispecific T cell engager. In some embodiments, the multispecific antigen-binding molecule is a bispecific antibody fragment, such as a bispecific T-engaging antibody (BiTE), a dual affinity retargeting molecule (DART), or a tandem diabody (TandAb). A bispecific T cell engager (BiTE) is a small fusion protein that contains two antibody binding sites. A DART is a bispecific engager that uses a diabody scaffold with an additional C-terminal disulfide bridge that improves stabilization. A tandem diabody (TandAb) is a type of bispecific antibody fragment. A tandAb is a tetravalent (2+2 antigen binding valency) bispecific molecule composed of Fv domains. A TandAb is usually expressed as a monomeric subunit (single chain diabody, scDb) with four variable domains from two parent antibodies.

[0167] The methods provided herein include methods for detecting at least a subset (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4%, 5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 7 .1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6% ,6.7%,6.8%,6.9%,7%,7.1%,7.2%,7.3%,7.4%,7.5%,7.6%,7.7%,7.8%,7.9%,8%,8.1%,8.2%,8.3%,8.4%,8.5%,8.6%,8.7%,8.8%,8.9%,9%,9.1%,9.2%, ,9.3%,9.4%,9.5%,9.6%,9.7%,9.8%,9.9%,10%,10.1%,10.2%,10.3%,10.4%,10.5%,10.6%,10.7%,10.8%,10.9%,11%,11.1%,11.2%,11.3%,11.4%,11 .5%, 11.6%, 11.7%, 11.8%, 11.9%, 12%, 12.1%, 12.2%, 12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6 %, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%, 15.9%, 16%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%、18%、18.1%、18.2%、18.3%、18.4%、18.5%、18.6%、18.7%、18.8%、18.9%、19%、19.1%、19.2%、19.3%、19.4%、19.5%、19.6%、19.7%、19.8%、19.9%、20%、20.1%、20.2%、20.3%、20.4%、20.5%、20.6%、20.7%、20.8%、20.9%、21%、21.1%、21.2%、21.3%、21.4%、21.5%、21.6%、21.7%、21.8%、21.9%、22%、22.1%、22.2%、22.3%、22.4%、22.5%、22.6%、22.7%、22.8%、22.9%、23%、23.1%、23.2%、23.3%、23.4%、23.5%、23.6%、23.7%、23.8%、23.9%、24%、24.1%、24.2%、24.3%、24.4%、24.5%、24.6%、24.7%、24.8%、24.9%、25%、25.1%、25.2%、25.3%、25.4%、25.5%、25.6%、25.7%、25.8%、25.9%、26%、26.1%、26.2%、26.3%、26.4%、26.5%、26.6%、26.7%、26.8%、26.9%、27%、27.1%、27.2%、27.3%、27.4%、27.5%、27.6%、27.7%、27.8%、27.9%、28%、28.1%、28.2%、28.3%、28.4%、28.5%、28.6%、28.7%、28.8%、28.9%、29%、29.1%、29.2%、29.3%、29.4%、29.5%、29.6%、29.7%、29.8%、29.9%、30%、30.1%、30.2%、30.3%、30.4%、30.5%、30.6%、30.7%、30.8%、30.9%、31%、31.1%、31.2%、31.3%、31.4%、31.5%、31.6%、31.7%、31.8%、31.9%、32%、32.1%、32.2%、32.3%、32.4%、32.5%、32.6%、32.7%、32.8%、32.9%、33%、33.1%、33.2%、33.3%、33.4%、33.5%、33.6%、33.7%、33.8%、33.9%、34%、34.1%、34.2%、34.3%、34.4%、34.5%、34.6%、34.7%、34.8%、34.9%、35%、35.1%、35.2%、35.3%、35.4%、35.5%、35.6%、35.7%、35.8%、35.9%、36%、36.1%、36.2%、36.3%、36.4%、36.5%、36.6%、36.7%、36.8%、36.9%、37%、37.1%、37.2%、37.3%、37.4%、37.5%、37.6%、37.7%、37.8%、37.9%、38%、38.1%、38.2%、38.3%、38.4%、38.5%、38.6%、38.7%、38.8%、38.9%、39%、39.1%、39.2%、39.3%、39.4%、39.5%、39.6%、39.7%、39.8%、39.9%、40%、40.1%、40.2%、40.3%、40.4%、40.5%、40.6%、40.7%、40.8%、40.9%、41%、41.1%、41.2%、41.3%、41.4%、41.5%、41.6%、41.7%、41.8%、41.9%、42%、42.1%、42.2%、42.3%、42.4%、42.5%、42.6%、42.7%、42.8%、42.9%、43%、43.1%、43.2%、43.3%、43.4%、43.5%、43.6%、43.7%、43.8%、43.9%、44%、44.1%、44.2%、44.3%、44.4%、44.5%、44.6%、44.7%、44.8%、44.9%、45%、45.1%、45.2%、45.3%、45.4%、45.5%、45.6%、45.7%、45.8%、45.9%、46%、46.1%、46.2%、46.3%、46.4%、46.5%、46.6%、46.7%、46.8%、46.9%、47%、47.1%、47.2%、47.3%、47.4%、47.5%、47.6%、47.7%、47.8%、47.9%、48%、48.1%、48.2%、48.3%、48.4%、48.5%、48.6%、48.7%、48.8%、48.9%、49%、49.1%、49.2%、49.3%、49.4%、49.5%、49.6%、49.7%、49.8%、49.9%、50%、50.1%、50.2%、50.3%、50.4%、50.5%、50.6%、50.7%、50.8%、50.9%、51%、51.1%、51.2%、51.3%、51.4%、51.5%、51.6%、51.7%、51.8%、51.9%、52%、52.1%、52.2%、52.3%、52.4%、52.5%、52.6%、52.7%、52.8%、52.9%、53%、53.1%、53.2%、53.3%、53.4%、53.5%、53.6%、53.7%、53.8%、53.9%、54%、54.1%、54.2%、54.3%、54.4%、54.5%、54.6%、54.7%、54.8%、54.9%、55%、55.1%、55.2%、55.3%、55.4%、55.5%、55.6%、55.7%、55.8%、55.9%、56%、56.1%、56.2%、56.3%、56.4%、56.5%、56.6%、56.7%、56.8%、56.9%、57%、57.1%、57.2%、57.3%、57.4%、57.5%、57.6%、57.7%、57.8%、57.9%、58%、58.1%、58.2%、58.3%、58.4%、58.5%、58.6%、58.7%、58.8%、58.9%、59%、59.1%、59.2%、59.3%、59.4%、59.5%、59.6%、59.7%、59.8%、59.9%、60%、60.1%、60.2%、60.3%、60.4%、60.5%、60.6%、60.7%、60.8%、60.9%、61%、61.1%、61.2%、61.3%、61.4%、61.5%、61.6%、61.7%、61.8%、61.9%、62%、62.1%、62.2%、62.3%、62.4%、62.5%、62.6%、62.7%、62.8%、62.9%、63%、63.1%、63.2%、63.3%、63.4%、63.5%、63.6%、63.7%、63.8%、63.9%、64%、64.1%、64.2%、64.3%、64.4%、64.5%、64.6%、64.7%、64.8%、64.9%、65%、65.1%、65.2%、65.3%、65.4%、65.5%、65.6%、65.7%、65.8%、65.9%、66%、66.1%、66.2%、66.3%、66.4%、66.5%、66.6%、66.7%、66.8%、66.9%、67%、67.1%、67.2%、67.3%、67.4%、67.5%、67.6%、67.7%、67.8%、67.9%、68%、68.1%、68.2%、68.3%、68.4%、68.5%、68.6%、68.7%、68.8%、68.9%、69%、69.1%、69.2%、69.3%、69.4%、69.5%、69.6%、69.7%、69.8%、69.9%、70%、70.1%、70.2%、70.3%、70.4%、70.5%、70.6%、70.7%、70.8%、70.9%、71%、71.1%、71.2%、71.3%、71.4%、71.5%、71.6%、71.7%、71.8%、71.9%、72%、72.1%、72.2%、72.3%、72.4%、72.5%、72.6%、72.7%、72.8%、72.9%、73%、73.1%、73.2%、73.3%、73.4%、73.5%、73.6%、73.7%、73.8%、73.9%、74%、74.1%、74.2%、74.3%、74.4%、74.5%、74.6%、74.7%、74.8%、74.9%、75%、75.1%、75.2%、75.3%、75.4%、75.5%、75.6%、75.7%、75.8%、75.9%、76%、76.1%、76.2%、76.3%、76.4%、76.5%、76.6%、76.7%、76.8%、76.9%、77%、77.1%、77.2%、77.3%、77.4%、77.5%、77.6%、77.7%、77.8%、77.9%、78%、78.1%、78.2%、78.3%、78.4%、78.5%、78.6%、78.7%、78.8%、78.9%、79%、79.1%、79.2%、79.3%、79.4%、79.5%、79.6%、79.7%、79.8%、79.9%、80%、80.1%、80.2%、80.3%、80.4%、80.5%、80.6%、80.7%、80.8%、80.9%、81%、81.1%、81.2%、81.3%、81.4%、81.5%、81.6%、81.7%、81.8%、81.9%、82%、82.1%、82.2%、82.3%、82.4%、82.5%、82.6%、82.7%、82.8%、82.9%、83%、83.1%、83.2%、83.3%、83.4%、83.5%、83.6%、83.7%、83.8%、83.9%、84%、84.1%、84.2%、84.3%、84.4%、84.5%、84.6%、84.7%、84.8%、84.9%、85%、85.1%、85.2%、85.3%、85.4%、85.5%、85.6%、85.7%、85.8%、85.9%、86%、86.1%、86.2%、86.3%、86.4%、86.5%、86.6%、86.7%, 86.8%, 86.9%, 87%, 87.1%, 87.2%, 87.3%, 87.4%, 87.5%, 87.6%, 87.7%, 87.8%, 87.9%, 88%, 88.1%, 88.2%, 88.3%, 88.4%, 88.5%, 88.6%, 88.7%, 88.8%, 88.9%, 89%, 89. .1%, 89.2%, 89.3%, 89.4%, 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 2.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98% , 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100% do not express the target antigen (e.g., the target antigen, the first target antigen, or the second target antigen).

[0168] The methods provided herein include methods for isolating at least a subset of tumor cells (e.g., at least 0.1%-1%, 1%-5%, 1%-10%, 5%-10%, 10%-15%, 10%-20%, 15%-20%, 15%-25%, 20%-25%, 25%-30%, 20%-30%, 25%-35%, 30%-35%, 30%-40%, 35%-40%, 40%-45%, 40%-50%, 45%-50%, The method may further include determining that 50%-55%, 50%-60%, 55%-60%, 60%-65%, 60%-70%, 65%-70%, 65%-75%, 70%-75%, 70%-80%, 75%-85%, 75%-80%, 80%-90%, 85%-90%, 85%-95%, 90%-95%, 90%-100%, or 95%-100% do not express the target antigen (e.g., the target antigen, the first target antigen, or the second target antigen).

[0169] It will be understood by those skilled in the art that the methods of the present invention may be utilized in subjects where the tumor does not express the target antigen (e.g., 100% of the tumor does not express or is not predicted to express the target antigen) where some cells within the tumor microenvironment express or are predicted to express the target antigen.

[0170] In some aspects, provided herein is a method of treating cancer in a subject having a tumor, inducing or effecting the killing of tumor cells in the tumor in the subject, and / or inducing T cell activation against tumor cells in the tumor in the subject, the method comprising administering to the subject a first multispecific antigen-binding molecule having an antigen-binding region specific for a first target antigen and an antigen-binding region specific for CD28 protein, and a second multispecific antigen-binding molecule having an antigen-binding region specific for a second target antigen and an antigen-binding region specific for CD3 protein, wherein the first target antigen is not the same antigen as the second target antigen. For example, both the first target antigen and the second target antigen can be selected from the antigens listed in Table 2, and the first target antigen and the second target antigen cannot be the same antigen.

[0171] In some embodiments, the first or second multispecific antigen-binding molecule is a bispecific T cell engager. In some embodiments, the first or second multispecific antigen-binding molecule is a bispecific antibody fragment, such as a bispecific T-engaging antibody (BiTE), a dual affinity retargeting molecule (DART), or a tandem diabody (TandAb).

[0172] Thus, in certain embodiments, the agent disclosed herein can be used alone or can be co-administered with another type of therapeutic agent.For example, different therapeutic agents can be administered in the same formulation, or can be administered simultaneously or sequentially in separate formulations.In certain embodiments, different therapeutic agents can be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or within 1 week of each other.Therefore, the subject who undergoes such treatment can benefit from the combined effect of different therapeutic agents.

[0173] In certain embodiments, methods are provided herein that comprise administering a multispecific antigen-binding molecule to a subject at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0174] In certain embodiments, the present invention provides a composition, such as a pharmaceutical composition, that contains at least one agent as described herein together with a pharma- ceutically acceptable carrier.In one embodiment, the composition comprises a combination of multiple agents as described herein (e.g., two or more, three or more, four or more, or five or more).

[0175] In some embodiments, the pharmaceutical composition is administered locally or systemically. In some embodiments, the pharmaceutical composition can be administered locally to the tumor or tumor microenvironment present in the subject. In some embodiments, the agent or pharmaceutical composition is administered together with a second cancer therapeutic agent.

[0176] The agents described herein can be administered with any other cancer treatment, including immunotherapy.Additional cancer treatments include immune checkpoint inhibition.In some embodiments, immune checkpoint inhibitors inhibit immune checkpoint proteins.Immune checkpoint inhibition broadly refers to inhibiting the checkpoints that cancer cells can form to prevent or downregulate immune response. Examples of immune checkpoint proteins are CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, A2aR, and combinations thereof.Immune checkpoint inhibitors include cemiplimab (REGN2810), nivolumab (BMS-936558, MDX-1106, ONO-4538), pembrolizumab (MK-3475, SCH900475), atezolizumab (MPDL3280A, RG7446, RO5541267), durvalumab (MEDI4736, MEDI-4736), avelumab (MSB0010718C), ipilimumab (BMS-734016, IBI310, MDX-010), and SHR12 10, sintilimab (IBI308), spartalizumab (PDR001), tislelizumab (BGB-A317), pidilizumab, BCD-100, toripalimab (JS001), BAY1905254, ASP8374, PF-06801591, AMP-224, AB122, AK105, AMG404, BCD-100, BI754091, F520, HLX10, HX008, JTX-4014, LZM009, MEDI0680, MGA012, Sym021, TSR- 042, PSB205, MGD019, MGD013, AK104, XmAb20717, RO7121661, CX-188, INCB086550, FS118, BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, HLX20, KN0 35, MDX-1105, MSB2311, SHR-1316, TG-1501, ZKAB001, INBRX-105, MCLA-145, KN046, M7824, LY3415244, INCB086550, CA-170, CX-072, ADU-1604, AGEN1181, AG It can be EN1884, MK-1308, REGN4659, XmAb22841, ATOR-1015, PSB205, MGD019, AK104, XmAb20717, BMS-986249, tremelimumab, BMS-986258, BGB-A425, INCAGN02390, Sym023, JNJ61610588, BI754111, LAG525, MK-4280, REGN3767, Sym022, TSR-033, leratolimab, JTX-2011, MGD009, BMS-986207, OMP-313M32, MK-7684, or TSR-022.

[0177] Additional cancer immunotherapy includes adoptive immunotherapy, such as autologous or allogeneic T cell therapy or autologous or allogeneic CAR T cell therapy. Adoptive immunotherapy is a therapeutic method designed to enhance a patient's immune response against tumor or cancer cells. The method involves removing immune cells from an individual, forming effector cells ex vivo, expanding the cells to clinically relevant numbers, and reinfusing the cells into the patient. Provided herein are methods that include co-administration of an agent disclosed herein and allogeneic or autologous CTLs that express a T cell receptor that specifically binds to a peptide presented on class I MHC (e.g., a cancer peptide or a peptide specific for the subject). In some embodiments, the CTLs are derived from a cell bank or from the subject to whom the CTLs are administered. In some embodiments, the MHC is class I MHC. In some embodiments, the class I MHC has an alpha chain polypeptide that is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, the class II MHC has a β chain polypeptide that is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some embodiments, the CTLs are stored in a cell library or cell bank before they are administered to a subject.

[0178] The additional cancer treatment can be a cell therapy. As used herein, cell therapy includes, for example, tumor infiltrating lymphocytes, modified TCR lymphocytes, or modified CAR lymphocytes. The methods disclosed herein also include treatment with T cell therapy and any other adoptive therapy, such as natural killer cell or macrophage therapy. The cell therapy can include unmodified cells, as in traditional TIL therapy, or genetically modified cells. The methods included herein include any method known in the art for achieving cell targeting of cell therapy to tumor targets. As an example, the cell therapy can include cells that include chimeric antigen receptors (CARs). Single chain antibodies can be used, and the CAR can also contain a costimulatory domain. The target of the CAR cell can be present on the membrane of the target cell, while the TCR modification can utilize an intracellular target.

[0179] In one embodiment, the cell therapy comprises a type of cell selected from the group consisting of T cells, CD8+ cells, CD4+ cells, NK cells, delta / gamma T cells, regulatory T cells, and peripheral blood mononuclear cells. In another embodiment, TILs, T cells, CD8+ cells, CD4+ cells, NK cells, delta / gamma T cells, regulatory T cells, or peripheral blood mononuclear cells constitute the cell therapy as disclosed herein. In a specific embodiment, the cell therapy comprises T cells. As used herein, "tumor infiltrating lymphocytes" or TILs refer to white blood cells that have left the bloodstream and migrated to a tumor. Lymphocytes can be classified into three groups, including B cells, T cells, and natural killer cells. In another specific embodiment, the cell therapy comprises T cells that have been modified with a target-specific chimeric antigen receptor or, in particular, a selected T cell receptor. As used herein, "T cells" include CD3+ cells, including, but not limited to, CD4+ helper cells, CD8+ cytotoxic T cells, and gamma delta T cells.

[0180] In some embodiments, the T cells are contacted with antigen-presenting cells (APCs) that present peptides specific to the cancer or tumor of interest. In some embodiments, the APCs are B cells, antigen-presenting T cells, dendritic cells, or artificial antigen-presenting cells (e.g., aK562 cells). The dendritic cells used in the method can be prepared by taking PBMCs from a patient sample and attaching them to plastic. Generally, the monocyte population is allowed to adhere and all other cells can be washed away. The adherent population is then differentiated with IL-4 and GM-CSF to generate monocyte-derived dendritic cells. These cells can be matured by the addition of IL-1β, IL-6, PGE-1, and TNF-α (which upregulate important costimulatory molecules on the surface of dendritic cells) and then transduced with one or more peptides provided herein. In some embodiments, the APCs are artificial antigen-presenting cells, such as aK562 cells. In some embodiments, the artificial antigen presenting cells are engineered to express CD80, CD83, 41BB-L, and / or CD86. Exemplary artificial antigen presenting cells, including aK562 cells, are described in U.S. Patent Publication No. 2003 / 0147869, which is incorporated herein by reference. Exemplary methods for producing antigen presenting cells can be found in WO2013088114, which is incorporated herein in its entirety.

[0181] Another exemplary adoptive immunotherapy protocol includes the administration of autologous tumor infiltrating lymphocytes (TILs). TIL cells have high killing power. TIL cells are effector cells differentiated in vivo in solid tumors (see U.S. Patent No. 5,126,132, which describes a method for generating TIL cells for adoptive immunotherapy of cancer). TIL cells can be generated, for example, by removing tumor samples from patients, isolating lymphocytes that have infiltrated the tumor samples, expanding these TIL cells ex vivo in the presence of IL-2, and reinjecting the cells with IL-2 into patients.

[0182] An additional cancer treatment can be CAR-T cell therapy. Chimeric antigen receptors (CARs) are molecules with specific anti-tumor cell immune activity that combine antibody-based specificity for tumor-associated surface antigens with a T cell receptor activating intracellular domain (Eshhar, 1997, Cancer Immunol Immunother 45(3-4) 131-136; Eshhar et al., 1993, Proc Natl Acad Sci USA 90(2):720-724; Brocker and Karjalainen, 1998, Adv Immunol 68:257-269). These CARs allow T cells to achieve MHC-independent primary activation by a single-chain Fv (scFv) antigen-specific extracellular domain fused to an intracellular domain that provides T cell activation and costimulatory signals. Second and third generation CARs also provide appropriate co-stimulatory signals through CD28 and / or CD137 (4-1BB) intracellular activation motifs, which enhance cytokine secretion and antitumor activity in various solid tumor and leukemia models (Pinthus, et al, 2004, J Clin Invest 114(12):1774-1781; Milone, et al., 2009, Mol Ther 17(8):1453-1464; Sadelain, et al., 2009, Curr Opin Immunol 21(2):215-223). Chimeric antigen receptor (CAR) T-cell therapy involves genetically modifying a patient's autologous T cells to express a CAR specific for a tumor antigen, followed by expanding the cells ex vivo and reinfusing them back into the patient. CARs are fusion proteins of selected single-chain fragment variable portions derived from specific monoclonal antibodies and one or more T-cell receptor intracellular signaling domains. The genetic modification of the T cells can be performed by either viral-based gene transfer methods or non-viral methods, such as DNA-based transposons, CRISPR / Cas9 technology, or direct introduction of in vitro transcribed mRNA by electroporation.

[0183] An additional cancer treatment can be natural killer cell therapy. Natural killer (NK) cells can recognize tumor cells as targets and therefore can be useful in cancer immunotherapy (Vivier et al., 2011, Science 331:44-49; Ruggeri et al., 2002, Science 295:2097-2100; Cooley et al., 2010, Blood 116:2411-2419; Miller et al., 2005, Blood 105:3051-3057; Rubnitz et al., 2010, J Clin Oncol.28:955-959). Infusion of NK cells has been used to treat patients with various types of cancer (Vivier et al., 2011, Science 331:44-49; Caligiuri, 2008, Blood 112(3):461-469; Ruggeri et al., 2002, Science 295:2097-2100; Miller et al., 2005, Blood 105:3051-3057). Methods are available that allow obtaining large amounts of human NK cells that exhibit higher anti-tumor capabilities than those of non-expanded NK cells (see U.S. Patent No. 7,435,596; Imai et al., 2005, Blood 106:376-83; Fujisaki et al., 2009, Cancer Res. 69:4010-4017; Cho et al., 2010, Clin Cancer Res. 16:3901-3909). NK cells expanded from primary peripheral blood mononuclear cells (PBMCs) are included herein. NK cells containing chimeric antigen receptors or other modifications are also included herein.

[0184] The additional cancer therapy can be a macrophage cell therapy. In some embodiments, the cell therapy comprises ex vivo expanded cytotoxic macrophages. Macrophage cells can recognize tumor cells as targets and therefore can be useful in cancer immunotherapy (Andreesen R, Hennemann B, Krause SW. Adoptive immunotherapy of cancer using monocyte-derived macrophages: rationale, current status, and perspectives. J Leukoc Biol. 1998 Oct; 64(4): 419-26). Macrophages are potent immune effector cells whose functional plasticity provides anti-tumor and tumor-promoting functions in different contexts, and this plasticity has led to significant efforts to deplete or redifferentiate tumor-associated macrophages. Alternatively, in some embodiments, macrophages are adoptively transferred, for example after ex vivo genetic modification (Anderson NR, Minutolo NG, Gill S, Klichinsky M. Macrophage-Based Approaches for Cancer Immunotherapy. Cancer Res. 2021 Mar 1;81(5):1201-1208).

[0185] Also provided herein is a method of treating cancer in a subject by obtaining a sample comprising T cells from the subject, isolating cytotoxic T lymphocytes (CTLs) from the sample, expanding the CTLs ex vivo, and administering the expanded CTLs to the subject with at least one agent (e.g., any agent disclosed herein). The cytotoxic T cells can be tumor-infiltrating lymphocytes. Expanding the CTLs can include contacting the CTLs with an antigen-presenting cell (APC) expressing a cancer-specific or tumor-specific antigen to generate antigen-specific CTLs. In some embodiments, the sample comprising the T cells or isolated CTLs is stimulated prior to administration to the subject. The method can further include contacting the CTLs with an anti-CD3 monoclonal antibody (OKT3) prior to administration to the subject. In other embodiments, the method further includes contacting the CTLs with human interleukin (IL)-2 prior to administration to the subject.

[0186] Additional cancer treatments may also include any known stimulatory agent of immune cells, such as agents that induce proliferation, expansion, or activation of such immune cells. Exemplary stimulatory methods include administration of stimulatory cytokines, such as IL-2, IL-12, IL-15, IL-18, and IL-21.

[0187] In some embodiments, the subject has been administered a chemotherapeutic agent prior to administration of the agent. The subject may exhibit resistance to the chemotherapeutic agent. The subject may be administered the chemotherapeutic agent subsequent to or concurrently with administration of the additional cancer treatment disclosed herein. Chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; emylerumine and memylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatasin and bullatasinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and biceresin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin duocarmycins (including synthetic analogs, KW-2189, and CBI-TMI); eloterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1 and calicheamicin phi 1); dynemycins, including dynemycin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adramycin™) (morpholinodoxorubicin, cyanomorpho doxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, queramycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-furan fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostanol, acetaminophen, testolactone; antiadrenal drugs, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; hestravucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformthine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine;Maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine, PSK (trademark); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracrine A) A), Roridin A, and Anguidine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel (Taxol®, Bristol Meyers Squibb Oncology, Princeton, NJ) and docetaxel (Taxoteret™, Rhone-Poulenc Rorer, Antony, France); chlorambucil, gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (Navelbine™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including Nolvadex™), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyfene, ketoxifene, LY117018, onapristone, and toremifene (Fareston™);Also included within the definition of "chemotherapeutic agent" are inhibitors of the aromatase enzyme, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate (Megace™), exemestane, formstein, fadrozole, vorozole (Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0188] As described in detail below, the pharmaceutical compositions and / or agents disclosed herein may be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets, such as those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, such as parenteral administration by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral, or epidural injection, such as sterile solutions or suspensions, or sustained release formulations. The method of preparing a pharmaceutical formulation or composition includes the step of mixing the agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulation is prepared by uniformly and intimately mixing the agent described herein with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0189] The pharmaceutical compositions provided herein may be formulated with suitable carriers, excipients, and other agents that provide improved transport, delivery, durability, etc. A number of suitable formulations may be found in the following formularies known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0190] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and size of the patient, the target disease, condition, route of administration, and the like.

[0191] Various delivery systems are known and can be used to administer the pharmaceutical compositions provided herein, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active substances. Administration can be systemic or local.

[0192] In some embodiments, the pharmaceutical compositions provided herein can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily used to deliver the pharmaceutical compositions disclosed herein. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device typically utilizes a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, a disposable pen delivery device is manufactured pre-filled with the pharmaceutical composition contained in a reservoir in the device. Once the pharmaceutical composition is dispensed and the reservoir is emptied, the entire device is discarded.

[0193] A number of reusable pen-type autoinjector delivery devices are used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN™, to name just a few. STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany). Examples of disposable pen delivery devices used for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA™ pen (Abbott Labs, Abbott Park IL), to name just a few.

[0194] In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are described in the review by Langer, 1990, Science 249:1527-1533.

[0195] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. The aqueous medium for injection may be, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, and the like, which may be used in combination with a suitable solubilizing agent, for example, alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. The oily medium may be, for example, sesame oil, soybean oil, and the like, which may be used in combination with a solubilizing agent, for example, benzyl benzoic acid, benzyl alcohol, and the like. Therefore, the injectable preparation is preferably filled in a suitable ampoule.

[0196] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for administration of the active ingredient.Such dosage forms of unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0197] The agents described herein may be administered in multiple doses, for example, an initial dose, a secondary dose, and a tertiary dose. The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of the antigen-binding molecules disclosed herein. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of the therapeutic agent described herein, but typically may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antigen-binding molecule contained in the initial, secondary, and / or tertiary doses varies from each other during the course of treatment (e.g., adjusted upwards or downwards as necessary). In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., maintenance doses) administered less frequently.

[0198] In an exemplary embodiment disclosed herein, each of the secondary and / or tertiary doses is administered 1 to 26 weeks (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, or more weeks) after the immediately preceding administration. As used herein, the phrase "immediately preceding administration" refers to a dose of an antigen-binding molecule administered to a patient prior to administration of the next dose in the sequence, without any intervening administrations.

[0199] The methods according to this aspect disclosed herein may include administering to the patient any number of secondary and / or tertiary doses of the therapeutic agent described herein. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0200] In embodiments including multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding administration. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding administration. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. Also, the frequency of administration may be adjusted during the course of treatment by the physician according to the needs of the individual patient after clinical testing.

[0201] Indications In some embodiments, the methods described herein may be used to treat any cancer, including any cancerous or precancerous tumor. Cancers that may be treated by the methods and compositions provided herein include, but are not limited to, bladder, blood, bone, bone marrow, brain, breast, colon, esophageal, gastrointestinal, gingival, head, kidney, liver, lung, nasopharyngeal, cervical, ovarian, prostate, skin, stomach, testis, tongue, or uterine cancer. In addition, the cancer may be of the following histological types, among others, but not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; familial polyposis coli adenocarcinoma; solid tumors; malignant carcinoid tumors bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary follicular adenocarcinoma; Sebaceous carcinoma; auditory canal adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; squamous Epithelial metaplastic adenocarcinoma;malignant thymoma;malignant ovarian stromal tumor;malignant thecoma;malignant granulosa cell tumor;malignant neuroblastoma;Sertoli cell carcinoma;malignant Leydig cell tumor;malignant lipocytoma;malignant paraganglioma;malignant extramammary paraganglioma;pheochromocytoma;angiocytoma;malignant melanoma;amelanotic melanoma;superficial spreading melanoma;malignant melanoma of giant pigmented nevus;epithelioid cell melanoma;malignant blue nevus;sarcoma;fibrosarcoma;malignant fibrous histiocytoma;myxosarcoma;liposarcoma;leiomyosarcoma;striated muscle Sarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;malignant mixed tumor;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian thyroid tumor;choriocarcinoma;malignant mesothelioma;angiosarcoma;malignant hemangioendothelioma;Kaposi's sarcoma;malignant hemangiopericytoma;lymphangiosarcoma;osteosarcoma;parosteal osteosarcoma;chondrosarcoma;malignant chondroblastoma;mesenchymal chondrosarcoma;giant cell tumor of bone;Ewing's sarcoma;Malignant odontogenic tumor;Ameloblastic odontosarcoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pinealoma;Chordoma;Malignant glioma;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Malignant meningioma;Neurofibrosarcoma;Malignant schwannoma;Malignant granular cell tumor;Malignant lymphoma;Hodgkin's Kin's disease;Hodgkin's lymphoma;paragranuloma;small lymphocytic lymphoma;diffuse large cell lymphoma;follicular lymphoma;mycosis fungoides;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0202] The composition or method of the present invention is useful for treating cancer or tumor that expresses CD20. The composition or method of the present invention is useful for treating B-cell malignancies, including non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom's macroglobulinemia, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma, or Burkitt's lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0203] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is an adenocarcinoma, an adrenal tumor, anal tumor, bile duct tumor, bladder tumor, bone tumor, blood-borne tumor, brain / CNS tumor, breast tumor, cervical tumor, colorectal tumor, endometrial tumor, esophageal tumor, Ewing's tumor, eye tumor, gallbladder tumor, gastrointestinal tumor, kidney tumor, laryngeal tumor or hypopharyngeal tumor, liver tumor, lung tumor, mesothelioma, multiple myeloma, muscle tumor, nasopharyngeal tumor, neuroblastoma, oral tumor, osteosarcoma, ovarian tumor, pancreatic tumor, penile tumor, pituitary tumor, primary tumor, prostate tumor, retinoblastoma, rhabdomyosarcoma, salivary gland tumor, soft tissue sarcoma, melanoma, metastatic tumor, basal cell carcinoma, Merkel cell tumor, testicular tumor, thymus tumor, thyroid tumor, uterine tumor, vaginal tumor, vulvar tumor, or Wilms' tumor.

[0204] In certain embodiments, the tumor being treated is one that is not expected to express a particular target antigen. Expression profiles of exemplary target antigens (e.g., exemplary TAAs) disclosed herein are known in the art and / or can be determined using the methods provided herein.

[0205] Expression profiles are known and can be found, for example, in the following databases: TANTIGEN 2.0 (found on the World Wide Web at projects.met-hilab.org / tadb / index.php; further details can be found in Zhang, G., Chitkushev, L., Olsen, LR et al. TANTIGEN 2.0: a knowledge base of tumor T cell antigens and epitopes. BMC Bioinformatics 22, 40 (2021)), Cancer Epitope Database and Analysis Resource (CEDAR) (further details can be found in Kosaloglu-Yalcin, Z. et al., The Cancer Epitope Database and Analysis Resource: A Blueprint for the Establishment of a New Bioinformatics Resource for Use by the Cancer Immunology Community, Frontiers in Immunology, 12 (2021)), Human Protein Atlas (found on the World Wide Web at v17.proteinatlas.org / ), dbPepNeo (found on the World Wide Web at biostatistics.online / dbPepNeo / search.php), and cBioPortal (found on the World Wide Web at cbioportal.org).

[0206] Exemplary expression profiles for certain exemplary target antigens are shown in Figures 18-26.

[0207] In some embodiments, a tumor is not predicted to express a target antigen based on a known expression profile (e.g., an expression profile disclosed herein). In some embodiments, a tumor type is expressed in 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13% of tumors analyzed. ,13.5%,14%,14.5%,15%,15.5%,16%,16.5%,17%,17.5%,18%,18.5%,19%,19.5%,20%,20.5%,21%,21.5%,22%,22.5%,23%,23.5%,24%,24.5%,25%,25.5%,26%,26.5%, %, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40% , 40.5%, 41%, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, or less than 50% express the target antigen, are predicted to not express the target antigen.

[0208] Primary tumors show different antigen expression patterns not only within tumors but also between patients. The variability of antigen expression between patients with the same histological tumor type can be large. Thus, the exemplary expression profiles disclosed herein are informative regarding the tumor types that can be treated using the methods disclosed herein, but are not limited to the cancers or tumor types that can be treated using the methods disclosed herein. In some embodiments, the tumor to be treated can be a tumor that does not express the target antigen or contains a subset of tumor cells that do not express the target antigen, even though the tumor has been examined and the antigen expression profile of the tumor indicates that the tumor type normally expresses the target antigen. In some embodiments, the target antigen is abundant in a particular tumor type, but the target antigen is not expressed in a subset of tumor cells of that particular tumor type in the patient. In some embodiments, the target antigen is normally expressed in various tumor types, but the target antigen is not expressed in a subset of tumor cells of the patient.

[0209] Screening, Diagnostic, and Prognostic Assays Screening, diagnostic and / or prognostic assays for identifying or quantifying the level of tumor-associated antigens on cancer or tumor cells in a subject's cancer are also provided.

[0210] In some embodiments, at least a subset of tumor cells within a tumor do not express the target antigen. The methods provided herein include methods for detecting at least a subset of tumor cells (e.g., at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8% ,8.9%,9%,9.1%,9.2%,9.3%,9.4%,9.5%,9.6%,9.7%,9.8%,9.9%,10%,10.1%,10.2%,10.3%,10.4%,10.5%,10.6%,10.7%,10.8%,10.9%,11%,11.1%,11.2%,11.3%,11.4%,11.5%,11.6%,11.7%,11.8%,11.9%,12%,12.1%,12.2%,12.3%,12.4%,12.5%,12.6%,12.7%,12.8%,12.9%,13%,13.1%,1 3.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%, 15.9%, 16%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%, 17.2%, 17.3%、17.4%、17.5%、17.6%、17.7%、17.8%、17.9%、18%、18.1%、18.2%、18.3%、18.4%、18.5%、18.6%、18.7%、18.8%、18.9%、19%、19.1%、19.2%、19.3%、19.4%、19.5%、19.6%、19.7%、19.8%、19.9%、20%、20.1%、20.2%、20.3%、20.4%、20.5%、20.6%、20.7%、20.8%、20.9%、21%、21.1%、21.2%、21.3%、21.4%、21.5%、21.6%、21.7%、21.8%、21.9%、22%、22.1%、22.2%、22.3%、22.4%、22.5%、22.6%、22.7%、22.8%、22.9%、23%、23.1%、23.2%、23.3%、23.4%、23.5%、23.6%、23.7%、23.8%、23.9%、24%、24.1%、24.2%、24.3%、24.4%、24.5%、24.6%、24.7%、24.8%、24.9%、25%、25.1%、25.2%、25.3%、25.4%、25.5%、25.6%、25.7%、25.8%、25.9%、26%、26.1%、26.2%、26.3%、26.4%、26.5%、26.6%、26.7%、26.8%、26.9%、27%、27.1%、27.2%、27.3%、27.4%、27.5%、27.6%、27.7%、27.8%、27.9%、28%、28.1%、28.2%、28.3%、28.4%、28.5%、28.6%、28.7%、28.8%、28.9%、29%、29.1%、29.2%、29.3%、29.4%、29.5%、29.6%、29.7%、29.8%、29.9%、30%、30.1%、30.2%、30.3%、30.4%、30.5%、30.6%、30.7%、30.8%、30.9%、31%、31.1%、31.2%、31.3%、31.4%、31.5%、31.6%、31.7%、31.8%、31.9%、32%、32.1%、32.2%、32.3%、32.4%、32.5%、32.6%、32.7%、32.8%、32.9%、33%、33.1%、33.2%、33.3%、33.4%、33.5%、33.6%、33.7%、33.8%、33.9%、34%、34.1%、34.2%、34.3%、34.4%、34.5%、34.6%、34.7%、34.8%、34.9%、35%、35.1%、35.2%、35.3%、35.4%、35.5%、35.6%、35.7%、35.8%、35.9%、36%、36.1%、36.2%、36.3%、36.4%、36.5%、36.6%、36.7%、36.8%、36.9%、37%、37.1%、37.2%、37.3%、37.4%、37.5%、37.6%、37.7%、37.8%、37.9%、38%、38.1%、38.2%、38.3%、38.4%、38.5%、38.6%、38.7%、38.8%、38.9%、39%、39.1%、39.2%、39.3%、39.4%、39.5%、39.6%、39.7%、39.8%、39.9%、40%、40.1%、40.2%、40.3%、40.4%、40.5%、40.6%、40.7%、40.8%、40.9%、41%、41.1%、41.2%、41.3%、41.4%、41.5%、41.6%、41.7%、41.8%、41.9%、42%、42.1%、42.2%、42.3%、42.4%、42.5%、42.6%、42.7%、42.8%、42.9%、43%、43.1%、43.2%、43.3%、43.4%、43.5%、43.6%、43.7%、43.8%、43.9%、44%、44.1%、44.2%、44.3%、44.4%、44.5%、44.6%、44.7%、44.8%、44.9%、45%、45.1%、45.2%、45.3%、45.4%、45.5%、45.6%、45.7%、45.8%、45.9%、46%、46.1%、46.2%、46.3%、46.4%、46.5%、46.6%、46.7%、46.8%、46.9%、47%、47.1%、47.2%、47.3%、47.4%、47.5%、47.6%、47.7%、47.8%、47.9%、48%、48.1%、48.2%、48.3%、48.4%、48.5%、48.6%、48.7%、48.8%、48.9%、49%、49.1%、49.2%、49.3%、49.4%、49.5%、49.6%、49.7%、49.8%、49.9%、50%、50.1%、50.2%、50.3%、50.4%、50.5%、50.6%、50.7%、50.8%、50.9%、51%、51.1%、51.2%、51.3%、51.4%、51.5%、51.6%、51.7%、51.8%、51.9%、52%、52.1%、52.2%、52.3%、52.4%、52.5%、52.6%、52.7%、52.8%、52.9%、53%、53.1%、53.2%、53.3%、53.4%、53.5%、53.6%、53.7%、53.8%、53.9%、54%、54.1%、54.2%、54.3%、54.4%、54.5%、54.6%、54.7%、54.8%、54.9%、55%、55.1%、55.2%、55.3%、55.4%、55.5%、55.6%、55.7%、55.8%、55.9%、56%、56.1%、56.2%、56.3%、56.4%、56.5%、56.6%、56.7%、56.8%、56.9%、57%、57.1%、57.2%、57.3%、57.4%、57.5%、57.6%、57.7%、57.8%、57.9%、58%、58.1%、58.2%、58.3%、58.4%、58.5%、58.6%、58.7%、58.8%、58.9%、59%、59.1%、59.2%、59.3%、59.4%、59.5%、59.6%、59.7%、59.8%、59.9%、60%、60.1%、60.2%、60.3%、60.4%、60.5%、60.6%、60.7%、60.8%、60.9%、61%、61.1%、61.2%、61.3%、61.4%、61.5%、61.6%、61.7%、61.8%、61.9%、62%、62.1%、62.2%、62.3%、62.4%、62.5%、62.6%、62.7%、62.8%、62.9%、63%、63.1%、63.2%、63.3%、63.4%、63.5%、63.6%、63.7%、63.8%、63.9%、64%、64.1%、64.2%、64.3%、64.4%、64.5%、64.6%、64.7%、64.8%、64.9%、65%、65.1%、65.2%、65.3%、65.4%、65.5%、65.6%、65.7%、65.8%、65.9%、66%、66.1%、66.2%、66.3%、66.4%、66.5%、66.6%、66.7%、66.8%、66.9%、67%、67.1%、67.2%、67.3%、67.4%、67.5%、67.6%、67.7%、67.8%、67.9%、68%、68.1%、68.2%、68.3%、68.4%、68.5%、68.6%、68.7%、68.8%、68.9%、69%、69.1%、69.2%、69.3%、69.4%、69.5%、69.6%、69.7%、69.8%、69.9%、70%、70.1%、70.2%、70.3%、70.4%、70.5%、70.6%、70.7%、70.8%、70.9%、71%、71.1%、71.2%、71.3%、71.4%、71.5%、71.6%、71.7%、71.8%、71.9%、72%、72.1%、72.2%、72.3%、72.4%、72.5%、72.6%、72.7%、72.8%、72.9%、73%、73.1%、73.2%、73.3%、73.4%、73.5%、73.6%、73.7%、73.8%、73.9%、74%、74.1%、74.2%、74.3%、74.4%、74.5%、74.6%、74.7%、74.8%、74.9%、75%、75.1%、75.2%、75.3%、75.4%、75.5%、75.6%、75.7%、75.8%、75.9%、76%、76.1%、76.2%、76.3%、76.4%、76.5%、76.6%、76.7%、76.8%、76.9%、77%、77.1%、77.2%、77.3%、77.4%、77.5%、77.6%、77.7%、77.8%、77.9%、78%、78.1%、78.2%、78.3%、78.4%、78.5%、78.6%、78.7%、78.8%、78.9%、79%、79.1%、79.2%、79.3%、79.4%、79.5%、79.6%、79.7%、79.8%、79.9%、80%、80.1%、80.2%、80.3%、80.4%、80.5%、80.6%、80.7%、80.8%、80.9%、81%、81.1%、81.2%、81.3%、81.4%、81.5%、81.6%、81.7%、81.8%、81.9%、82%、82.1%、82.2%、82.3%、82.4%、82.5%、82.6%、82.7%、82.8%、82.9%、83%、83.1%、83.2%、83.3%、83.4%、83.5%、83.6%、83.7%、83.8%、83.9%、84%、84.1%、84.2%、84.3%、84.4%、84.5%、84.6%、84.7%、84.8%、84.9%、85%、85.1%、85.2%、85.3%、85.4%、85.5%、85.6%、85.7%、85.8%、85.9%、86%、86.1%、86.2%、86.3%、86.4%、86.5%、86.6%、86.7%、86.8%、86.9%、87%、87.1%、87.2%、87.3%、87.4%、87.5%、87.6%、87.7%、87.8%、87.9%、88%、88.1%、88.2%、88.3%. ,88.4%,88.5%,88.6%,88.7%,88.8%,88.9%,89%,89.1%,89.2%,89.3%,89.4%,89.5%,89.6%,89.7%,89.8%,89.9%,90%,90.1%,90.2%,90.3%,90.4%,90.5%,90.6%,90.7%,90.8%,90.9%,91%,91.1%,91.2%,91.3%,91.4%,91.5%,91.6%,91 .7%, 91.8%, 91.9%, 92%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 5.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4 %, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 100%) of the tumors do not express the target antigen, and administering an agent or agents disclosed herein to the subject if the determined subset / percentage does not express the target antigen. It will be understood by those skilled in the art that the methods of the present invention can be utilized for subjects whose tumors do not express the target antigen (e.g., about 100% of tumors do not express or are not predicted to express the target antigen) when some cells in the tumor microenvironment express or are predicted to express the target antigen.

[0211] The methods provided herein can further include determining that at least a subset of tumor cells within the tumor do not express the target antigen, hi some embodiments, the tumor cells do not express the target antigen if expression of the target antigen is below the level of detection or below the signal to noise ratio.

[0212] Provided herein is a method for screening a subject by measuring or calculating the amount or level of a tumor-associated antigen in the subject's cancer. The tumor-associated antigen can be measured by any method known in the art. For example, a biological sample can be taken from a patient. The sample can be taken by any means known in the art. The sample can be taken directly from a cancer, tumor, or tumor microenvironment. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject.

[0213] In some embodiments, the determination of the percentage of tumor cells expressing the target antigen may be determined by measuring the level or expression of the target antigen in a biological sample (e.g., a biopsy of the tumor or tumor microenvironment) taken from the subject. In some embodiments, the percentage may then be extrapolated to quantify the expected overall percentage of cells expressing the target antigen in the tumor and / or tumor microenvironment in the subject.

[0214] The detection methods encompassed by the present disclosure can be used to detect tumor-associated antigen mRNA, protein, or genomic DNA, or biologically active fragments thereof, in a biological sample in vitro and in vivo. For example, in vitro techniques for detecting mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting genomic DNA include Southern hybridization. Additionally, in vivo techniques for detecting protein include introducing into a subject a labeled antibody against the protein of interest to be detected. For example, the antibody can be labeled with a radioactive marker, and its presence and location in a subject can be detected by standard imaging techniques.

[0215] The assays described herein may include measuring the level of tumor-associated antigens after isolation from cells (e.g., after biopsy or isolation of a biological sample). These may be performed in a cell-free format using known components of gene expression of tumor-associated antigens. It may be desirable to immobilize certain components of the assay, and such embodiments may benefit from the use of well-known applications of biomolecule immobilization, e.g., microtiter plates, beads, test tubes, microcentrifuge tubes, in combination with derivatizable moieties, e.g., fusion protein domains, biotinylation, antibodies, etc.

[0216] To determine whether a subject suffers from a condition disclosed herein, whether he / she is at risk of developing such a condition, or whether he / she may benefit from the administration of an agent disclosed herein, a biological sample may be taken from the subject, and the biological sample may be contacted with a compound or agent capable of detecting a tumor-associated antigen protein or a polynucleotide (e.g., mRNA or genomic DNA) encoding the tumor-associated antigen in the biological sample. An agent for detecting mRNA or genomic DNA may comprise a labeled nucleic acid probe capable of hybridizing to the mRNA or genomic DNA. The nucleic acid probe may be, for example, an oligonucleotide having a sequence complementary to a nucleic acid encoding a tumor-associated antigen or a portion thereof, for example, at least 15, 20, 25, 30, 25, 40, 45, 50, 100, 250, or 500 nucleotides in length, sufficient to specifically hybridize to the mRNA or genomic DNA of interest under stringent conditions. Other suitable probes for use in the diagnostic assays encompassed by the present disclosure are described herein.

[0217] In another embodiment, the method further comprises obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting the protein, mRNA, or genomic DNA, such that the presence of the protein, mRNA, or genomic DNA of interest in the biological sample is detected, and comparing the presence of the protein, mRNA, or genomic DNA in the control sample with the presence of the protein, mRNA, or genomic DNA in the control sample.

[0218] In some embodiments, the assays described herein can be performed in a cell-free format using known components of gene expression of tumor-associated antigens. It may be desirable to immobilize certain components of the assay, and such embodiments can benefit from the use of well-known applications of biomolecule immobilization, such as microtiter plates, beads, test tubes, microcentrifuge tubes, in combination with derivatizable moieties, such as fusion protein domains, biotinylation, antibodies, etc.

[0219] The analysis of one or more regions of the nucleic acid of tumor-associated antigen in a subject can be useful for predicting whether the subject will benefit from the method disclosed herein or will likely benefit from the method disclosed herein.For example, the detection of tumor-associated antigen in a part of a cancer, such that the cancer is classified as heterogeneous cancer, indicates that the subject will benefit from the method disclosed herein.Similarly, the analysis of the genome copy number of tumor-associated antigen in a subject can be useful for predicting whether the subject will benefit from the method disclosed herein.In some embodiments, the method encompassed by the present disclosure can be characterized by comprising detecting the presence or absence of one or more polymorphic regions of the gene encoding tumor-associated antigen in a cell sample from a subject.

[0220] Other detection methods require first amplifying at least a portion of the nucleic acid to be detected in the biological sample. Amplification can be carried out according to methods known in the art, for example, by PCR and / or LCR (see Wu and Wallace, (1989) Genomics 4:560). In one embodiment, the genomic DNA of the cell is exposed to two PCR primers and amplified for a number of cycles sufficient to generate the required amount of amplified DNA.

[0221] Alternative amplification methods include autonomous sequence replication (Guatelli, JC et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification system (Kwoh, DY et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Qβ-replicase (Lizardi, PM et al., 1988, Bio / Technology 6:1197), and autonomous sequence replication (Guatelli et al., (1989) Proc. Nat. Acad. Sci. 87:1874), and nucleic acid sequence-based amplification (NABSA), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detection of nucleic acid molecules when such molecules are present in very low numbers.

[0222] In one embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the nucleic acid in a biological sample by comparing the sequence of the sample sequence with the corresponding reference (control) sequence. Exemplary sequencing reactions include those based on the technology developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). In carrying out the subject assays (Biotechniques (1995) 19:448), sequencing by mass spectrometry (see, e.g., U.S. Pat. No. 5,547,835 and International Patent Application Publication No. WO 94 / 16101, entitled "DNA Sequencing by Mass Spectrometry" by H. Koster, U.S. Pat. No. 5,547,835 and International Patent Application Publication No. WO 94 / 21822, entitled "DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation" by H. Koster, and U.S. Pat. No. 5,605,798 and International Patent Application No. PCT / US96 / 03651, entitled "DNA Diagnostics Based on Mass Spectrometry" by H. Koster, Cohen et al. (1996) Adv Chromatogr 36:127-162, and Griffin et al. (1993) Appl Biochem Biotechnol 19:1999-2002, both of which are incorporated herein by reference) may be used. It is also contemplated that any of a variety of automated sequencing methods can be utilized, including those described in the literature (see, for example, 38:147-159). In certain embodiments, it is clear to those skilled in the art that the occurrence frequency of only one, two or three of the nucleic acid bases needs to be determined in sequencing reaction. For example, A-track, etc., can be performed, in which only one nucleotide is detected.

[0223] Antibodies directed against tumor-associated antigens may also be used in the diagnosis and prognosis of disease. In addition, such diagnostic methods may be used to detect abnormalities in the expression levels of such polypeptides, or in the structure and / or tissue, cellular, or subcellular location of such polypeptides. Structural differences may include, for example, differences in size, electronegativity, or antigenicity of the mutant polypeptide compared to the normal polypeptide. Analyzed proteins from tissues or cell types may be easily detected or isolated using techniques well known to those of skill in the art, including, but not limited to, Western blot analysis. For a detailed description of methods for performing Western blot analysis, see Chapter 18 of Sambrook et al., 1989, supra. Protein detection and isolation methods used herein may be, for example, those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is incorporated herein by reference in its entirety.

[0224] This can be accomplished, for example, by immunofluorescence using fluorescently labeled antibodies (see below) in combination with light microscopic, flow cytometric, or fluorescent detection. Additionally, useful antibodies (or fragments thereof) according to the present disclosure can be utilized in histological examinations for in situ detection of tumor-associated antigens, as in immunofluorescence or immunoelectron microscopy. In situ detection can be accomplished by removing a tissue specimen from a subject and applying thereto a labeled antibody as disclosed herein. The antibody (or fragment) can be applied by overlaying the labeled antibody (or fragment) on a biological sample. Using such procedures, it is possible to determine not only the presence of tumor-associated antigens, but also their distribution in the examined tissue. Those skilled in the art will readily appreciate that any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0225] In many cases, a solid phase support or carrier is used as a support capable of binding antigens or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the carrier can be soluble to some extent or insoluble for the purposes achieved by the present disclosure. The support material can have virtually any possible structural configuration so long as the bound molecule can bind to the antigen or antibody. Thus, the shape of the support can be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as, for example, a sheet, test strip, etc. Supports include, but are not limited to, polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to ascertain such using routine experimentation.

[0226] One means for labeling antibodies is by linking them to an enzyme and using them in an enzyme immunoassay (EIA) (Voller, "The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic Horizons 2:1-7, 1978, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller, et al., J. Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, FL, 1980; Ishikawa, et al., (eds.) Enzyme Immunoassay, Kugaku Shoin, Tokyo, 1981). The enzyme bound to the antibody reacts with an appropriate substrate, e.g., a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric, or visual means. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, Δ5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Detection can be achieved by colorimetric methods using a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the extent of enzymatic reaction of the substrate compared to similarly prepared standards.

[0227] Detection can also be achieved by using any of a variety of other immunoassays.For example, by radioactively labeling antibody or antibody fragment, it is possible to detect the wild-type or mutant peptide of fingerprint gene by using radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference).Radioisotopes can be detected by means such as using a gamma counter, a scintillation counter, or by autoradiography.

[0228] It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can then be detected by fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. The antibody may be 152 They may also be detectably labeled by using fluorescence-emitting metals, such as Eu or others of the lanthanide series, which may be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0229] An antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0230] Similarly, a bioluminescent compound can be used to label the antibodies encompassed by the present disclosure. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling are luciferin, luciferase, and aequorin.

[0231] The methods described herein can be carried out, for example, by utilizing a prepackaged diagnostic kit, such as those described above, that includes at least one probe or primer nucleic acid described herein, which can be conveniently used, for example, to determine whether a subject has or is at risk of developing a disease associated with a particular allele variant of interest. The nucleic acid sample analyzed by any of the diagnostic and prognostic methods described above can be taken from any cell type or tissue of the subject. For example, the subject's body fluid (e.g., blood) can be taken by known techniques (e.g., venipuncture). Alternatively, nucleic acid testing can be performed on a dry sample (e.g., hair or skin). Fetal nucleic acid samples can be taken from maternal blood, as described in International Patent Application WO91 / 07660 belonging to Bianchi. Alternatively, amniotic cells or chorionic villi can be taken to perform prenatal testing.

[0232] Diagnostic methods may be performed in situ directly on tissue sections (fixed and / or frozen) of tissue of interest obtained by biopsy or resection, such that purification of nucleic acids is not required. Nucleic acid reagents may be used as probes and / or primers for such in situ methods (see, e.g., Nuovo, GJ, 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY).

[0233] In addition to methods that primarily focus on the detection of a single nucleic acid sequence, profiles may be evaluated with such detection schemes. Fingerprint profiles may be generated, for example, by utilizing differential display methods, Northern analysis, and / or RT-PCR.

[0234] Also provided herein is a method for predicting whether tumor cells in a subject express a target antigen based on the target antigen expression profile of the tissue or tumor. For example, a subject may be diagnosed with cancer, and if the known expression profile of the tissue or tumor does not normally express the target antigen, the tumor cells in the tumor in the subject are not predicted to express the target antigen. Additionally, a subject may be diagnosed with cancer and a known expression profile of a tissue or tumor is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ...9 Tumor cells within a tumor in a subject are not predicted to express the target antigen if less than 2%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or less than 75% do not express the target antigen.

[0235] The present disclosure further relates to novel agents identified by the above screening assays. Thus, screening, diagnostic and / or prognostic assays are provided to identify antibodies specific for a tumor-associated antigen that induce / reduce killing of tumor cells that do not express the tumor-associated antigen or activate T cells against the tumor cells.

[0236] In one embodiment, the disclosure provides an assay for screening candidate or test compounds that are substrates for or interact with a tumor-associated antigen.

[0237] In one embodiment, the assay is a cell-based assay in which cells, such as cancer cells, are contacted with a test agent and the ability of the test compound to kill tumor cells or activate T cells is measured. Measuring the ability of a test agent to perform a described function can be accomplished by monitoring biomarkers as described herein, such as biopsy, biomarker expression, physical assays, etc.

[0238] The ability of a test agent to modulate the binding of an antibody to its target substrate can also be measured. Measuring the ability of a test agent to bind can be achieved, for example, by conjugating the substrate with a radioisotope or enzyme label, so that the binding of the substrate to the antibody can be measured by detecting the labeled substrate in the complex. The target substrate can be conjugated to a radioisotope or enzyme label to monitor the ability of a test agent to modulate the binding to the substrate in the complex. Measuring the ability of a test agent to bind to a target protein can be achieved, for example, by conjugating the agent with a radioisotope or enzyme label, so that the binding can be measured by detecting the labeled agent in the complex. For example, such an agent can be 125 I, 35 S, 14 C, or 3 Agents may be directly or indirectly labeled with H and the radioisotope may be detected by direct counting of radioactive emission or by scintillation counting. Agents may be further enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label may be detected by identifying conversion of an appropriate substrate to product.

[0239] In certain embodiments, the ability of an agent to bind is measured with or without labeling any of the reactants.For example, a microphysiometer can be used to detect interactions without labeling any of the components (McConnell,HMet al.(1992) Science 257:1906-1912).As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that uses a light-addressable potentiometric sensor (LAPS) to measure the rate at which a cell acidifies its environment.

[0240] In one aspect, cell-based system as described herein can be used to identify the agent for treating cancer disclosed herein.For example, such cell system can be exposed to agent at sufficient concentration and for sufficient time to induce such improvement of disease symptoms in exposed cells.After exposure, cells can be examined to determine whether one or more of disease phenotypes have changed to resemble more normal or more wild-type phenotypes.

[0241] In addition, animal or animal-based disease systems, such as those described herein, can be used to identify such agents. Such animal models can be used as test substrates to identify drugs, pharmaceuticals, therapies, and treatments that may be effective in binding to tumor-associated antigens or other targets herein, for example, to treat or prevent the cancers disclosed herein. EXAMPLES

[0242] Example 1: A CD28 bispecific antibody targeted to CD22 enhances the antitumor efficacy of odronextamab in a treatment-resistant diffuse large B-cell lymphoma model Although many patients with diffuse large B-cell lymphoma (DLBCL) can achieve complete responses with state-of-the-art chemoimmunotherapy, patients with relapsed / refractory (r / r) disease usually have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through activation of the T cell receptor / CD3 complex, has shown early promising activity in highly refractory DLBCL patients in Phase I trials, but not all patients achieve complete responses and many relapse. This represents a large unmet medical need. In this example, it is investigated whether the addition of a costimulatory "signal 2" by binding to the CD28 receptor on T cells can enhance the activity of odronextamab. The data disclosed herein demonstrate for the first time that REGN5837, a bispecific antibody that crosslinks CD22-expressing tumor cells with CD28-expressing T cells, enhances the activity of odronextamab by enhancing T cell activation and cytolytic function. In preclinical studies of DLBCL tumors using animals that mimic the human immune system, REGN5837 promotes the antitumor activity of odronextamab, inducing intratumoral proliferation of reprogrammable plastic T cells while deflecting them from a dysfunctional state. REGN5837 monotherapy has shown activity in primate studies, exhibiting no toxicity, and enhancing T cell activation when administered in combination with odronextamab. In addition, analysis of non-Hodgkin's lymphoma (NHL) clinical samples has shown increased expression of CD28 following odronextamab administration. + CD8 + We demonstrated an increase in T cells, demonstrating the presence of a population that can be targeted by CD22xCD28 antibodies. Collectively, these data demonstrate that REGN5837 enhances the antitumor activity of odronextamab in preclinical NHL models, and that the combination of these two bispecific antibodies may provide a novel chemotherapy-free approach for the treatment of r / r DLBCL.

[0243] A therapeutic strategy in the field of cancer immunotherapy is to use bispecific antibodies to redirect T cells to tumors to enhance antitumor activity. This has led to the approval of Blincyto, a CD19xCD3 T cell engager for the treatment of acute lymphoblastic leukemia, and Removab, an EpCAMxCD3 bispecific antibody for the treatment of malignant ascites. These T cell redirecting therapeutics are designed to engage a tumor antigen with one antigen binding fragment (Fab) arm and a T cell activating receptor with the other Fab arm. Odronextamab is a CD20xCD3 bispecific antibody that efficiently induces T cell-mediated killing of cells expressing CD20 in preclinical tumor models. Clinical data from phase 1 trials show promising activity with manageable safety in patients with highly resistant and relapsed B-NHL. However, there is still room to improve efficacy for patients with high-grade lymphomas, as many pretreated patients invariably relapse. For optimal T cell activation, T cells require engagement of the T cell receptor complex (TCR) to provide "signal 1" and further engagement of costimulatory receptors to provide "signal 2." Odronextamab activates T cells by providing signal 1 through crosslinking of CD3, but further enhancement of T cell effector function and activation can be provided by the addition of costimulatory signals. In this study, it is investigated whether the addition of a CD22xCD28 bispecific antibody can enhance the antitumor activity of odronextamab.

[0244] REGN5837 or CD22xCD28 is a hinge-stabilized human IgG4-based antibody designed to enhance T cell responses against tumors expressing CD22 (i.e., NHL) by crosslinking CD22-expressing cells with CD28-expressing T cells. CD22 is a transmembrane protein that binds sialic acid and is found on normal B cells and malignant B cells, such as DLBCL tumors. Although the exact role of human CD22 is not completely clear, CD22 has been suggested to regulate B cell antigen receptor signaling, B cell migration, and maintenance of peripheral B cell homeostasis and survival. Crosslinking CD22 on tumor cells with CD28 on T cells may deliver a CD28 costimulatory signal that is important for enhancing CD20xCD3-induced T cell activation. This study demonstrates that by combining with REGN5837, potent amplification of odronextamab's antitumor activity against DLBCL tumor cells can be achieved both in vitro and in vivo. Characterization of intratumoral T cell responses reveals that CD28 engagement in combination with odronextamab not only results in curative antitumor effects in preclinical models of DLBCL, but also expands intratumoral T cells and induces reprogrammable T cells. Thus, these data provide support for promising clinical studies of the combination of REGN5837 and odronextamab in r / r DLBCL patients.

[0245] result CD28 expression was correlated with intratumoral CD8+ / -CD28 expression at baseline and after odronextamab treatment in patients with r / r NHL. + It is detected in T cells. CD22 is a well-validated tumor target for the treatment of B-cell leukemia and lymphoma and is widely but variably expressed in DLBCL patient samples (Figures 7A-C). In addition, CD28, which can be targeted by the CD22xCD28 bispecific antibody, was identified from untreated DLBCL patient resection samples. +A significant population of CD28 cells was observed (Figures 8A and 8B). Analysis of baseline samples from a dose-finding Phase I study of odronextamab (ClinicalTrials.gov Identifier: NCT02290951) demonstrated that this CD28 expression was consistent with intratumoral CD8 + Predominantly found in T cells, CD4 + We demonstrated that CD28 T cells, but to a lesser extent CD48 T cells, were positive for CD48 T cells (Figures 1A and 1B). In addition, although limited in number, several matched patient samples before and after odronextamab treatment showed CD28 + CD8 + The presence of CD8 T cells was assessed in follicular lymphoma and DLBCL patient samples, consistent with a persistent intratumoral T cell population that could be targeted by REGN5837 to enhance effector memory differentiation and further enhance antitumor activity. + CD28 + A specific increase in the density of T cells was observed 5 weeks after the initiation of odronextamab treatment (Figure 1C). Although CD86 and to a lesser extent CD80 (two ligands for CD28) were also expressed in DLBCL patient resection samples, the expression of the co-inhibitory molecule CTLA4, which has a higher affinity for CD80 and CD86 and can abrogate CD28 costimulatory activity, was simultaneously observed (Figures 8A and 8B). Thus, the use of a CD22xCD28 bispecific antibody may allow for specific engagement of CD28 and a potent enhancement of odronextamab activity.

[0246] REGN5837 enhances odronextamab-mediated T cell activation and effector function in vitro To evaluate the ability of REGN5837 to enhance the cytolytic and T cell activation activities of odronextamab, lymphocyte-enriched human peripheral blood mononuclear cells (PBMCs) were co-cultured with WSU-DLCL2 target cells or DLBCL cells expressing high levels of CD20 and CD22 (Figure 9B) with increasing doses of odronextamab as a single agent (4.8 fM-10 nM) or odronextamab in the presence of a fixed concentration of REGN5837 (ranging from 77 pM-10 uM). Because bispecific CD28, unlike CD28 superagonists such as TeGenero, cannot confer costimulatory activity in the absence of signal 1 or TCR / CD3 complex engagement, REGN5837 activity was analyzed in the presence of odronextamab (Figure 9A). Application of costimulation with REGN5837 enhanced odronextamab-mediated cytotoxicity of WSU-DLCL2 cells (Figure 2A) in a concentration-dependent manner (Table 6), and suppressed CD4 + T cells (Figure 2B) and CD8 + Upregulation of activation markers CD25 on T cells (Figure 2D) and CD4 + T cells (Figure 2C) and CD8 + induced proliferation of T cells (Figure 2E). [Table 5]

[0247] Analysis of co-culture supernatants revealed that REGN5837 also increased odronextamab-mediated cytokine release (Figure 2F) in a dose-dependent manner (Table 7). The ability of REGN5837 to enhance odronextamab activity was additionally demonstrated using B-cell acute lymphoblastic leukemia (NALM6) and Burkitt's lymphoma (Raji CD80 / Cd86dko) cell lines as targets (Figure 9C). [Table 6]

[0248] CD22 expression is variable within DLBCL patient samples, and tumors express CD22 +and CD22 - Because it contains a mixture of tumor cells (Figure 1A-C), CD22 + and CD22 - The ability of REGN5837 to enhance the ability of T cells to kill targets was also evaluated. Purified T cells were incubated with CRISPR-edited CD22-deficient and CD22 wild-type WSU-DLCL2 cells and treated with a fixed concentration of odronextamab (5 pM) and 4.63 × 10 -10 ~1.67×10 -8 T cells and CD22 + Incubation of T cells with target cells expressing CD22 and REGN5837 enhanced tumor cell lysis mediated by odronextamab, whereas incubation of T cells with target cells not expressing CD22 did not result in enhanced cytotoxicity, as expected (Figure 2G). However, incubation of T cells with 20–80% CD22 + Culture with a mixed population of targets upregulates CD22 in a REGN5837 dose-dependent manner. + and CD22 - This resulted in enhanced killing of both target populations (Figure 2G), as well as enhanced CD4 and CD8 T cell activation (Figure 2H). - Target cells were CD22+ at the highest concentration of REGN5837 evaluated. + Although they were not lysed to the same extent as target cells (when mixed at a 4:1 ratio), CD22 + 11% of cells viable vs. CD22 - 21% of cells survived), CD22 + The presence of targets was the lowest CD22 + / CD22 - CD22 even in the proportion of cells - Thus, these data demonstrate that REGN5837 enhancement of odronextamab in vitro cytotoxic activity is not hindered by variability in CD22 expression on DLBCL tumor cells.

[0249] REGN5837-mediated costimulation enhances the antitumor efficacy of odronextamab in xenogeneic DLBCL tumor models The ability of REGN5837 to enhance the antitumor activity of odronextamab in vivo was evaluated in the WSU-DLCL2 tumor model, a heterogeneous DLBCL tumor in which odronextamab alone was not sufficient to cause tumor elimination, thus modeling patients who did not respond completely to bispecific CD3 antibody treatment. With increasing doses of odronextamab, the WSU-DLCL2 tumor model initially showed efficacy in killing mediated by CD20xCD3 in vivo (Figure 10B and Figure 10C), but it became clear that this treatment failed to cause sustained tumor rejection, and all animals eventually succumbed to tumor burden despite treatment with high doses of odronextamab (10 mg / kg) (Figure 10B and Figure 10D). Therefore, we evaluated whether the addition of REGN5837 could further promote antitumor activity (Figure 3A). As expected, prophylactic treatment with REGN5837 alone had no effect on tumor growth (Fig. 3B,C). Although odronextamab monotherapy initially suppressed tumor growth (Fig. 3B,C left) and provided a survival advantage (Fig. 3C right) compared to animals treated with isotype control, all animals eventually succumbed to tumor growth. However, the combination of REGN5837 with odronextamab enhanced antitumor immunity, resulting in 86% (6 / 7) of animals rejecting their tumors (Fig. 3B) and significantly prolonging the overall survival of animals compared to the group treated with monotherapy (Fig. 3C right).

[0250] CD22xCD28-mediated costimulation enhances CD20xCD3 antitumor efficacy in preclinical models of B cell malignancies To investigate how combination therapy enhances antitumor immunity, modulation of intratumoral T cell responses was evaluated 26 days after implantation in different treatments (Figure 11A). At this time point, treatment with odronextamab significantly reduced tumor volume, and combination therapy further suppressed tumor growth (Figure 11B). Advanced dimensionality reduction analysis of intratumoral immune subsets (Figure 3D) revealed enrichment and depletion of specific populations in response to bispecific CD3 or combination therapy. Administration of odronextamab treatment significantly reduced intratumoral CD4 + and CD8 + The percentage (%) of WSU-DLCL2 tumor cells was decreased while preferentially expanding T cells (FIG. 11C). The addition of REGN5837 to odronextamab treatment further increased the frequency of T cells and concomitantly decreased the frequency of tumor cells compared to bispecific CD3 monotherapy (FIG. 11C). The densities of tumor and T cell subsets were counted, which demonstrated that odronextamab monotherapy favored CD4 + T cells (Figure 11D) and CD8 + We found that the combination therapy significantly increased the intratumoral density of CD8 T cells (Figure 3E) compared with odronextamab alone. + This further increased the density of T cells while simultaneously decreasing the number of WSU-DLCL2 cells / mg tumor (Figure 3E). Examination of the induction of memory subsets revealed that treatment with odronextamab significantly drove the expansion of effector and central memory CD8 T cells (Figure 3F, left) as well as effector and central memory CD4 T cells (Figure 11E) compared to isotype control. Interestingly, the addition of REGN5837 significantly increased the proliferation of central and effector memory CD8 T cells compared to groups treated with odronextamab or CD22xCD28 monotherapy. + The density of CD8 T cells was significantly increased (Fig. 3F, right). + This was caused by an increase in the density of the T cell population (Figure 3E, right). Combination therapy did not further alter the ratio of memory to naive cells. Since we observed a strong synergistic effect of the combination of REGN5837 and odronextamab in the prophylactic treatment of WSU-DLCL2 tumors, we decided to investigate the efficacy of the combination in the context of delayed or therapeutic treatment. A one-week delay in the administration of the study drug abrogated the activity of odronextamab monotherapy, such that tumor growth was no longer suppressed (Figure 4B, C). However, the combination treatment still resulted in a strong antitumor activity, significantly suppressing tumor growth (Figure 4C), with an overall rejection rate of 40%. These were associated with a significant survival benefit (50% survival at 125 days after tumor implantation) compared to odronextamab alone (0% survival at day 75). In addition, the efficacy of REGN5837 in enhancing odronextamab activity was evaluated in a systemic tumor model of B-cell acute lymphoblastic leukemia (B-ALL). Immunocompromised animals were pre-transplanted with human PBMCs and 12 days later intravenously implanted with NALM-6 B-ALL cells engineered to express luciferase to allow in vivo tracking using bioluminescence imaging (BLI). Delaying treatment until 8 days after implantation resulted in a non-significant trend toward reduced tumor burden in the odronextamab-treated group (Figure 4E and Figure 4F). However, the combination of REGN5837 and odronextamab induced a significant inhibition of tumor growth (Figure 4F).

[0251] The combination of REGN5837 and odronextamab inhibited peripheral and intratumoral CD8+ / -regulation in human immune-reconstituted animals bearing DLBCL tumors. + Boosting T cell responses To evaluate the in vivo efficacy of REGN5837 in a more physiologically relevant context, WSU-DLCL2 tumors were cultured using SIRPA h / h TPO h / m Rag2 - / - Il2rg - / - Mouse or fetal liver CD34 +The cells were implanted subcutaneously into transplanted human immune system (HIS) animals. Animals were randomized into the indicated treatment groups based on fetal liver donor, human T cell engraftment frequency, and sex. Monotherapy with 0.4 mpk odronextamab did not suppress tumor growth, but combination therapy with REGN5837 significantly suppressed tumor growth (Figure 5C, left) and improved survival (80% survival 64 days after transplant) compared to bispecific CD3 antibody alone (0% survival 64 days after transplant) or isotype control (0% survival 61 days after transplant) (Figure 5C, right). REGN5837 monotherapy also induced tumor rejection (Figures 5B and 5C). This was because allogeneicity between donor T cells providing "signal 1" and WSU-DLCL2 tumor cells was the basis of this antitumor response. In vitro, REGN5837 enhanced T cell activation and effector function mediated by allogeneic signal 1 (FIGS. 12A and 12B). Thus, this enhanced antitumor activity in vivo may be due to costimulation of the allogeneic response.

[0252] Serum cytokines were evaluated to investigate the ability of the combination to enhance peripheral T cell activation and cytokine release after combination therapy. Tumor-bearing human immune system mice were bled before and after dosing for evaluation of lymphocyte subset and serum cytokine induction. As expected, treatment with REGN5837 monotherapy did not show single-agent activity in peripheral blood, as no T cell activation was observed after dosing. However, treatment with odronextamab monotherapy efficiently reduced circulating B cells (Figure 5D right) and increased CD8 in blood after initial T cell margination. + T cells (Figure 5D, left), which was also observed in response to other bispecific CD3 antibodies in preclinical and clinical trials. The combination of REGN5837 with odronextamab increased blood CD8 T cells compared to odronextamab monotherapy. + T cells (Fig. 5D, left) and CD4 +REGN5837 further promoted the proliferation of T cells (Figure 13A). Examination of serum cytokines after treatment revealed that, as expected, REGN5837 did not induce any serum cytokines due to the absence of signal 1 from either the allogeneic tumor or CD20xCD3 treatment. However, consistent with previous studies, induction of serum cytokines was observed after the first dose with odronextamab treatment (Figure 5E and Figure 13B). The combination treatment induced significant post-first dose cytokine release (TNFa, IL-2, IL-10) compared to odronextamab monotherapy, while there was a trend toward increased IL-6. Overall, the enhanced, but transient, induction of serum cytokines and proliferation of T cells in peripheral blood are evidence of enhanced T cell activation induced by CD28 costimulation.

[0253] The combination of odronextamab and REGN5837 enhances antitumor activity by enhancing intratumor T cell accumulation and biasing T cells toward a reprogrammable state and away from a dysfunctional phenotype. To determine how REGN5837 enhanced the antitumor activity of odronextamab, immune profiling of intratumoral, splenic, and blood T cell responses was performed 30 days after implantation. At this time point, the combination of odronextamab and REGN5837 significantly suppressed tumor growth compared to animals treated with isotype and REGN5837 monotherapy, and tended to reduce tumor growth compared to odronextamab monotherapy (Figure 13C). All live cells from tumor, spleen, and blood were analyzed using FlowSOM, an unsupervised clustering algorithm to identify distinct cell populations (metaclusters), and these metaclusters were mapped onto UMAP (Uniform Manifold Approximation and Projection for Dimension Reduction) plots (Figure 5F left). UMAP plots of spleen (Figure 5F right), tumor (Figure 5F right), and blood (Figure 14D) revealed striking differences in the relative proportions of different immune subsets in response to different treatments. In the spleen (FIGS. 14A and 14C) and blood (FIGS. 14B and 14E), odronextamab monotherapy was sufficient to result in significant depletion of B cells compared to treatment with isotype and REGN5837 monotherapy. Importantly, combination treatment tended to reduce splenic B cells to the same extent as odronextamab monotherapy and expand splenic (FIGS. 14A and C) and blood (FIGS. 14B and E) T cell populations compared to isotype-treated animals. In addition, combined administration of odronextamab and REGN5837 resulted in a significant reduction in the frequency of tumor cells within the tumor (FIG. 13D, left) and concomitant expansion of intratumoral T cells (FIG. 13D, right) compared to odronextamab monotherapy.

[0254] Absolute tumor cell counts revealed significant tumor growth inhibition in response to odronextamab or combination treatment compared to isotype-treated animals (Fig. 5G, bottom). At this time point, there was no significant difference in tumor volume (Fig. 13C) or absolute WSU-DLCL2 cell counts between combination or odronextamab treatment (Fig. 5G, bottom), but CD4+ cells were significantly increased in response to combination therapy compared to odronextamab or REGN5837 monotherapy. +and CD8 + T cells were significantly expanded (Figure 5G, top). Investigation of the induction of memory subsets showed that treatment with odronextamab or the combination therapy significantly increased the number of effector memory (EM) CD8 + T cells (Figure 13E) and CD4 + It was shown that EM formation in response to combination treatment was slightly increased compared to odronextamab (CD8 + T cells: 96% vs. 91%; CD4 + T cells: 93% vs. 90%), however, there was a greater increase in effector and central memory CD8 T cells due to an overall increase in total intratumoral T cells. + T cells (Figure 13G) and CD4 + The density of T cells (FIG. 13H) was significantly increased.

[0255] To better understand how different treatments modulated intratumoral T cell phenotype, we analyzed intratumoral CD4 + and CD8 + T cell populations were analyzed by FlowSOM and 11 metaclusters were identified (Figure 5H, left). The UMAP plots show that the combination treatment significantly increased intratumoral CD8 + This resulted in a marked enrichment of metacluster 1 and suppression of metacluster 3 in T cells, and intratumoral CD4 + In T cells, we found that metacluster 11 was biased toward metacluster 10 (Figure 5H and J). Metacluster 1 was biased toward a reprogrammable phenotype (CD38 lo CD101 lo ), or CD8 with a reversible dysfunctional state previously described in the literature + While metacluster 3 is a population of CD8+ T cells, metacluster 4 is a highly proliferative but dysfunctional population of CD8+ T cells (PD1+) that is associated with lack of effector cytokine production and tumor progression. hi CD28 + Ki67 +) (Figure 5I). Metacluster 11 downregulates CD38 in metacluster 10 due to the maintenance of stemness and effector functions and prevention of metabolic exhaustion. hi CD4 + PD-1 has been reported to show better tumor control compared to T cells + CD38 lo CD4 + T cell population (Figure 5I). Thus, these data demonstrated that costimulation with CD22xCD28 bispecific antibody in combination with odronextamab not only expanded intratumoral T cells but also prevented the induction of dysfunctional T cells, thereby resulting in enhanced antitumor immunity.

[0256] REGN5837 monotherapy does not activate T cells in cynomolgus monkey studies, but the bispecific CD28 antibody enhances odronextamab-mediated T cell activation. A study in cynomolgus monkeys was performed to evaluate the tolerability of REGN5837 alone or in combination with odronextamab. Three monkeys per group received a single slow intravenous bolus of REGN5837 alone (10 mpk), odronextamab alone (0.001, 0.01, 0.1, or 1 mpk), or REGN5837 (1 or 10 mpk) followed by odronextamab (0.001, 0.01, or 0.1 mpk). Evaluations of tolerability and pharmacological activity during the survival phase of the study included body weight, clinical findings, veterinary physical examination (which included assessment of heart rate, temperature, and respiratory rate), neuromuscular / musculoskeletal findings, clinical pathology (hematology, blood chemistry [including C-reactive protein], coagulation, and urinalysis). In addition, blood samples were collected at 5 hours post-dose, and at the end of the study on days 1, 4, and 8 for cytokine profiling and immunophenotypic analysis by flow cytometry. Extensive gross and microscopic evaluations were performed at necropsy. Single or repeated intravenous administration of REGN5837 or odronextamab as monotherapy or in combination was not associated with any moribundity or premature death. In addition, there were no adverse test article-related clinical findings, including no signs of cytokine release syndrome (CRS) (e.g., vomiting or fecal changes).

[0257] As previously described with other CD28-based bispecific costimulatory antibodies, treatment with REGN5837 as a single agent increased peripheral CD4+ T cell activation in agreement with in vitro data demonstrating that bispecific CD28 antibodies do not induce T cell activation in the absence of signal 1 (Figure 9A). + or CD8 + REGN5837 did not induce T cell proliferation or activation (Figure 6B, C, and Figure 15A-B). In addition, REGN5837 monotherapy had no effect on B cell numbers in the periphery (Figure 6A). However, consistent with previously published data on bispecific CD3 antibodies, odronextamab promoted initial lymphocyte margination, as indicated by upregulation of ICOS and induction of proliferation, followed by CD4 +T cells (Figure 15A, B) and CD8 + induced a trend towards proliferation and activation of T cells (Figures 6B and 6C). At all concentrations of odronextamab evaluated, sustained depletion of peripheral B cells occurred concomitant with T cell activation (Figure 6A). The addition of REGN5837 to odronextamab depleted peripheral B cells as efficiently as odronextamab alone. Importantly, the combination with 1mpk REGN5837 significantly reduced peripheral CD8 T cells compared to odronextamab monotherapy and placebo controls. + T cells (Figures 6B and 6C) and CD4 + 10 mpk of REGN5837 also induced a trend towards enhanced T cell activation mediated by odronextamab, however, the addition of this concentration of costimulatory antibody did not significantly enhance T cell activity to the same extent as REGN5837 administered at 1 mpk. This may be due to the binding of one arm of the bispecific CD28 antibody at high concentrations, which results in a bell-shaped curve when investigating parameters of T cell activation.

[0258] Odronextamab monotherapy resulted in a dose-dependent increase in serum cytokines (Figure 6D), correlating with previous observations with another tumor-targeting bispecific CD3 antibody. The combination of odronextamab (0.1 mpk) and REGN5837 (1 mpk) significantly increased IL-2 production compared to placebo, with a trend toward increased serum IL-6, reflecting the enhanced T cell activation observed with the combination treatment (Figures 6C and 15B). Interestingly, the combination of odronextamab at 0.1 or 0.01 mpk with REGN5837 (1 mpk) did not induce IL-6 as strongly as odronextamab alone administered at 1 mpk, but the combination treatment increased CD4 expression to the same extent as odronextamab monotherapy at 1 mpk. + and CD8 +T cells expanded and activated (FIG. 6B and FIG. 15A). Overall, these results demonstrate that the addition of REGN5837 to odronextamab enhanced T cell activation in cynomolgus monkey animals by inducing increased serum cytokines and peripheral T cell proliferation.

[0259] conclusion In this example, it was demonstrated that REGN5837 can enhance the activity mediated by odronextamab by promoting in vitro cytotoxicity of DLBCL and other NHL cell lines, as well as by potently enhancing antitumor activity against DLBCL tumors that cannot be eliminated by treatment with bispecific CD3 antibodies. Odronextamab as a single agent suppressed WSU-DLCL2 tumor growth, but only combination treatment with REGN5837 resulted in a curative effect in a preclinical in vivo model and increased overall survival. In addition, the combination of REGN5837 and odronextamab not only maintained cytotoxicity against cells expressing CD20, but also resulted in a significant expansion of intratumoral T cells and enhanced the induction of effector memory cells compared to odronextamab monotherapy. Further characterization of the intratumoral immune compartment from HIS mice showed that the addition of CD28 costimulation to odronextamab enhanced the expression of CD8 + Reprogrammable T cell populations into a phenotype (CD38 lo CD101 lo ) or into a plastic dysfunctional state that can be reversed under normal conditions, allowing the reactivated T cells to produce high levels of proinflammatory cytokines and mediate antitumor immunity. Concomitant with this proliferation was the expression of CD28 + Ki67 + CD8 +There was a concomitant reduction in the induction of dysfunctional T cells, a hyperactivated population previously described as deficient in effector function, proliferating in melanoma and late-stage NSCLC and associated with resistance to PD-1 blockade therapy. The addition of REGN5837 maintained effector function, promoted T cell persistence, and upregulated CD38 T cells due to metabolic reprogramming driven by enhanced glutaminolysis. hi PD-1 has been reported to show superior tumor control compared to T cells + CD38 lo CD4 + It also expanded the population of T cells. Thus, the data suggest that REGN5837 not only expands intratumoral T cells but also prevents the induction of dysfunctional T cells, thereby allowing for enhanced antitumor immunity.

[0260] Toxicity studies in cynomolgus monkeys and analysis of peripheral blood from HIS mice demonstrated that REGN5837 had little activity as a monotherapy and showed no toxicity due to the lack of signal 1, despite the expression of CD22 on peripheral B cells. Peripheral T cell activation and proliferation was observed only when REGN5837 was administered in combination with odronextamab, which mediates TCR clustering. Importantly, in the cynomolgus monkey study, the addition of REGN5837 to low-dose odronextamab was able to result in similar levels of peripheral T cell proliferation as 10- to 100-fold higher doses of the bispecific CD3 antibody, but this proliferation was accompanied by a lower induction of serum cytokines compared to treatment with high-dose monotherapy of the bispecific CD3 antibody in the cynomolgus monkey study. Thus, treatment with either the combination or high-dose bispecific CD3 antibody was well tolerated without any signs of cytokine release syndrome (CRS) in the animals.

[0261] Odronextamab is currently being evaluated in phase 1 and 2 clinical trials for the treatment of r / r B-cell NHL. The patient population in this trial is heavily pretreated and has failed at least two prior therapies. Early results from odronextamab clinical trials have shown promising activity, with refractory follicular lymphoma patients receiving at least 5 mg of odronextamab weekly having an ORR of 92.9% and a complete response rate (CR) of 75.0%. Heavily pretreated r / r DLBCL patients receiving 80 mg or more and not receiving prior CAR T therapy have an ORR and CR rate of 60%, and patients refractory to CAR T therapy have an ORR of 33.3% and a CR rate of 23.8%. Overall, the safety profile appears to be manageable and consistent with the class of CD20xCD3 bispecific agents. Although encouraging, these data reveal that there is still room for improvement regarding the treatment of r / r DLBCL patients, especially in the post-CAR T setting. Collectively, the data show that REGN5837, a novel CD22xCD28 bispecific antibody, can enhance the antitumor activity of odronextamab and provide support for the study of the combination of REGN5837 with odronextamab in difficult-to-treat patients with aggressive lymphomas.

[0262] Example 2: Materials and Methods from Example 1 Study design This study demonstrated that tumor-targeted bispecific CD28 antibodies can expand T cell activation mediated by odronextamab to enhance antitumor activity. Control and experimental treatments were administered to age- and sex-matched mice. Sample sizes were empirically selected to ensure adequate statistical power and were consistent with standards in the field of technology used in the study. All animals were randomized into different treatment groups based on tumor volume before treatment initiation, and investigators were blinded to all treatment groups. The number of experimental replicates is shown in the figure legends.

[0263] cell line For testing in WSU-DLCL2, DLBCL cell lines were obtained from DSMZ (ACC 575) and maintained in RPMI-1640 containing 10% FBS (Seradigm) supplemented with penicillin, streptomycin, glutamine, and 1 mM HEPES (Gibco). CRISPR-edited CD22-deficient lines were generated by electroporation of Cas9 ribonucleoprotein (RNP) using the Neon™ Transfection System 100 μL Kit (Invitrogen) with TrueCut™ Cas9 Protein v2 (Invitrogen) and High Scoring TrueGuide™ Synthetic sgRNA targeting human CD22 (Invitrogen, guide RNA: CCGGTGCACCTCAATGACAG). CD22-deficient cells were bulk-sorted 96 hours after electroporation.

[0264] For experiments with NALM6-luc tumors, the NALM6 cell line (DSMZ:ACC 128) was modified with EF1a-luciferase-2A-GFP-Puro lentivirus (GenTarget) to image tumor cell growth in vivo. The cell line was maintained in RPMI containing 10% FBS supplemented with PSG (penicillin, streptomycin, and glutamine) and puromycin selection (1ug / ml).

[0265] Animal testing All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Experimental protocols were approved by the Regeneron Pharmaceuticals Institutional Animal Care and Use Committee (IACUC), and all animals were maintained under pathogen-free conditions.

[0266] For NSG experiments, WSU-DLCL2 cells (3 × 10 6 5 × 10 cells were collected. 5The cells were mixed with PBMCs (ReachBio) and resuspended in a 1:1 mixture of PBS and GFR Matrigel (Corning). The cell mixture was injected subcutaneously into the right flank of female NSG mice (Jackson Laboratory). For human immune system reconstitution experiments, WSU-DLCL2 cells (3 × 10 6 ) were cultured using fetal liver CD34 + SIRPA cells transplanted h / h TPO h / m Rag2 - / - Il2rg - / - Mice were implanted subcutaneously. Animals were divided so that each treatment group had a similar distribution of fetal liver donors, human immune cell engraftment frequency, and gender. Mice were randomized to receive blinded treatment of either isotype control (EGFRV3xCD3 or MUC16xCD28) or test article (REGN5837, odronextamab), which was administered by intraperitoneal injection at the specified concentrations as monotherapy or combination on days 1, 8, and 15 after implantation for prophylactic treatment, and days 8, 15, and 22 for therapeutic treatment. Tumor growth was monitored over time using X and Y diameter measurements (perpendicular measurements of length and width) with a digital caliper (VWR). Tumor volume was calculated (X*Y*(X / 2), where X is the shorter diameter). Mice were euthanized when tumors reached the designated tumor endpoint (tumor diameter >20 mm or tumor ulceration). This specified endpoint complies with IACUC standards.

[0267] For NALM6-luc tumor experiments, 4 × 10 6 Successfully engrafted animals were transplanted with 5 × 10 human PBMCs (ReachBio). 6Single NALM6-luc cells were injected intravenously 12 days after PBMC transplantation. Mice were randomized to receive blinded treatment of either isotype control (EGFRV3xCD3 or MUC16xCD28) or test article (REGN5837, odronextamab), which was administered by intraperitoneal injection at the specified concentrations as monotherapy or in combination on days 8, 15, and 22 after transplantation. Mice transplanted with NALM6-luc were imaged twice weekly using an IVIS Spectrum (Perkin Elmer) after intraperitoneal injection of luciferin substrate (PerkinElmer). Bioluminescence (total light) was quantified using Living Image software. According to IACUC criteria, experiments were terminated when mice began to show signs of GVHD (weight loss ≥ 20%).

[0268] Measurement of serum cytokines in mice At the indicated time points, blood was collected from the submandibular vein into Microtainer serum tubes (BD 365967). Cytokine concentrations were analyzed using the V-plex Human ProInflammatory-10 Plex kit according to the manufacturer's instructions (Meso Scale Diagnostics).

[0269] Flow cytometry For immunophenotyping experiments, tumors, spleens, and blood were harvested on the indicated days. Single-cell suspensions were prepared and live / dead cell discrimination was performed using the Live / Dead Fixable Blue Dead Cell Staining Kit (Thermo Fisher Scientific). To quantify tissue cell numbers, a fixed number of CountBright absolute counting beads (Thermo Fisher Scientific) were added to each sample before capture. Samples were captured on a Symphony (BD Biosciences) and analyzed using FlowJo (TreeStar) or OMIQ.

[0270] FlowSOM in OMIQ was performed on samples acquired on a Symphony (BD Biosciences) for automated identification of T cell clusters based on selected markers. Analysis was performed on an equal number of events per sample. Event range was determined by the sample containing the fewest cells. Intratumoral CD8 + FlowSOM clustering in Omiq.ai for selected parameters of T cells (activation / dysfunction and memory markers) was performed with default settings. To visualize the clusters identified by FlowSOM, an advanced dimensionality reduction method, UMAP, was performed on all samples within Omiq.ai. Samples from each treatment group were concatenated and FlowSOM clusters were overlaid on the UMAP plot.

[0271] Wet plate coating assay Antibodies diluted to 10 μg / ml in PBS were wet coated onto polypropylene plates overnight. Diluted antibodies (100 μl) were added to 96-well polypropylene assay plates in triplicate. Plates were stored overnight at 4° C. to allow antibody adsorption and plates were washed twice with PBS before use in proliferation assays. PBMCs were isolated from leukocyte-enriched peripheral blood (New York Blood Center) collected from four healthy independent donors. PBMCs from each donor were resuspended in RPMI medium (Irvine Scientific) containing 10% human AB serum (GemCell) and penicillin / streptomycin / glutamine (100 units / ml, 100 μg / ml, 292 μg / ml, Gibco, respectively) and then added to 96-well assay plates at 100,000 cells / well in a final volume of 200 μl / well. Assay plates were then incubated at 37° C.+5% CO2 for 54 hours. At 54 hours, assay plates were centrifuged and 100 μl of supernatant was removed for cytokine analysis. Cytokine concentrations of IFNg, IL1B, IL2, IL4, IL6, IL8, IL10, IL13, and TNFa were measured using the V-PLEX Proinflammatory Panel1 Human Kit according to the manufacturer's instructions. Mean and range of concentrations (pg / mL) obtained from four donors were plotted. Individual data points represent the average concentration for each individual donor obtained with assays performed in triplicate wells.

[0272] To assess proliferation, 100 μl of 1 mCi / ml tritiated thymidine (Perkin Elmer) was added to each well and incubated for a further 18 hours at 37° C.+5% CO2. Each 96-well assay plate was harvested using a Filtermate Harvester (Perkin Elmer) and analyzed in a TopCount scintillation counter (Perkin Elmer). The amount of radioactivity was measured as counts per minute (CPM) per well and was proportional to the number of proliferating cells. CPM values ​​are shown. Four data points represent the average radioactivity counts for each individual donor obtained from assays performed in triplicate wells.

[0273] In vitro T cell activation assay Human CD3(III) isolated from white blood cell packs of healthy donors by density gradient centrifugation in 50 mL SepMate™ tubes and previously frozen + T cells were thawed on the day of assay in stimulation medium (X-VIVO 15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing 50 U / ml Benzonase nuclease and diluted at 1 × 10 5 NALM6 cells were plated in a 96-well round-bottom plate at a concentration of 10 × 10 cells / well. NALM6 cells were diluted to 10 × 10 with 10 ug / mL mitomycin C. 6 After 1 h of incubation at 37°C and 5% CO2, the mitomycin C-treated cells were washed three times with D-PBS containing 2% FBS and then purified with CD3+. + Add 5 x 10 cells per well to wells containing T cells. 4A final concentration of 1.25 μM of IL-2 was added to the wells. An irrelevant hIgG1 mAb (100 nM / well) was added to all wells to block Fc receptors. A fixed concentration of odronextamab or 500 pM of non-binding isotype control was added to the wells along with increasing doses of REGN5837 or non-binding isotype control from 3.1 pM to 200 nM. Plates were incubated at 37°C and 5% CO2 for 72 hours, at which point 50 μl of culture supernatant was harvested. 5 μl of this harvested supernatant was tested in the Human IL-2 (AL221F) AlphaLISA Assay (Perkin Elmer) according to the manufacturer's protocol. Measurements were taken on an Envision multilabel plate reader (Perkin Elmer). A standard curve of known concentrations was generated to estimate the concentration of produced IL-2 in the assay wells. To assess T cell proliferation, 1.25 μM [ 3 The medium was supplemented with thymidine (Perkin Elmer) at a final concentration of [H] and cells were incubated for 16 h at 37° C., 5% CO2. Plates were harvested using a Microbeta Filermat-96 Cell Harvester (Perkin Elmer), 30 μL of MicroScint-20 (Perkin Elmer) was added, and [ 3 [H]thymidine incorporation was measured using a TopCount NXT microplate scintillation counter (Perkin Elmer). All serial dilutions were tested in triplicate. EC 50 Values ​​were determined by a four-parameter logistic equation for a 10-point dose-response curve using GraphPad Prism software.

[0274] In vitro cytotoxicity assay Thaw human PBMCs and culture at 1 × 10 in complete medium (RPMI cell culture medium supplemented with 10% FBS, penicillin-streptomycin-glutamine). 6WSU-DLCL2 cells were plated at 1000 cells / mL and incubated overnight at 37°C to enrich for lymphocytes by depleting adherent cells, e.g., macrophages, dendritic cells, and some monocytes. The next day, PBMCs were harvested and labeled with 1 μM Violet Cell Tracker fluorescent tracking dye. WSU-DLCL2 cells were labeled with 1 μM Vybrant CFDA-SE fluorescent dye. After labeling, 5,000 labeled target cells were plated in a round-bottom 96-well plate at a 1:5 ratio with the labeled PBMCs. Serial dilutions of R5837 were combined with serial dilutions of R1979 and added to the labeled target and effector cells, and the plate was incubated at 37°C for 72 hours. After incubation, cells were washed and stained with a live / dead cell stain in PBS, followed by a cocktail of fluorophore-conjugated antibodies against CD2, CD4, CD8, and CD25 for analysis of viable target cells, T cell activation, and proliferation. Counting beads (20 μL per well) were added immediately prior to sample analysis on a BD Celesta flow cytometer.

[0275] Target cell killing was assessed by calculating the number of viable target cells / well labeled with CFDA-SE by normalizing to the number of beads / well collected. Percent viability was normalized to the number of viable target cells in control conditions (target cells in the presence of PBMCs only). T cell activation was assessed by recording %CD25 on CD4+ and CD8+ T cells. T cell proliferation was assessed by recording the percentage of cells with reduced MFI of Violet Cell Tracker dye.

[0276] Supernatants from this assay were collected for analysis of cytokine levels. Concentrations of IL-17a, IFNγ, TNFα, IL-10, IL-6, IL-4, and IL-2 were analyzed using a cytometric bead array (CBA) kit according to the manufacturer's instructions. Briefly, 1 / 15 dilutions of supernatants were incubated with standards in 96-well assay plates containing cytokine-specific bead arrays for 3 hours at room temperature. After incubation, samples were washed twice and analyzed by flow cytometry on a BD FACS Canto II. Cytokine levels were interpolated from the MFI of the kit standards and reported as pg / mL.

[0277] Antibody EC50 values ​​were determined by a four-parameter logistic equation for nine-point dose-response curves using GraphPad Prism software. Maximum responses for percent cytotoxicity, T cell activation, proliferation, and cytokine release were obtained as plateau values ​​generated by Prism curve fitting. Fold changes in EC50 were calculated as EC50. R5837なし / EC50 [M]R5837 The fold change in maximum cytokine release was calculated as the maximum [M]R5837 / Maximum R5837なし It was calculated as:

[0278] Chromogenic IHC Chromogenic IHC assays were performed on formalin-fixed paraffin-embedded DLBCL patient samples (Tristar) on a Ventana Discovery ULTRA platform. Antigen retrieval was performed using Tris-EDTA (pH 9) buffer and slides were incubated with primary antibodies listed below. An additional amplification step was required for some markers to visualize the signal using the OptiView DAB detection system. Slides were counterstained with Novolink Hematoxylin (Leica Microsystems, Inc) and coverslipped (Corning coverslips, #1) with Cytoseal 60 (Thermo Scientific / Richard Allen Scientific). Once the mounting medium was completely dry, slides were scanned at 40x on a Leica Aperio AT2 scanner. Images were analyzed using Indica HALO software. [Table 7]

[0279] multiplex IHC A fully automated multiplex immunohistochemistry assay was performed on the Ventana Discovery ULTRA platform (Ventana Medical Systems, Tucson, AZ). Five rounds of sequential application of primary and horseradish peroxidase-conjugated secondary antibodies were performed. Heat denaturation between each step to completely remove bound primary and secondary antibodies was performed to eliminate downstream cross-reactivity. This allowed the use of primary antibodies raised in the same species. The fluorochromes used were carefully selected to ensure spectral separation and result in optimal staining. The combination and order of application of primary antibodies and tyramide fluorophores were optimized to ensure that both epitopes and fluorophores could withstand repeated heat denaturation steps. The optimal concentration of each antibody was determined and they were applied in the following order and detected with the indicated fluorophores: [Table 8]

[0280] After staining, tissues were counterstained and coverslipped with Invitrogen ProLong Gold Antifade Mountant with NucBlue. Imaging of whole slides was performed on a Zeiss Axioscan equipped with a Colibri light source and appropriate filters to visualize these specific fluorophores. Quantitative image analysis was performed using the HALO Indica Labs Hyperplex module (IndicaLabs, Albuquerque, NM). The number of positive cells for each immune subset and their density across the tumor area were measured.

[0281] Cynomolgus monkey toxicity test Cynomolgus monkey studies were conducted in facilities accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care, with Animal Welfare Assurance Numbers issued by the Office of Laboratory Animal Welfare, and registered with the United States Department of Agriculture and the IACUC. Male cynomolgus monkeys (Macaca fascicularis) (three animals per group) were used to conduct the study at Altasciences Preclinical Seattle (formerly SNBL USA). Subjects received a single dose of each test article by intravenous bolus (REGN5837) or intravenous infusion (approximately 30-minute odronextamab infusion). Combination treatments were administered sequentially, with odronextamab infusions beginning 5-10 minutes after administration of REGN5837. Evaluation of treatment-related effects included body weight measurements, clinical findings, veterinary physical examinations (which included assessment of heart rate, temperature, and respiratory rate), neuromuscular / musculoskeletal findings, and clinical pathology (hematology). Blood and / or tissue samples were collected for cytokine analysis, immunophenotypic analysis, histopathology, and toxicokinetic evaluation. For peripheral blood flow cytometry, blood was collected into potassium EDTA tubes, hemolyzed, stained for CD3, CD4, CD8, CD14, CD16, CD20, CD28, and Ki67 (BD Biosciences), and CD278 (BioLegend), and analyzed on a FACSCanto II flow cytometer. For cytokine analysis, blood was collected into serum separator tubes containing anticoagulant. Serum was separated by centrifugation at 1000g-2000g for 10-15 min at 4°C and analyzed using the MSD U-Plex platform (IL-1β, IL-2, IL-6, MCP-1, and TNF-α, and IFN-γ).

[0282] statistical analysis Sample sizes were empirically selected to ensure adequate statistical power and were consistent with standards in the techniques used in the study. Statistical significance was determined using one-way and two-way analysis of variance (ANOVA), and the Kaplan-Meier method was used with the log-rank test to compare survival times between groups. Graph generation and statistical analysis were performed using GraphPad Prism (version 8).

[0283] Example 3: REGN5837 enhances cytotoxicity mediated by human T cells activated with the CD20xCD3 bispecific antibody REGN1979 against B cell lymphoma cells with or without surface expression of CD22. T cells were isolated from freshly thawed PBMCs using the EasySep Human T-Cell Isolation Kit and used immediately. WSU-DLCL2 / CD22 WT Cells were labeled with 1 μM Vybrant CFDA-SE fluorescent dye. WSU-DLCL2 / CD22 KO Cells were labeled with 1 μM CellTrace Far Red fluorescent dye. WT and WSU-DLCL2 / CD22 KO Target cells were plated in 96-well round-bottom plates at different ratios (0:100, 20:80, 40:60, 60:40, 80:20, 100:0) and unlabeled T cells were added at a final effector to target ratio of 5:1. Target and effector cells were incubated with 5pM CD20xCD3 (REGN1979) alone or in combination with different concentrations of REGN5837 (16.7nM, 2.77nM, 463pM) and plates were incubated at 37°C for 72 hours.

[0284] After incubation, cells were washed and stained with a live / dead cell stain in PBS, followed by staining with a cocktail of fluorophore-conjugated antibodies against CD2, CD4, CD8, and CD25 for analysis of T cell activation. Counting beads (20 μL per well) were added immediately prior to sample analysis on a BD Celesta flow cytometer.

[0285] WSU-DLCL2 / CD22 WT Killing of WSU-DLCL2 / CD22 was assessed by calculating the number of viable target cells / well labeled with CFDA-SE by normalizing to the number of beads / well collected. KOCell killing was assessed by calculating the number of viable target cells / well labeled with Far-Red by normalizing to the number of beads / well collected. Percent viability was normalized to the number of viable target cells in control conditions (target cells in the presence of effector cells only). T cell activation was assessed by recording the MFI of CD25 on CD4+ and CD8+ T cells.

[0286] Summary of Results The ability of REGN5837 to enhance cytotoxicity mediated by human T cells activated with the CD20xCD3 bispecific antibody REGN1979 against B cell lymphoma cells with or without surface expression of CD22 was assessed using flow cytometry. Additionally, T cell activation, as measured by upregulation of CD25 on T cells, was assessed. REGN1979 at 5 pM activates human T cells and inhibits WSU-DLCL2 / CD22 WT and WSU-DLCL2 / CD22 KO REGN1979 alone induced CD22 T cells to kill to a similar extent (Table 8). T cell activation in the presence of 5 pM REGN1979 alone induced CD22 T cells to kill to a similar extent (Table 8). KO :CD22 WT The results were comparable regardless of the cell ratio (Table 11). REGN5837 inhibits WSU-DLCL2 / CD22 mediated by REGN1979 WT Enhanced cell killing in a dose-dependent manner. WSU-DLCL2 / CD22 WT Cell killing was determined by the expression of WSU-DLCL2 / CD22 in culture. KO The results were similar regardless of the presence of . REGN5837 inhibited WSU-DLCL2 / CD22 by more than 20% WT REGN1979-mediated WSU-DLCL2 / CD22 expression in cultures containing cells KO REGN5837 enhanced cell killing by inhibiting any WSU-DLCL2 / CD22 in the cultures. WT REGN1979-mediated WSU-DLCL2 / CD22 in the absence of cells KOIt did not enhance cell killing (Table 10). ·WSU-DLCL2 / CD22 WT In cultures containing cells, REGN5837 enhanced REGN1979-mediated T cell activation in a dose-dependent manner, resulting in a higher ratio of WSU-DLCL2 / CD22 WT Stronger T cell activation was observed when WSU-DLCL2 / CD22 cells were present in the cultures. WT The absence of cells did not enhance REGN1979-mediated T cell activation (Table 11).

[0287] In summary, costimulation with REGN5837 enhanced REGN1979-mediated killing of target cells lacking CD22 expression, as long as cells expressing CD22 were present in the culture. [Table 9] [Table 10]

[0288] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0289] Any polynucleotide and polypeptide sequences that reference accession numbers that correlate to entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web and / or those maintained by the National Center for Biotechnology Information (NCBI) on the World Wide Web, are also incorporated by reference in their entirety.

[0290] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition for use in causing the killing of tumor cells in a target, inducing the killing of tumor cells in a target, or inducing T cell activation in tumor cells in a target, It comprises a multispecific antigen-binding molecule having a first antigen-binding domain specific to the target antigen and a second antigen-binding domain specific to the CD28 protein, A pharmaceutical composition wherein the tumor cells do not express the target antigen, or are not expected to express it.

2. A pharmaceutical composition for use in the treatment of cancer in subjects with tumors, It comprises a multispecific antigen-binding molecule having a first antigen-binding domain specific to the target antigen and a second antigen-binding domain specific to the CD28 protein, A pharmaceutical composition wherein at least a subset of tumor cells within the tumor do not express the target antigen.

3. It is determined that at least a subset of the tumor cells within the tumor do not express the target antigen, The pharmaceutical composition according to claim 1 or 2, wherein tumor cells optionally do not express the target antigen if the expression of the target antigen is below a detection level or below a signal-to-noise ratio.

4. A pharmaceutical composition for use in methods of cancer treatment, It contains a multispecific antigen-binding molecule that includes a first antigen-binding domain specific to the target antigen and a second antigen-binding domain specific to the CD28 protein. The method described above is (i) Determining that the subject includes a tumor containing tumor cells that do not express the target antigen, (ii) administering the pharmaceutical composition to the subject, A pharmaceutical composition in which, optionally, tumor cells do not express the target antigen if the expression of the target antigen in the tumor cells is below a detection level or below a signal-to-noise ratio, thereby selecting targets for cancer treatment.

5. The aforementioned target antigen is (i) Tumor-associated antigens (TAAs), (ii) an antigen associated with the tumor microenvironment of the tumor, wherein the antigen associated with the tumor microenvironment is optionally an antigen associated with the tumor stroma, an antigen associated with the extracellular matrix of the tumor, an antigen associated with blood vessels in the tumor microenvironment, or an antigen associated with cancer-associated fibroblasts, or (iii) Immune antigen The pharmaceutical composition according to any one of claims 1, 2, and 4.

6. The pharmaceutical composition according to claim 5, wherein the target antigen is a TTA selected from CD38, EGFR, CD22, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7.

7. (i) The target antigen is an antigen associated with tumor stroma selected from PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin isoform EDB; (ii) The target antigen is an antigen associated with the extracellular matrix of the tumor, selected from nectin, versican (VACN), fibronectin, and carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) proteins; (iii) The target antigen is an antigen associated with cancer-associated fibroblasts selected from α-smooth muscle actin (α-SMA), fibroblast-activating protein (FAP), S100A4, platelet-derived growth factor receptor (PDGFRα / β), vimentin, PDPN, CD70, CD10, GPR77, CD10, CD74, CD146, CAV1, Saa3-, and CD49e; or (iv) The target antigen is an antigen associated with the blood vessels in the tumor microenvironment, selected from DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, and VEGFR2. The pharmaceutical composition according to claim 5.

8. The pharmaceutical composition according to claim 6, wherein the target antigen is CD22.

9. The pharmaceutical composition according to claim 5, wherein the immune antigen is an antigen expressed on the surface of an immune cell, and the immune cell is selected from macrophages, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, natural killer cells, T cells, and B cells.

10. The pharmaceutical composition according to claim 5, wherein the target antigen is an immune antigen selected from any one of the immune antigens listed in Table 4.

11. The tumor comprises immune cells, and the immune cells are B cells or cells expressing CD20; and / or The tumor cells that do not express the target antigen also do not express CD20; and / or The tumor microenvironment of the tumor includes cells expressing the target antigen; and / or At least 10% to 100% of the tumor cells within the tumor do not express the target antigen. A pharmaceutical composition according to any one of claims 1, 2, and 4.

12. The pharmaceutical composition according to claim 5, wherein the target antigen is TTA, and the tumor is a heterogeneous tumor further comprising tumor cells expressing TAA.

13. (i) The multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, wherein the bispecific antibody fragment is a bispecific T-engaging antibody (BiTE), a biaffinity retargeting molecule (DART), or a tandem diabody (TandAb); or (ii) The multispecific antigen-binding molecule is a bispecific antibody selected from the bispecific antibodies listed in Table 3. A pharmaceutical composition according to any one of claims 1, 2, and 4.

14. The pharmaceutical composition according to any one of claims 1, 2, and 4, wherein the multispecific antigen-binding molecule is administered to the subject together with a second multispecific antigen-binding molecule having a first antigen-binding region specific to a second target antigen and a second antigen-binding region specific to the CD3 protein.

15. (i) The second target antigen is selected from any of the TAAs listed in Table 2; (ii) The second target antigen is the CD20 protein, The pharmaceutical composition according to claim 14.

16. The pharmaceutical composition according to claim 14, wherein the second multispecific antigen-binding molecule is selected from any of the multispecific antigen-binding molecules in Table 5.

17. The multispecific antigen-binding molecule exhibits a co-stimulatory effect when administered together with the second multispecific antigen-binding molecule. The aforementioned co-stimulatory effect is one or more of the following: activation of T cells, induction of IL-2 release, induction of CD25+ upregulation in PBMCs, and enhancement of T cell-mediated cytotoxicity. The pharmaceutical composition according to claim 14.

18. The tumor cells are B-cell carcinoma tumor cells, The aforementioned B-cell carcinoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenström macroglobulinemia), pilocytic cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma). A pharmaceutical composition according to any one of claims 1, 2, and 4.

19. A combination pharmaceutical for use in treating cancer in subjects with tumors, inducing the killing of tumor cells within a tumor in a subject, or inducing T cell activation against tumor cells within a tumor in a subject, A first multispecific antigen-binding molecule having an antigen-binding domain specific to a first target antigen and an antigen-binding domain specific to the CD28 protein, The present invention comprises a second multispecific antigen-binding molecule having an antigen-binding domain specific to a second target antigen and an antigen-binding domain specific to the CD3 protein, The first target antigen is not the same antigen as the second target antigen. A combination pharmaceutical in which at least a subset of the tumor cells within the tumor do not express the first target antigen.

20. The first target antigen is (i) Tumor-associated antigens (TAAs), (ii) an antigen associated with the tumor microenvironment of the tumor, wherein the antigen associated with the tumor microenvironment is optionally an antigen associated with the tumor stroma, an antigen associated with the extracellular matrix of the tumor, an antigen associated with blood vessels in the tumor microenvironment, or an antigen associated with cancer-associated fibroblasts, or (iii) It is an immune antigen, The combination pharmaceutical product according to claim 19.

21. The combination pharmaceutical according to claim 19 or 20, wherein the first target antigen is a TTA selected from CD38, EGFR, CD22, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7.

22. The combination pharmaceutical according to claim 21, wherein the first target antigen is CD22.

23. (i) The first target antigen is an antigen associated with the tumor stroma, selected from PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin isoform EDB; (ii) The first target antigen is an antigen associated with the extracellular matrix of the tumor, selected from nectin, versican (VACN), fibronectin, and carcinoembryonic antigen-associated cell adhesion molecule (CEACAM) proteins; (iii) The first target antigen is an antigen associated with cancer-associated fibroblasts selected from α-smooth muscle actin (α-SMA), fibroblast-activating protein (FAP), S100A4, platelet-derived growth factor receptor (PDGFRα / β), vimentin, PDPN, CD70, CD10, GPR77, CD10, CD74, CD146, CAV1, Saa3-, and CD49e; or (iv) The first target antigen is an antigen associated with the blood vessels in the tumor microenvironment, selected from DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, and VEGFR2. The combination pharmaceutical product according to claim 19 or 20.

24. The combination pharmaceutical according to claim 19 or 20, wherein the first target antigen is an immune antigen expressed on the surface of an immune cell, and the immune cell is selected from macrophages, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, natural killer cells, T cells, and B cells.

25. The combination drug according to claim 19 or 20, wherein the tumor is a heterogeneous tumor comprising cells expressing the first target antigen and cells expressing the second target antigen.

26. (i) The first multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, wherein the bispecific antibody fragment is a bispecific T-engaging antibody (BiTE), a biaffinity retargeting molecule (DART), or a tandem diabody (TandAb); or (ii) The first multispecific antigen-binding molecule is selected from the bispecific antibodies listed in Table 3. The combination pharmaceutical product according to claim 19 or 20.

27. ​​(i) The second target antigen is selected from any of the antigens listed in Table 2; (ii) The second target antigen is CD20; or (iii) The second target antigen is an immune tumor antigen. The combination pharmaceutical product according to claim 19 or 20.

28. The combination pharmaceutical according to claim 27, wherein the immunotumor antigen is CD22, CD20, CD72, CD19, CD21, CD24, or CD79.

29. (i) The second multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, wherein the bispecific antibody fragment is a bispecific T-engaging antibody (BiTE), a biaffinity retargeting molecule (DART), or a tandem diabody (TandAb); or (ii) The second multispecific antigen-binding molecule is selected from the bispecific antibodies listed in Table 5. The combination pharmaceutical product according to claim 19 or 20.

30. The tumor is derived from a B-cell carcinoma, and the B-cell carcinoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenström macroglobulinemia), pilocytic cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma). The combination pharmaceutical product according to claim 19 or 20.

31. The use of a multispecific antigen-binding molecule having a first antigen-binding domain specific to a target antigen and a second antigen-binding domain specific to the CD28 protein in the manufacture of a pharmaceutical for causing the killing of tumor cells in a target, inducing the killing of tumor cells in a target, or inducing T cell activation against tumor cells in a target, Use in which the tumor cells do not express or are not expected to express the target antigen.

32. The use of a multispecific antigen-binding molecule having a first antigen-binding domain specific to a target antigen and a second antigen-binding domain specific to the CD28 protein in the manufacture of a pharmaceutical for the treatment of cancer in a subject having a tumor, Use in which at least a subset of tumor cells within the tumor do not express the target antigen.

33. Use of a multispecific antigen-binding molecule comprising a first antigen-binding domain specific to a target antigen and a second antigen-binding domain specific to the CD28 protein in the manufacture of a pharmaceutical for a method of cancer treatment, The method described above is (i) Determining that the subject includes a tumor containing tumor cells that do not express the target antigen, (ii) administering to the subject a multispecific antigen-binding molecule comprising a first antigen-binding domain specific to the target antigen and a second antigen-binding domain specific to the CD28 protein, Optionally, if the expression of the target antigen in the tumor cells is below the detection level or below the signal-to-noise ratio, the tumor cells will not express the target antigen, thereby allowing for the selection of targets for cancer treatment.

34. A pharmaceutical product for the treatment of cancer in a subject having a tumor, for inducing the killing of tumor cells within a tumor in a subject, or for inducing T cell activation against tumor cells within a tumor in a subject, A first multispecific antigen-binding molecule having an antigen-binding domain specific to a first target antigen and an antigen-binding domain specific to the CD28 protein, A second multispecific antigen-binding molecule having an antigen-binding domain specific to a second target antigen and an antigen-binding domain specific to the CD3 protein, In use, The first target antigen is not the same antigen as the second target antigen. Use wherein at least a subset of the tumor cells within the tumor do not express the first target antigen.

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