Compositions and methods for universal tumor cell killing

Abstract

Provided herein are methods of mediating killing of tumor cells by administering to a subject a multispecific antigen-binding molecule having a first antigen-binding region specific for a first target antigen and a second antigen-binding region specific for a CD28 protein. In some embodiments, the tumor cell does not express the target antigen. In some embodiments, the tumor cells are predicted not to express the target antigen.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 329,762, filed on April 11, 2022, and U.S. Provisional Application No. 63 / 347,330, filed on May 31, 2022, the entire contents of each of which are incorporated herein by reference. Background Art

[0003] Cancer is one of the leading causes of death in North America. Despite decades of research, many cancers remain unresponsive or resistant to traditional chemotherapy and other therapeutic agents. Recently, advances in immunotherapy have produced some promising therapies, but even these agents tend to be effective only in specific patients and cancer types. Therefore, new cancer treatments are needed. Summary of the invention

[0004] The methods and compositions provided herein are based in part on the unexpected discovery that CD28 bispecific antibodies targeting tumor-associated antigens, antigens targeting cells in the tumor microenvironment, or immune antigens (e.g., antigens expressed on the surface of immune cells in a tumor or tumor microenvironment) can induce killing of cancer or tumor cells that lack expression of such antigens.

[0005] In certain aspects, provided herein are methods and compositions for mediating killing of tumor cells in a tumor of a subject by administering to the 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, wherein the tumor cells do not express or are predicted not to express the target antigen. In some embodiments, at least a subpopulation of tumor cells in the tumor do not express the target antigen.

[0006] In some aspects, provided herein are methods and compositions for inducing killing of tumor cells in a subject and / or inducing T cell activation against tumor cells in a tumor, the method 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 one subpopulation of tumor cells in the tumor does not express the target antigen.

[0007] In some aspects, methods and compositions for treating cancer in a subject having a tumor are also provided herein, 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 subpopulation of tumor cells in the tumor do not express the target antigen.

[0008] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigen is an antigen associated with a tumor microenvironment (e.g., a tumor microenvironment in a subject). For example, in some embodiments, the target antigen is an antigen on an immune cell, on a tumor cell matrix, or on an extracellular matrix within a 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.

[0009] In some embodiments, the methods provided herein may further include determining that at least one subpopulation of tumor cells in the tumor does not express the target antigen. In some embodiments, if the expression of the target antigen is below the detection level or below the signal-to-noise ratio, the tumor cells do not express the target antigen.

[0010] In some aspects, provided herein are methods of treating cancer in a subject, comprising i) determining that the subject comprises 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.

[0011] In some aspects, provided herein are methods of selecting a subject for cancer therapy, the method comprising: i) determining that the subject contains a tumor containing 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 if expression of the target antigen is below the level of detection or below the signal-to-noise ratio, the tumor cells do not express the target antigen, thereby selecting the subject for cancer therapy.

[0012] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the tumor may be a heterogeneous tumor that also includes tumor cells expressing TAA. In some embodiments, the tumor microenvironment of the tumor includes cells expressing 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 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 TAA.

[0013] In some embodiments, the target antigen is an antigen associated with the tumor microenvironment of a tumor, such as an antigen associated with a tumor stroma, an antigen associated with the extracellular matrix of a tumor, an antigen associated with blood vessels in the tumor microenvironment, or an antigen associated with cancer-associated fibroblasts.

[0014] In some embodiments, the target antigen is an antigen associated with a tumor stroma selected from the group consisting of PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin-EDB isoform.

[0015] In some embodiments, the target antigen is an antigen associated with the extracellular matrix of the tumor selected from connexin (e.g., connexin-3 or connexin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) proteins.

[0016] In some embodiments, the tumor microenvironment of a tumor comprises cells that express the target antigen.

[0017] In some embodiments, the target antigen is an antigen expressed on the surface of cancer-associated fibroblasts (e.g., 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-, or CD49e).

[0018] In some embodiments, the target antigen is an antigen expressed on the surface of blood vessels in the tumor microenvironment, such as DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, or VEGFR2.

[0019] 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, 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 has infiltrated into a tumor or a tumor microenvironment of a tumor. The immune antigen can be selected from any one of the immune antigens listed in Table 4.

[0020] 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% of a tumor , 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 the 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 a tumor do not express the target antigen.

[0021] 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 can be selected from CD38, EGFR, CD22, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7. The target antigen can be CD22.

[0022] 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 cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb). In some embodiments, the multispecific antigen-binding molecule is administered to a subject in combination 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 cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb).

[0023] The second target antigen can be selected from any one of the antigens listed in Table 2. The second target antigen can be a CD20 protein. In some embodiments, the second multispecific antigen-binding molecule is selected from any one of the multispecific antigen-binding molecules in Table 5. When administered in combination with a second multispecific antigen-binding molecule, the multispecific antigen-binding molecule can exhibit a co-stimulatory effect. In some embodiments, the co-stimulatory effect is one or more of: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in PBMCs, and increasing 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 (ocular lymphoma).

[0024] In certain embodiments, the tumor is a solid tumor. The tumor can be adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a hematogenous tumor, a brain / CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing's tumor, an eye tumor, a gallbladder tumor, a gastrointestinal tumor, a kidney tumor, a laryngeal or hypopharyngeal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penis tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymic tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms' tumor.

[0025] The multispecific antigen binding molecule can be administered systemically, intravenously, subcutaneously or intramuscularly. The multispecific antigen binding molecule can be administered to a subject in a pharmaceutically acceptable formulation. In some embodiments, the method further comprises administering an additional anticancer agent. The additional anticancer agent can be a chemotherapeutic agent, an immune checkpoint inhibitor, a CAR-T cell or a tumor vaccine. The immune checkpoint inhibitor can be an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody or an anti-LAG3 antibody.

[0026] In some embodiments, the subject has a refractory cancer.

[0027] In some aspects, provided herein is a method of treating cancer in a subject having a tumor, inducing or mediating killing of tumor cells in a subject's tumor, and / or inducing T cell activation against tumor cells in a subject's tumor, 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 a 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 a CD3 protein, wherein the first target antigen is not the same antigen as the second target antigen. In some embodiments, the first target antigen and / or the second target antigen is a tumor-associated antigen (TAA).

[0028] In some embodiments, the method further comprises determining that the tumor comprises a subpopulation of tumor cells that do not express the first target antigen.

[0029] In some aspects, provided herein is a method of treating cancer in a subject having a tumor, inducing or mediating killing of tumor cells in a subject's tumor, and / or inducing T cell activation against tumor cells in a subject's tumor, the method comprising determining whether the tumor comprises 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 a 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 a CD3 protein, wherein the first target antigen is not the same antigen as the second target antigen. In some embodiments, the first target antigen and / or the second target antigen is a tumor-associated antigen (TAA).

[0030] In some embodiments, at least one subpopulation of tumor cells in a tumor does 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 the tumor cells in a tumor do not express the first target antigen. In some embodiments, at least one subpopulation of tumor cells in a tumor does not express the second target antigen. In some embodiments, the tumor is a heterogeneous tumor comprising cells expressing the first target antigen and cells expressing the second target antigen. In some embodiments, the tumor cells in the tumor do not express both the first target antigen and the second target antigen.

[0031] In some embodiments, the first target antigen is an antigen associated with the tumor microenvironment of the tumor, such as 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.

[0032] In some embodiments, the first target antigen is an antigen associated with a tumor stroma selected from the group consisting of PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin-EDB isoform.

[0033] In some embodiments, the first target antigen is an antigen associated with the extracellular matrix of the tumor selected from connexin (e.g., connexin-3 or connexin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) protein.

[0034] In some embodiments, the tumor microenvironment of a tumor comprises cells that express the target antigen.

[0035] In some embodiments, the first target antigen is an antigen expressed on the surface of cancer-associated fibroblasts (e.g., 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-, or CD49e).

[0036] In some embodiments, the first target antigen is an antigen expressed on the surface of blood vessels in the tumor microenvironment, such as DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, and VEGFR2.

[0037] 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 has infiltrated into a tumor or a tumor microenvironment of a tumor. The immune antigen can be selected from any one of the immune antigens listed in Table 4.

[0038] 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%, , 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% of the tumor cells do not express the first target antigen or the 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 a tumor do not express the first target antigen and / or the second target antigen.

[0039] The first target antigen may be selected from any one 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 cell engagement antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem double antibody (TandAb). In some embodiments, the first multispecific antigen-binding molecule is selected from the bispecific antibodies listed in Table 3. In some embodiments, the second target antigen is selected from any one 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 immune antigen is CD22, CD20, CD72, CD19, CD21, CD24, or CD79. The second multispecific antigen-binding molecule may 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-cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb).

[0040] In some embodiments, the first multispecific antigen-binding molecule exhibits a co-stimulatory effect when administered in combination with the second multispecific antigen-binding molecule. The co-stimulatory effect can be one or more of the following: activation of T cells, induction of IL-2 release, induction of CD25+ upregulation in PBMCs, and increase in T cell-mediated cytotoxicity.

[0041] The tumor may be 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 (ocular lymphoma).

[0042] In certain embodiments, the tumor is a solid tumor. The tumor can be adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a hematogenous tumor, a brain / CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing's tumor, an eye tumor, a gallbladder tumor, a gastrointestinal tumor, a kidney tumor, a laryngeal or hypopharyngeal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penis tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymic tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms' tumor.

[0043] In some embodiments, the first multispecific antigen-binding molecule is administered systemically, intravenously, subcutaneously, or intramuscularly. The second multispecific antigen-binding molecule can be administered systemically, intravenously, subcutaneously, or intramuscularly. The first multispecific antigen-binding molecule and / or the second multispecific antigen-binding molecule can be administered to a subject in a pharmaceutically acceptable formulation.

[0044] In some embodiments, the method further comprises administering an additional anticancer agent. In some embodiments, the additional anticancer agent is a chemotherapeutic agent, an immune checkpoint inhibitor, a CAR-T cell, or a tumor vaccine. In some embodiments, the additional anticancer 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.

[0045] In some embodiments, the subject may have a refractory cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1A-Figure 1C showed that CD28 was expressed on intratumoral CD8 T cells in r / r NHL patients before and after odronextamab treatment. Figure 1A Representative images of multiplex IHC staining (CD3, CD8, CD28, and DAPI) of baseline DLBCL patient samples from the Phase I study of onitumumab are shown. Figure 1B Densities of CD4+CD28+ and CD8+CD28+ cells from baseline DLBCL and FL samples are shown. N=64. Figure 1C Representative images of DLBCL patient samples at baseline (upper left) and 5 weeks after starting onitumab treatment (upper right) are shown. Density of CD8+Cd28+ and CD8+CD28+ cells from paired DLBCL and FL samples at baseline and 5 weeks of treatment.

[0047] Figure 2A-2H The REGN5837 bispecific antibody was shown to enhance onitumab-mediated T cell activation, cytotoxicity, and effector function in vitro. WSU-DLCL2 cells were incubated with lymphocyte-enriched human PBMCs with titrated onitumab doses and fixed REGN5837 concentrations (ranging from 7.72x10-" ​​to 1.00x10 -7 M). Figure 2A Tumor cell killing was calculated by the percentage of dead cells. Figure 2B and Figure 2D Activation of CD4 and CD8 T cells was shown to be indicated by upregulation of CD25. Figure 2C and Figure 2E Proliferation is shown calculated by the percentage of dividing CD4 and CD8 T cells. Figure 2F The release of IL-2, IL-4, IL-6, IL-10, TNF-a, IFN-y, and IL-17A cytokines in the supernatant was evaluated. Arrows indicate the highest REGN5837 concentration (1.00x10 -7 M) Fold change in EC50 or fold change in maximum cytokine concentration between with and without REGN5837. Figure 2G We show that purified human T cells were inoculated with mixed CD22+ and CD22- WSU-DLCL2 tumor cells at different ratios and a fixed concentration of REGN5837 (ranging from 4.63x10 -10 Up to 1.67x10 -8 M) and 5pM onituzumab were incubated together to determine the enhancement of T cell killing of CD22-negative cells by REGN5837 (R5837). Killing of CD22+ targets is depicted in red shading, while killing of CD22- targets is depicted in blue shading. Figure 2H It was shown that activation of T cells in mixed WSU-DLCL2CD22+ and CD22- cultures was determined by upregulation of CD25.

[0048] Figure 3A-Figure 3F We showed that REGN5837 enhanced the antitumor efficacy of onitumumab and expanded intratumoral CD8 T cells in the WSU-DLCL2 tumor model in a preventive treatment setting. Figure 3A Treatment regimen for WSU-DLCL2 tumors implanted in NSG animals is shown. Figure 3B Plot showing the volume of individual tumors. Ratios represent the number of tumor-free mice. Figure 3CMean tumor growth (left) and survival rate (right) are shown. Statistical significance of mean tumor growth was calculated using two-way ANOVA with Tukey's multiple comparisons. Statistical significance of survival was calculated using the Kaplan-Meier method and log-rank test. *P<0.05,"P<0.01, ***P<0.001. Animals bearing WSU-DLCL2 tumors were sacrificed 26 days after implantation to analyze the immunophenotype of intratumoral T cell responses. UMAP plots of all live cells in the tumor were overlaid with color-coded immune cell subsets identified by FlowSOM ( Figure 3D Left), and the density UMAP map ( Figure 3D Right) reveals biases in how certain groups respond to the combination treatment. Figure 3E The density of WSU-DLCL2 cells (left) and the density of CD8+ T cells within the tumor (right) are shown. Figure 3F Pie charts are shown depicting the proportion of activated and memory subsets of intratumoral CD8 T cells in response to treatment (left). Density of effector memory and central memory intratumoral CD8+ T cells (right). Statistics were calculated using one-way ANOVA and Tukey's test. ***P<0.001, ****P<0.0001

[0049] Figure 4A-4F REGN5837-mediated costimulation was shown to enhance the antitumor efficacy of onitumumab against B-cell malignancies in a therapeutic treatment setting. Figure 4A Treatment regimen for treatment of WSU-DLCL2 tumors implanted in NSG animals is shown. Figure 4B Plot showing the volume of individual tumors. Ratios represent the number of tumor-free mice. Figure 4C Mean tumor growth (left) and survival rate (right) are shown. Statistical significance of mean tumor growth was calculated using two-way ANOVA and Tukey's multiple comparisons. Statistical significance of survival rate was calculated using the Kaplan-Meier method and log-rank test. *P<0.05, **P<0.01. Figure 4D Treatment regimens for NALM6-luc tumors implanted in PBMC-engrafted NSG animals are shown. Figure 4E BLI is shown, demonstrating tumor burden in individual mice. Figure 4F Mean NALM6-luc tumor growth is shown. Significance was calculated using two-way ANOVA with Tukey's multiple comparisons. *P<0.05, ***P<0.001.

[0050] Figure 5A-5J The combination of REGN5837 and onitumumab was shown to enhance peripheral and intratumoral T cell responses in human immune system reconstituted animals bearing WSU-DLCL2 tumors. Figure 5AShowing treatment options for WSU-DLCL2 tumors implanted in animals reconstituted with the human immune system. Figure 5B Plots showing individual tumor volumes. Figure 5C Mean tumor growth (left) and survival rate (right) are shown. Two-way ANOVA and Tukey's multiple comparisons were used to calculate the statistical significance of mean tumor growth. The statistical significance of survival rate was calculated using the Kaplan-Meier method and log-rank test. *P<0.05, "P<0.01. Figure 5D Shown are the time courses of peripheral CD8 T cell counts (left) and peripheral B cell counts (right) in response to treatment. Figure 5E The time course of serum cytokines induced in response to treatment is shown. Human immune system animals bearing WSU-DLCL2 tumors were sacrificed 30 days after implantation to analyze the immunophenotype of intratumoral T cell responses. UMAP plots of all live cells in blood, spleen, and tumor are overlaid with color-coded immune cell subsets identified by FlowSOM ( Fig. 5F Left), and the density UMAP map ( Fig. 5F Right) reveals biases in how certain groups respond to the combination treatment. Figure 5G The density of intratumoral CD4 T cells (top right), CD8 T cells (top left), and WSU-DLCL2 cells (bottom) are shown. Figure 5H A UMAP plot showing all intratumoral T cells overlaid with color-coded metaclusters identified by FlowSOM (left), and a density UMAP plot (right) revealing biases in certain metaclusters. Fig.5I Heatmap showing T cell activation, memory, and dysfunctional markers used by FlowSOM to identify T cell metaclusters. Figure 5J The frequencies of enrichment or depletion of selected T cell clusters in response to combined treatment are shown. Statistics were calculated using one-way ANOVA and Tukey's test. P<0.05,"P<0.01,****P<0.0001.

[0051] Figure 6A-6D Shown is the synergistic activation of CD8 T cells in the peripheral blood of cynomolgus monkeys when REGN5837 is combined with onitumumab. Cynomolgus monkeys received a single dose of REGN5837 (indicated in parentheses) at 1 mg / kg or 10 mg / kg in combination with dose-titrated onitumumab. Blood was collected at the indicated times (hours) after dosing. Fig. 6A B cell counts are shown 5 hours after dosing (left) and for the duration of the experiment (right). Figure 6B Peripheral CD8+ T cell counts are shown 5 hours after dosing (left) and for the duration of the experiment (right). Figure 6C Shown is ICOS upregulation on peripheral CD8+ T cells 5 hours after dosing (left) and proliferation 4 days after dosing (right). Fig.6D Serum cytokines induced 5 hours after administration are shown. Statistical data were calculated using one-way ANOVA and Tukey's test. Black asterisks indicate significance compared with placebo. n = 3 animals / group. *P < 0.05, **13 < 0.01, ***13 < 0.001, ****P < 0.0001.

[0052] Figure 7A-7C showed that CD22 expression is variable in resected DLBCL patient samples. Fig. 7A Chromogenic immunohistochemical staining of CD20 and CD22 on resected untreated DLBCL patient samples is shown. Representative images of high (left), moderate (middle), and low (right) CD22 expression. Figure 7B Representative images of DLBCL patient samples with high (top) and low (bottom) percentage of CD22 expression by multiplex IHC staining for PAX5, CD20, and CD22 are shown. Figure 7C Shown are the percentages of cells positive for B-cell markers in samples from patients with DLBCL who had not received treatment.

[0053] Figure 8A-8B CD28, CTLA4, CD80, and CD86 are shown to be detectable by chromogenic IHC in DLBCL patient samples. Fig. 8A Representative images showing chromogenic staining of CD28, CTLA4, CD80 and CD86 on untreated resected DLBCL patient samples (Tristar). Figure 8B Shown are the densities of CD28', CTLA4', CD86', and CD80' cells from 25 DLBCL patient samples.

[0054] Figure 9A-9D The REGN5837 bispecific antibody was shown to enhance onitumumab-mediated T cell cytotoxicity and proliferation. Fig. 9A REGN5837, non-targeted control antibody or CD28 superagonist (REGN2329) were anchored to the test plate using wet coating. Human PBMCs were incubated in the antibody-coated test plate and cytokine release was measured after 50-54 hours. The data shown are from 4 individual donors. Indicates cytokines for which significant release responses were observed compared to the non-binding control group (Tukey post hoc test). Fig. 9B Shown are the quantities of CD22 (left) and CD20 (right) epitopes per cell on the WSU-DLCL2 cell line, reported as antibody binding capacity. Fig. 9CShown are profiles of REGN5837-mediated proliferation of human CD4 and CD8 T cells when cultured with NALM-6 or Raji CD80 / CD86 DKO cells in the presence or absence of onitumumab. NC, not calculated. Fig.9D Shown are the expression of CD22 (left) and CD20 (right) on WSU-DLCL2CD22ko (red) and WSU-DLCL2CD22wt (green) cell lines as measured by flow cytometry.

[0055] Figures 10A-10D It was shown that onitumumab monotherapy treatment inhibited tumor growth in the WSU-DLBCL (DLBCL) tumor model but failed to mediate complete tumor rejection. Fig. 10A Treatment regimen for WSU-DLCL2 tumors implanted in NSG animals is shown. Fig. 10B Individual tumor growth curves in response to dose titration of onitumumab are shown. Fig. 10C Shows mean tumor growth, and Fig. 10D Survival rates are shown. Statistical significance of survival rates between isotype treatments and R1979 titrations was calculated using the Kaplan-Meier method with a log-rank test. ***P<0.001, ****P<0.0001.

[0056] Figure 11A-11E REGN5837 in combination with onitumumab was shown to promote intratumoral T cell expansion and activation and killing of WSU-DLBCL2 tumor cells in vivo. Fig.11A Shown is the treatment regimen for analyzing the immunophenotype of WSU-DLCL2 tumors in NSG mice 26 days after implantation. Fig. 11B Shown are graphs of tumor mass for each treatment group 26 days after implantation. Fig. 11C The percentages of the indicated cell populations of total viable cells are shown, demonstrating expansion of CD4 and CD8 T cells and a shift away from WSU-DLCL2 cells. Fig.11D A plot showing the density of CD4 T cells within the tumor. Fig.11E Figure 2 shows the percentage of memory subsets of intratumoral CD4 T cells. Statistical data were calculated using one-way ANOVA and Tukey's test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0057] Figure 12A-12B REGN5837 enhances IL-2 production and T cell proliferation in the presence of onitumumab-mediated alloreactivity signal 1. The ability of REGN5837 to mediate IL-2 release and T cell proliferation in the presence of a human B-cell leukemia cell line was determined using enriched human primary T cells and allogeneic NALM-6 cells. Fig. 12A The assay is shown in the presence of onitumab (500 pM) to provide a signal 1, or in the absence of onitumab, where the signal 1 is provided only by the allogeneic reaction and dose titration of REGN5837 or non-binding CD28 bispecific control antibody. The plate was incubated for 72 hours, at which time the culture supernatant was collected for IL-2 analysis. To assess proliferation, tritium was added and the cells were incubated for another 16 hours. Fig. 12B Profiles showing concentration-dependent increases in REGN5837-mediated IL-2 release and T-cell proliferation in the presence and absence of onitumumab.

[0058] Figures 13A-13H The combination of REGN5837 and onitumumab was shown to expand peripheral and intratumoral T cells in human immune system-reconstituted animals bearing DLBCL tumors. Fig.13A Shown is the time course of peripheral CD4 T-cell counts. Fig. 13B The time course of serum IL-10 induced in response to treatment is shown. Fig. 13C Mean growth of mean WSU-DLCL2 tumors before sacrifice for immunophenotyping is shown. Statistical significance between combination treatment and isotype (black asterisks) or combination treatment and REGN5837 monotherapy (red asterisks) was calculated using two-way ANOVA with Tukey's multiple comparisons. Fig.13D The percentages of WSU-DLCL2 cells (left) and T cells (right) among all viable intratumoral cells are shown. Fig.13E and Fig.13F The results showed that CD8( Fig.13E ) and CD4( Fig.13F )The percentages of memory and activated T cell subsets. Figure 13G and Fig.13H Display memory CD8( Figure 13G ) and CD4( Fig.13H ) Intratumoral density of T cells. Statistical data were calculated using one-way ANOVA and Tukey's test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0059] Figure 14A-14E showed that in animals reconstituted with the human immune system bearing DLBCL tumors, onitumumab effectively depleted spleen and blood B cells while promoting effector memory T cell induction. Fig.14A and Fig. 14B The spleen was shown 30 days after WSU-DLCL2 tumor implantation. Fig.14A ) or blood( Fig. 14B ) The percentage of B cells (left) and T cells (right) in the human body. Fig. 14C Counts of B-cell and T-cell subsets in the spleen are shown. Fig.14D UMAP plot showing the density of all viable cells in the blood (right). Fig.14E Displays counts of B-cell and T-cell subsets in the blood.

[0060] Figure 15A-15B showed synergistic activation of CD4 T cells in peripheral blood of cynomolgus monkeys when REGN5837 was combined with onitumumab. Fig.15A Peripheral CD4 T cell counts are shown 4 days after dosing (left) and for the duration of the experiment. Fig. 15B ICOS upregulation on peripheral CD4 T cells 5 hours after administration (left) and proliferation 4 days after administration (right) are shown. Statistical data were calculated using one-way ANOVA and Tukey's test. Black asterisks indicate significance compared to placebo. n = 3 animals / group. *P<0.05, "P<0.01, ***P<0.001, ****P<0.0001.

[0061] Fig.16 CD22 expression was shown to be more variable than CD20 expression in DLBCL patient samples.

[0062] Fig.17 We show that in mixed cultures of CD22wt and CD22ko tumor cells, the combination of CD22xCD28 and CD20xCD3 mediates enhanced T cell killing of bystander CD22ko target cells.

[0063] Fig.18 Exemplary expression profiles of EGFR target antigens in several cancers are shown.

[0064] Fig.19 Exemplary expression profiles of PMSA target antigens in several cancers are shown.

[0065] Fig. 20 Exemplary expression profiles of the CA9 target antigen in several cancers are shown.

[0066] Fig.21 Exemplary expression profiles of the HER2 target antigen in several cancers are shown.

[0067] Fig. 22 Exemplary expression profiles of SLAMF7 target antigens in several cancers are shown.

[0068] Fig.23 Exemplary expression profiles of the MUC16 target antigen in several cancers are shown.

[0069] Fig.24 Exemplary expression profiles of FOLR1 target antigens in several cancers are shown.

[0070] Fig.25Exemplary expression profiles of the CD38 target antigen in several cancers are shown.

[0071] Fig.26 Exemplary expression profiles of the CD22 target antigen in several cancers are shown. DETAILED DESCRIPTION

[0072] As shown in this article, T cells expressing CD22 + The disclosure herein is based in part on the discovery that combination therapy with anti-CD28 / anti-CD22 bispecific antibodies increases CD3 bispecific-mediated CD22 cell lysis. + and CD22 - A similar approach also increased CD22 + and CD22 - Activation of CD4 and CD8 T cells in the target population. Thus, Applicants have shown that anti-CD28 bispecific antibodies targeting tumor-associated antigens can increase the killing of tumor cells that lack tumor-associated antigens.

[0073] Provided herein are methods and compositions for mediating killing of tumor cells in a tumor of a subject by 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, wherein the tumor cells do not express or are predicted not to express the target antigen. In some embodiments, at least a subpopulation of tumor cells in the tumor do not express the target antigen.

[0074] In some aspects, provided herein are methods and compositions for inducing killing of tumor cells in a subject and / or inducing T cell activation against tumor cells in a tumor, the method 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 one subpopulation of tumor cells in the tumor does not express the target antigen.

[0075] 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 subpopulation of tumor cells in the tumor do not express the target antigen.

[0076] As used herein, when referring to tumor cells in a tumor "not expressing the target antigen", the phrase includes experimental detection or confirmation that at least a subpopulation of tumor cells do not express the target antigen, as well as methods that predict (i.e., with or without further experimental confirmation) that the tumor cells do not express the target antigen.

[0077] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigen is an antigen associated with a tumor microenvironment (e.g., a tumor microenvironment in a subject). For example, in some embodiments, the target antigen is an antigen on an immune cell, on a tumor cell matrix, or on an extracellular matrix within a tumor microenvironment. Examples of extracellular matrix antigens include connexins (e.g., connexin-3 or connexin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM).

[0078] definition

[0079] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0080] As used herein, "administering" or "administration" means providing an agent or composition to a subject, and includes, but is not limited to, administration by a medical professional or self-administration. Such agents may contain, for example, a bispecific antibody or bispecific antibody fragment provided herein.

[0081] As used herein, the term "antibody" may refer to both intact antibodies and antigen-binding fragments thereof. An intact antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as V L ) and the light chain constant region. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl 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.

[0082] 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) the smallest recognition unit consisting of amino acid residues of a hypervariable region that mimics an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide) or a restrictive FR3-CDR3-FR4 peptide. 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, divalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also included in the expression "antigen-binding fragment" as used herein.

[0083] In addition, the term "antibody" may include (unless otherwise specified or clear from the context) any construct or form known in the art that utilizes the structural and / or functional characteristics of antibodies, including but not limited to intrabodies, domain antibodies, antibody mimetics, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or groups thereof, single chain antibodies, single chain Fv (scFv), disulfide-linked Fv (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof), camelid antibodies, camelized antibodies, masked antibodies (e.g., ), affybodies, anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), small modular immunopharmaceuticals ("SMIPsTM"), single-chain or tandem diabodies VHH, Mini-antibodies, Ankyrin repeat protein or DART, TCR-like antibodies, MicroProteins and CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0084] The antibody used in the present invention can be a bispecific antibody. Bispecific antibodies have binding sites for two different antigens in a single antibody polypeptide. Antigen binding can be simultaneous or sequential. Triomas and hybridomas are examples of two cell lines that can secrete bispecific antibodies. U.S. Patent No. 4,474,893 discloses examples of bispecific antibodies produced by hybridomas or triomas. Bispecific antibodies have been constructed by chemical methods (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. Fragments of bispecific antibodies are described in US Patent 5,798,229.

[0085] Bispecific agents can also be produced by fusing hybridomas or other cells that produce different antibodies to prepare heterohybridomas, then identifying clones that produce and co-assemble two antibodies. They can also be produced by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof (such as Fab and Fv sequences).

[0086] Antibodies can also be "humanized," which is intended to include antibodies produced by non-human cells whose variable and constant regions have been altered to more closely resemble antibodies produced by human cells. For example, amino acids found in human germline immunoglobulin sequences are incorporated by altering the non-human antibody amino acid sequence. The humanized antibodies 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 mutations in vivo). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted onto human framework sequences.

[0087] As used herein, "cancer-associated fibroblasts" (CAFs) include fibroblasts found in and around tumor tissues, which are activated by normal resident tissue fibroblasts or transdifferentiated from non-fibroblast lineages (such as epithelial cells and adipocytes) due to stimulation of 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. More detailed information about antigens and biomarkers can be found in Han, C., Liu, T. & Yin, R. Biomarkers for cancer-associated fibroblasts. Biomark Res8, 64 (2020).

[0088] "Cancer" broadly refers to the uncontrolled abnormal growth of the host's own cells, which causes them to invade surrounding tissues and may invade tissues distal to the initial site of abnormal cell growth in the host. The main categories include carcinomas, which are cancers of epithelial tissues (such as skin, squamous cells); sarcomas, which are cancers of connective tissues (such as bone, cartilage, fat, muscle, blood vessels, etc.); leukemias, which are cancers of hematopoietic tissues (such as bone marrow tissue); lymphomas and myelomas, which are cancers of immune cells; and central nervous system cancers, which include cancers of brain and spinal cord tissues. "Cancer" and "neoplasm" are used interchangeably herein. As used herein, "cancer" refers to all types of cancers or neoplasms or malignant tumors, including new or recurrent leukemias, carcinomas and sarcomas. Specific examples of cancer are: carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed tumors. Non-limiting examples of cancer include new or recurrent cancers of the brain, melanoma, bladder, breast, cervical, colon, head and neck, kidney, lung, non-small cell lung cancer, mesothelioma, ovarian, prostate, sarcoma, stomach, uterine, and medulloblastoma. In some embodiments, cancer comprises a solid tumor. In some embodiments, cancer comprises a metastasis.

[0089] The term "chimeric antigen receptor" (CAR) refers to a molecule that combines a binding domain for a component present on a target cell (e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen) with a T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-target cell immune activity. In general, CAR-T is composed of an extracellular single-chain antigen binding domain (scFv) fused to the intracellular signaling domain of the ζ chain of the T cell antigen receptor complex, and when expressed in T cells, is able to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0090] As used herein, the phrase "administered in combination" or "administered in combination" refers to the administration of two or more different therapeutic agents in any form such that the second agent is administered while the previously administered therapeutic agent is still effective in vivo (e.g., the two agents are effective simultaneously in the subject's body, which may include a synergistic effect of the two agents). For example, the different therapeutic agents may be administered in the form of the same formulation or separate formulations, simultaneously or sequentially. In certain embodiments, the different therapeutic agents may be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about a week of each other. Thus, a subject receiving such treatment may benefit from the combined effects of the different therapeutic agents.

[0091] "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 of 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.

[0092] "Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including but not limited to proliferation activation, differentiation, etc. Co-stimulatory ligands may include but are not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, an agonist or antibody that binds to a Toll ligand receptor, and a ligand that specifically binds to B7-H3.

[0093] A "co-stimulatory signal" refers to a signal that, in combination with the primary signal, results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0094] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (called a paratope) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on the antigen and may have different biological effects. An epitope may be conformational or linear. Conformational epitopes are produced by spatially juxtaposing amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include a sugar, phosphoryl or sulfonyl moiety on an antigen.

[0095] As used herein, "immune cells" include any white blood cell that develops from stem cells in the 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 a tumor or tumor microenvironment.

[0096] As used herein, the phrase "pharmaceutically acceptable" refers to those agents, compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0097] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which participates in carrying or transporting an agent from one organ or part of the body to another. 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 serve as pharmaceutically 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, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10 ) glycols, such as propylene glycol; (11) polyols, such as glycerol, 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) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other nontoxic compatible substances used in pharmaceutical formulations.

[0098] As used herein, a therapeutic agent that "prevents" a disorder refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the incidence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.

[0099] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or fragment thereof means that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, at least about 95%, more preferably at least about, 96%, 97%, 98% or 99% of the nucleotide bases have nucleotide sequence identity as measured by any well-known sequence identity algorithm such as FASTA, BLAST or Gap, as discussed below. In some cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having an identical or substantially similar amino acid sequence to a polypeptide encoded by the reference nucleic acid molecule.

[0100] When applied to polypeptides, the term "substantially similar" or "substantially similar" means that two peptide sequences share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity when optimally aligned using default gap weights, such as by the programs GAP or BESTFIT. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are substitutions in which an amino acid residue is substituted by another amino acid residue having a similar chemical property (e.g., charge or hydrophobicity) in the side chain (R group). In general, conservative amino acid substitutions will not substantially change the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity percentage or similarity may be adjusted to correct for the conservativeness of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331, which is incorporated herein by reference. Examples of amino acid groups having 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: aspartate and glutamate, 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, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change with 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 with a non-negative value in the PAM250 log-likelihood matrix.

[0101] As used herein, the term "specifically binds" or "specific binding" in reference 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 agents. Thus, under specified conditions (e.g., immunoassay conditions in the case of an antibody), a particular ligand or antibody "specifically binds" to its specific "target" (e.g., an antibody specifically binds to an antigen) when it does not bind to other proteins present in the sample or other proteins that the ligand or antibody may contact in the organism. Typically, a first molecule that "specifically binds" to a second molecule has an affinity constant (Ka) of greater than about 10 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 Or greater). For example, with respect to the ability of a PIG-specific CAR to bind to a peptide presented on an MHC (e.g., class I MHC or class II MHC); typically, the CAR specifically binds to its peptide / MHC with a KD affinity of at least about 10-4 M or less, and binds to a predetermined antigen / binding partner with an affinity (expressed as KD) that is at least 1 / 10, at least 1 / 100, or at least 1 / 1000 of its binding affinity to a nonspecific and unrelated peptide / MHC complex (e.g., a complex comprising a BSA peptide or a casein peptide).

[0102] As used herein, "subject" means a human or non-human animal 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 in need of the methods provided herein, such as a subject suffering from cancer.

[0103] "Tumor microenvironment" refers to the cellular environment in which a tumor exists, and includes, for example, the stroma, interstitial fluid surrounding the tumor, surrounding blood vessels, immune cells, other cells, fibroblasts, signaling molecules, and extracellular matrix.

[0104] As used herein, the term "treatment" refers to a clinical intervention in the clinical pathology process that is intended to change the natural course of the individual being treated. Desired effects of treatment include reducing the rate of progression, improving or alleviating the pathological state, and alleviating or improving the prognosis of a particular disease, disorder, or condition. For example, if one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated, then the individual is successfully "treated."

[0105] As used herein, the phrases "therapeutically effective amount" and "effective amount" mean an amount of an agent effective to produce the desired therapeutic effect in at least one subpopulation of cells in a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0106] Therapeutic antibodies

[0107] Overview

[0108] In certain aspects, the methods and compositions provided herein involve the use of a therapeutic antibody (e.g., a bispecific antibody disclosed herein, such as a bispecific antibody having an antigen binding region specific for a first target antigen and an antigen binding region specific for a CD28 protein, optionally administered in combination with a second anti-CD3 bispecific antibody and / or an immune checkpoint inhibitor).

[0109] As described above, as used herein, the term "antibody" encompasses both intact antibody molecules and antigen-binding fragments of intact 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) the minimum recognition unit consisting of amino acid residues (e.g., isolated complementary determining regions (CDRs), such as CDR3 peptides) or restrictive FR3-CDR3-FR4 peptides that mimic the hypervariable region of an antibody. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, double antibodies, three antibodies, four antibodies, mini antibodies, nano antibodies (e.g., monovalent nano antibodies, divalent nano antibodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains are also included in the expression "antigen-binding fragment" as used herein.

[0110] The antigen-binding fragment of an antibody will generally comprise at least one variable domain. The variable domain may have any size or amino acid composition, and will generally comprise at least one CDR that is adjacent to or in frame with one or more framework sequences. In an antigen-binding fragment having a VH domain associated with a VL domain, the VH and VL domains may be positioned relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain a VH-VH, VH-VL, or VL-VL dimer. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0111] In certain embodiments, the 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 the antigen-binding fragments of antibodies 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 domains and constant domains, including any of the exemplary configurations listed above, the variable domains and constant domains may be directly connected to each other or may be connected by a complete or partial hinge or linker region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which produce a flexible or semi-flexible linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies disclosed herein may include homodimers or heterodimers (or other multimers) of any variable domain and constant domain configuration listed above, which are non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0112] As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific or trispecific). Multispecific antigen-binding fragments of antibodies will generally contain at least two different variable domains, each of which is capable of specifically binding to a separate antigen or a different epitope on the same antigen. Using conventional techniques available in the art, any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use with the antigen-binding fragments of antibodies disclosed herein.

[0113] In certain embodiments provided herein, at least one variable domain of the multispecific antibody is capable of specifically binding to a T cell co-stimulatory domain, such as CD28. In certain embodiments provided herein, at least one variable domain of the multispecific antibody disclosed herein is capable of specifically binding to CD3.

[0114] In some embodiments, the antibodies provided herein can function by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of cells expressing antigens by 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 (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcR) recognize the antibodies bound on target cells and thus cause the lysis of target cells. CDC and ADCC can be measured using analyses known and available in the art. (See, e.g., U.S. Patent 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 an antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether it is desired that the antibody mediate cytotoxicity.

[0115] 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 include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. Human antibodies disclosed herein may include, for example, in CDR, particularly in CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutations). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from germlines of another mammalian species (such as mice) have been transplanted to human framework sequences.

[0116] In some embodiments, the antibodies provided herein may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, formed or separated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (further described below); antibodies separated from recombinant, combinatorial human antibody libraries (further described below); antibodies separated from animals (e.g., mice) that are transgenic animals for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295); or antibodies prepared, expressed, formed or separated by any other means involving splicing of human immunoglobulin gene sequences to 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 are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgenic for human Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0117] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of about 150-160 kDa, in which the dimer is bound together by interchain heavy chain disulfide bonds. In the second form, the dimer is not connected via interchain disulfide bonds, but forms a molecule of about 75-80 kDa composed of covalently coupled light and heavy chains (half antibodies). These forms are extremely difficult to separate even after affinity purification.

[0118] The frequent 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. Single amino acid substitutions in the hinge region of the human IgG4 hinge can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30: 105), reaching levels typically observed with human IgG1 hinges. The present disclosure encompasses antibodies with one or more mutations in the hinge, CH2 or CH3 regions, which may be required, for example, to improve the yield of the desired antibody form in preparation.

[0119] Sequence variants

[0120] In some embodiments, the monospecific or multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may include one or more amino acid substitutions, insertions and / or deletions in the framework region and / or CDR region of the heavy chain variable domain and the light chain variable domain compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof, which are derived from any amino acid sequence disclosed herein, wherein one or more amino acid mutations in one or more framework regions and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or the corresponding residues of another human germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein, one of ordinary skill in the art can easily generate many antibodies and antigen-binding fragments comprising 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 mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4 or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of one or more framework and / or CDR residues are mutated to one or more corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences from which the original antibody is derived). In addition, the antibodies disclosed herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, wherein certain single residues are mutated to the corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to the corresponding residues of different germline sequences. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., KD), improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0121] In some embodiments, the multispecific (e.g., bispecific or trispecific) antibodies provided herein comprise variants of any 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 CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein have HCVR, LCVR and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions relative to any HCVR, LCVR and / or CDR amino acid sequences disclosed herein.

[0122] Fc variants

[0123] According to certain embodiments provided herein, antibodies and multispecific antigen-binding molecules comprising an Fc domain containing one or more mutations are provided, wherein the mutations enhance or weaken the binding of the antibody to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. For example, the disclosure includes antibodies comprising mutations in the CH2 or CH3 region of the Fc domain, wherein the mutations increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome having a pH range of about 5.5 to about 6.0). Such mutations may increase the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., modifications at positions 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, 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).

[0124] For example, the present disclosure includes multispecific antigen-binding molecules (e.g., anti-CD28 / anti-TAA bispecific or anti-CD3 / anti-TAA bispecific antibodies) comprising an Fc domain containing one or more pairs or one or more 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 foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are contemplated herein.

[0125] Bioequivalent

[0126] Provided herein are antigen binding molecules having amino acid sequences that differ from the exemplary molecules disclosed herein but retain the ability to bind to the same one or more antigens. Such variant molecules may comprise one or more amino acid additions, deletions or substitutions when compared to the parent sequence, but exhibit biological activity that is substantially equivalent to the described bispecific antigen binding molecules.

[0127] The present disclosure includes antigen binding molecules that are bioequivalent to any one of the exemplary antigen binding molecules set forth herein. If, for example, two antigen binding proteins or antibodies are drug equivalents or drug substitutes that do not show significant differences in absorption rate and degree when applied with the same molar dose (single dose or multiple doses) under similar experimental conditions, they are considered to be bioequivalent. If some antigen binding proteins are equivalent in terms of their absorption extent, but not equivalent in terms of their absorption rate, and can be considered to be bioequivalent, because such differences in absorption rate are intentional and reflected in labeling, it is not necessary to reach effective in vivo drug concentration after, for example, long-term use, and it is considered to be medically unimportant for the specific drug product studied, so they will be considered to be equivalents or drug substitutes.

[0128] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0129] In one embodiment, two antigen binding proteins are bioequivalent if patients can switch one or more times between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity or diminished effectiveness, compared to continued therapy without such switching.

[0130] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action for one or more conditions of use (where such mechanisms are known).

[0131] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo tests that measure the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids over time in humans or other mammals; (b) in vitro tests that correlate with in vivo bioavailability data in humans and are appropriately predictive of in vivo bioavailability in humans; (c) in vivo tests that measure appropriate acute pharmacological effects of the antibody (or its target) over time in humans or other mammals; and (d) fully controlled clinical trials that determine the safety, efficacy or bioavailability or bioequivalence of the antigen-binding protein.

[0132] Bioequivalent variants of the exemplary bispecific antigen binding molecules described herein can be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not required for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or inappropriate intramolecular disulfide bridges after renaturation. In other cases, bioequivalent antigen binding proteins may include variants of the exemplary bispecific antigen binding molecules described herein that include amino acid changes that modify the glycosylation characteristics of the molecule, such as mutations that eliminate or remove glycosylation.

[0133] Antibody Binding

[0134] As used herein, the term "binding" in the context of an antibody, immunoglobulin, antibody binding fragment or Fc-containing protein binding to, for example, a predetermined antigen such as a cell surface protein or a fragment thereof, generally refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.

[0135] For example, when an antigen is used as a ligand and an antibody, Ig, antibody binding fragment or Fc-containing protein is used as an analyte (or antiligand) in a BIAcore 3000 instrument by, for example, surface plasmon resonance (SPR) technology, the binding affinity generally corresponds to a KD value of about 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less. Cell-based binding strategies such as fluorescence activated cell sorting (FACS) binding assays are also routinely used, and FACS data correlate well with other methods such as radioligand competition 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).

[0136] Thus, the antibodies or antigen binding proteins provided herein bind to a predetermined antigen or cell surface molecule (receptor) with an affinity corresponding to a KD value that is at most one-tenth of its affinity for binding to a non-specific antigen (e.g., BSA, casein). According to the present disclosure, an antibody with an affinity corresponding to a KD value that is equal to or one-tenth of a non-specific antigen can be considered to be undetectable binding, however such antibodies can be paired with a second antigen binding arm to generate a bispecific antibody disclosed herein.

[0137] The term "KD" (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody binding fragment bound to an antigen. There is an inverse relationship between KD and binding affinity, so the smaller the KD value, the higher the affinity, i.e., the stronger. Therefore, the term "higher affinity" or "stronger affinity" is related to a higher ability to form interactions and therefore to a smaller KD value, while on the contrary the term "lower affinity" or "weaker affinity" is related to a lower ability to form interactions and therefore to a larger KD value. In some cases, a specific molecule (such as an antibody) has a higher binding affinity (or KD) to its interaction partner molecule (such as antigen X) than a molecule (such as an antibody) to another interaction partner molecule (such as antigen Y) The binding affinity can be expressed as a binding ratio determined by dividing a larger KD value (lower or weaker affinity) by a smaller KD (higher or stronger affinity), for example, it is 5 times or 10 times as the case may be.

[0138] The term "kd" (seconds-1 or 1 / s) refers to the dissociation rate constant of a specific 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.

[0139] The term "ka" (M-1 x second-1 or 1 / M) refers to the association rate constant for a specific antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment.

[0140] The term "KA" (M-1 or 1 / M) refers to the association equilibrium constant for a particular antibody-antigen interaction, or the association equilibrium constant for an antibody or antibody binding fragment. The association equilibrium constant is obtained by dividing ka by kd.

[0141] The term "EC50" or "EC50" refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between the baseline and the maximum after a specified exposure time. EC50 generally represents the antibody concentration at which 50% of the maximal effect is observed. In certain embodiments, the EC50 value is equal to the concentration at which the antibodies disclosed herein give half-maximal binding to cells expressing CD28 or a tumor-associated antigen (e.g., an antigen disclosed in Table 2) as determined by, for example, a FACS binding assay. Thus, decreased or weaker binding is observed with increased EC50 or half-maximal effective concentration values.

[0142] In one embodiment, decreased binding of a CD28 multispecific antibody can be defined as an increase in the EC50 antibody concentration that achieves binding to half maximal number of target cells.

[0143] In another embodiment, the EC50 value represents the concentration of the CD28 multispecific antibodies disclosed herein that elicits half-maximal depletion of target cells by T cell cytotoxic activity. Thus, an increase in cytotoxic activity (e.g., T cell-mediated tumor cell killing) is observed at a reduced EC50 or half-maximal effective concentration value.

[0144] Preparation of antigen binding domains and construction of multispecific molecules

[0145] Antigen binding domains specific for specific antigens can be prepared by any antibody production technology known in the art. After obtaining two different antigen binding domains specific for two different antigens (e.g., CD28 and human tumor antigens), they can be appropriately arranged relative to each other to produce bispecific antigen binding molecules disclosed herein using conventional methods. In certain embodiments, one or more of the individual components (e.g., heavy chains and light chains) of the multispecific antigen binding molecules disclosed herein are derived from chimeric antibodies, humanized antibodies, or fully human antibodies. Methods for preparing such antibodies are well known in the art. For example, VELOCIMMUNE can be used TM One or more of the heavy chains and / or light chains of the bispecific antigen binding molecules disclosed herein are prepared using VELOCIMMUNE TMThe invention relates to the use of the invention to obtain a chimeric antibody having a human variable region and a mouse constant region for a specific antigen (e.g., CD28 or a human tumor-associated antigen) using the human antibody generation technology (or any other human antibody generation technology). The antibodies are characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen-binding molecules disclosed herein.

[0146] Genetically engineered animals can be used to prepare human bispecific antigen binding molecules.For example, genetically modified mice can be used, and the genetically modified mice cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences, wherein the mice only express one or two human light chain variable domains encoded by human immunoglobulin sequences operably connected to mouse κ constant genes at endogenous mouse κ locus.Such genetically modified mice can be used to produce fully human bispecific antigen binding molecules, and the fully human bispecific antigen binding molecules include two different heavy chains associated with the same light chain, and the same light chain includes a variable domain derived from one of two different human light chain variable region gene segments.(See, for example, US 2011 / 0195454).Fully human refers to antibodies or their antigen-binding fragments or immunoglobulin domains comprising an amino acid sequence encoded by a DNA derived from a human sequence on the entire length of each polypeptide of an antibody or its antigen-binding fragment or immunoglobulin domain.In some cases, fully human sequences are derived from proteins endogenous to humans.In other cases, fully human proteins or protein sequences include chimeric sequences that each component sequence is derived from a human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are typically designed to minimize the formation of immunogenic epitopes in the junctions of the component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

[0147] CD28 multispecific antigen binding molecules

[0148] In certain embodiments, the methods and compositions provided herein relate to CD28 antigen binding molecules (i.e., antigen binding molecules comprising at least one antigen binding region that binds to CD28). In certain embodiments, the CD28 multispecific 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, such as a tumor associated antigen (TAA)).

[0149] In certain embodiments, provided herein is a second antigen binding molecule that is a CD3 multispecific antigen binding molecule provided herein, further comprising an antigen binding domain that binds to a target antigen (e.g., an antigen expressed on cancer cells, such as a tumor associated antigen (TAA)).

[0150] In some embodiments, the target antigen is a tumor-associated antigen (TAA). In some embodiments, the target antigen is an antigen associated with a tumor microenvironment (e.g., a tumor microenvironment in a subject). For example, in some embodiments, the target antigen is an antigen on an immune cell, on a tumor cell matrix, or on an extracellular matrix within a tumor microenvironment. Examples of extracellular matrix antigens include connexins (e.g., connexin-3 or connexin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM).

[0151] As used herein, the expression "multispecific antigen-binding molecule" refers to a protein, polypeptide or molecular complex comprising at least a first antigen-binding region and a second antigen-binding region. In some embodiments, each antigen-binding domain within a multispecific antigen-binding molecule may comprise at least one single CDR, or a combination of at least one CDR and one or more additional CDRs and / or FRs, specifically binding to a specific antigen. 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 second different antigen (e.g., a tumor-associated antigen).

[0152] In some embodiments, the CD28 multispecific antigen binding molecule is a CD28 multispecific antibody, such as a CD28 bispecific antibody. The CD28 multispecific antibodies provided herein may be, for example, bispecific or trispecific. The multispecific antibody may be specific for different epitopes of a target polypeptide or may contain an antigen binding domain that is specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147: 60-69; Kufer et al., 2004, Trends Biotechnol. 22: 238-244. The CD28 bispecific antibodies provided herein may be connected to another functional molecule (e.g., another peptide or protein) or co-expressed with it. For example, an antibody or a fragment thereof may be functionally connected (e.g., by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0153] As used herein, the term "CD28" refers to cluster of differentiation 28, which is one of the proteins expressed on T cells and can provide co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation through CD28 can provide a powerful signal for the production of various interleukins. Human CD28 contains the amino acid sequence shown below.

[0154] Table 1: Human protein sequence of CD28

[0155] >NP_001230006.1; GeneID = 940; Isoform = 2 precursor

[0156] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSWKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0157] >NP_001230007.1; GeneID = 940; Isoform = 3 precursor

[0158] MLRLLLALNLFPSIQVTGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0159] >NP_006130.1; GeneID = 940; Isoform = 1 precursor

[0160] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF

[0161] WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA

[0162] PPRDFAAYRS

[0163] >XP_011510496.1; Gene ID = 940; Isoform = X1

[0164] MPCGLSALIMCPKGMVAVVVAVDDGDSQALAGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLG NESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTP

[0165] RRPGPTRKHYQPYAPPRDFAAYRS

[0166] >XP_011510497.1;Gene ID=940;Isotype=X2

[0167] MPCGLSALIMCPKGMVAVVVAVDDGDSQALAGNKILVKQSPMLVAYDNAVNLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0168] >XP_011510499.1;Gene ID=940;Isotype=X3

[0169] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0170] Unless expressly specified as being from a non-human species, all references to proteins, polypeptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, polypeptide, or protein fragment. Thus, unless specified as being from a non-human species, e.g., "mouse CD28," "monkey CD28," etc., the expression "CD28" means human CD28.

[0171] As used herein, the expression "CD28 expressed on the cell surface" refers to one or more CD28 proteins that are expressed on the cell surface 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 accessible to the antigen-binding portion of the antibody. The CD28 expressed on the cell surface includes the CD28 protein contained in the membrane of the cell in the case of a functional T cell receptor. The CD28 expressed on the cell surface may include or consist of a CD28 protein expressed on the surface of a cell that normally expresses the CD28 protein. Alternatively, the CD28 expressed on the cell surface may include or consist of a CD3 protein expressed on the surface of a cell that normally does not express human CD28 but has been artificially engineered to express CD28 on the surface.

[0172] In some embodiments, the disclosure includes bispecific antibodies in which one arm of the immunoglobulin binds CD28 and the other arm of the immunoglobulin 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 arm of the immunoglobulin binds CD28, a second arm of the immunoglobulin is specific for a tumor antigen, and a third arm of the immunoglobulin binds an additional T cell antigen (e.g., CD3) or an additional tumor antigen.

[0173] In some embodiments, the CD28 multispecific antibody may include any of the antibodies disclosed in US2020 / 0239576. In some embodiments, the CD28 binding arm may include 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 weakly binds to human CD28 and induces human T cell activation. In other embodiments, in the case of bispecific or multispecific antibodies, the CD28 binding arm weakly binds to human CD28 and induces killing of cells expressing tumor-associated antigens.

[0174] In certain embodiments, the multispecific antibodies or antigen-binding fragments used in the present disclosure comprise 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.

[0175] In certain embodiments, a CD28 multispecific antigen-binding molecule comprises an antigen-binding domain that is specific for a tumor-associated antigen.

[0176] 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.

[0177] The tumor-associated antigen may be any one of the antigens selected from Table 2 below.

[0178] Table 2: Exemplary tumor-associated antigens

[0179]

[0180]

[0181] In certain embodiments, the tumor-associated antigen is selected from AIM-2, ALDH1A1, α-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, CLP P, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, 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-1,2,8, GAGE-3,4,5,6,7, GAS7, phosphatidylinositol glycan-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-H N-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-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-poly peptide, 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, secernin1, SIRT2, SNRPD1, SOX10, Sp 17, SPA17, SSX-2, SSX-4, STEAP1, STEAP2, Survivin, SYT-SSX1 or -SSX2 fusion protein, 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.

[0182] In some embodiments, tumor-associated antigens may include ADAM 17, 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, FOLR1, 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.

[0183] In some embodiments, the tumor-associated antigen may be a non-immune-associated antigen selected from CD38, EGFR, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7.

[0184] In some embodiments, the tumor-associated antigen may be an immune-associated antigen selected from CD22, CD20, CD72, CD19, CD21, CD24, and CD79.

[0185] CD22 (Siglec 2) is a receptor expressed on the cell 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 to sialylated glycoproteins; one of which is CD45. It also preferentially binds to α-2,6-linked sialic acids. It also plays a positive regulatory role by interacting with Src family tyrosine kinases and can also act as an inhibitory receptor by recruiting cytoplasmic phosphatases through its SH2 domain that block signal transduction by dephosphorylation of signaling molecules.

[0186] CD20 is a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. CD20 is considered to be a B cell tumor-associated antigen because more than 95% of B cell non-Hodgkin's lymphomas (NHL) and other B cell malignancies express this antigen, but it is not present in precursor B cells, dendritic cells, and plasma cells. The human CD20 protein has the amino acid sequence shown in SEQ ID NO:5 of U.S. Patent Application Publication No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0187] MUC16 refers to mucin 16. MUC16 is a highly glycosylated integral membrane glycoprotein with a single transmembrane domain that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is listed in SEQ ID NO: 1899 of U.S. Patent Application Publication No. US2018 / 0118848A1, the entire contents of which are incorporated herein by reference in their entirety.

[0188] BCMA refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells, which plays a central role in regulating B cell maturation and differentiation into plasma cells that produce immunoglobulins. The amino acid sequence of human BCMA protein is shown in SEQ ID NO: 115 of U.S. Patent Application Publication No. US2020 / 0024356, the entire contents of which are incorporated herein by reference in their entirety. It can also be found in GenBank Accession No. NP_001183.2.

[0189] PSMA refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is an intact, non-shedding 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 shown in SEQ ID NO: 7 of U.S. Patent Application Publication No. US2020 / 0129617, the entire contents of which are incorporated herein by reference in their entirety.

[0190] In some embodiments, the CD28 multispecific antibody can 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.

[0191] In some embodiments, the CD28 multispecific antibody can 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, or a bispecific CD28xSLAM7 antibody.

[0192] In certain embodiments, the multispecific antigen binding molecule is a multispecific antibody or its antigen binding fragment.Each antigen binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR).In the case where a bispecific antigen binding molecule comprises a first antigen binding domain and a second antigen binding domain (e.g., a bispecific antibody), the prefix "A1" can be used to specify the CDR of the first antigen binding domain and the prefix "A2" can be used to specify the CDR of the second antigen binding domain.Therefore, the CDR of the first antigen binding domain may be referred to as A1-HCDR1, A1-HCDR2 and A1-HCDR3 herein; and the CDR of the second antigen binding domain may be referred to as A2-HCDR1, A2-HCDR2 and A2-HCDR3 herein. In the case where a bispecific antigen binding molecule comprises a first antigen binding domain, a second antigen binding domain, and a third antigen binding domain (e.g., a trispecific antibody), the prefix "A1" may be used to designate the CDRs of the first antigen binding domain, the prefix "A2" may be used to designate the CDRs of the second antigen binding domain, and the prefix "A3" may be used to designate the CDRs of the third antigen binding domain. 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.

[0193] The bispecific antigen binding molecules discussed above or herein can be bispecific antibodies. In some cases, the bispecific antibodies include a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is an isotype IgG1. In some cases, the human IgG heavy chain constant region is an isotype IgG4. In various embodiments, the bispecific antibodies include a chimeric hinge that reduces Fcγ receptor binding relative to a wild-type hinge of the same isotype.

[0194] In some embodiments, the CD28 multispecific antibody is any one of the multispecific antibodies in Table 3.

[0195] Table 3: CD28 multispecific antibodies

[0196] name Target REGN7075 EGFR / CD28 REGN5678 PSMA / CD28 SAR442257 CD38 / CD28xCD3 REGN5668 MUC16 / CD28 REGN7945 CD38 / CD28 REGN5837 CD22 / CD28

[0197] 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 antigens on cells within the matrix, interstitial fluid around the tumor, blood vessels around the tumor, and extracellular matrix. Antigens associated with immune cells, other cells, fibroblasts, or signaling molecules in the tumor microenvironment are also included.

[0198] In some embodiments, the target antigen is an antigen associated with a tumor stroma selected from the group consisting of PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin-EDB isoform.

[0199] In some embodiments, the target antigen is an antigen associated with the extracellular matrix of the tumor selected from connexin (e.g., connexin-3 or connexin-4), versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) proteins.

[0200] In some embodiments, the target antigen is an antigen expressed on the surface of cancer-associated fibroblasts (e.g., 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-, or CD49e).

[0201] In some embodiments, the target antigen is an antigen expressed on the surface of blood vessels in the tumor microenvironment, such as DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1, and VEGFR2.

[0202] 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, 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 has infiltrated into a tumor or a tumor microenvironment of a tumor. The immune antigen can be selected from any one of the immune antigens listed in Table 4.

[0203] Table 4: Exemplary Immunizing Antigens

[0204]

[0205]

[0206]

[0207] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form a bispecific antigen binding molecule disclosed herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerization domain. The association of a multimerization domain with another multimerization domain contributes to the association between the two antigen-binding domains, thereby forming a bispecific antigen binding molecule. As used herein, "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid with the ability to associate with the second multimerization domain of the same or similar structure or structure. For example, the multimerization domain can be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerization component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), for example, an Fc domain of an IgG selected from isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype in each isotype group.

[0208] The bispecific antigen binding molecules disclosed herein will generally comprise two multimerization domains, e.g., two Fc domains each individually being part of a separate antibody heavy chain. The first multimerization domain and the second multimerization domain may belong to the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimerization domain and the second multimerization domain may belong to different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0209] In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence of 1 to about 200 amino acids in length and containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue, or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0210] Any bispecific antibody form or technology can be used to prepare bispecific antigen binding molecules disclosed herein. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally connected (e.g., by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment with a second antigen binding specificity to produce a bispecific antigen binding molecule. Specific exemplary bispecific forms that can be used in the methods provided herein include, but are not limited to, for example, scFv-based or double antibody bispecific forms, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-in-hole structures, common light chains (e.g., common light chains with knobs-in-hole structures, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab2 bispecific forms (for a review of the aforementioned forms, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0211] In the case of bispecific antigen binding molecules provided herein, the multimerization domain (e.g., Fc domain) may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to wild-type, naturally occurring forms of the Fc domain. For example, the present disclosure includes bispecific antigen binding molecules that include one or more modifications in the Fc domain so that the modified Fc domain has a modified binding interaction (e.g., enhanced or reduced) between Fc and FcRn. In one embodiment, the bispecific antigen binding molecule includes a modification in the CH2 or CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome at a pH value in the range of about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, e.g., modifications at positions 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, 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).

[0212] In certain embodiments, the bispecific antigen binding molecules provided herein comprise 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 at least one amino acid difference reduces the 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 protein A and the second Ig CH3 domain contains a mutation that reduces or eliminates protein A binding, such as an H95R modification (according to IMGT exon numbering; according to EU numbering, H435R). The second CH3 may further comprise a Y96F modification (according to IMGT; according to EU, Y436F). See, e.g., U.S. Patent No. 8,586,713. Other modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I in the case of IgG1 antibodies (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I according to EU); N44S, K52N and V82I in the case of IgG2 antibodies (IMGT; N384S, K392N and V422I according to EU); and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I in the case of IgG4 antibodies (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU).

[0213] In certain embodiments, the Fc domain may be a chimeric combined Fc sequence derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include part or all of a CH2 sequence derived from a human IgG1, human IgG2, or human IgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgG1, human IgG2, or human IgG4. A chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may include a combination of an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region and a "lower hinge" sequence derived 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 one of the antigen binding molecules described herein includes from N-terminus to C-terminus: [IgG4CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG4CH3]. 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. Publication No. 2014 / 0243504, published on August 28, 2014, which is incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements, as well as variants thereof, may have altered Fc receptor binding, which in turn affects Fc effector functions.

[0214] The CD28 multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domain and the light chain variable domain compared to the corresponding germline sequence from which the antibody is derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof, which are derived from any amino acid sequence disclosed herein, wherein one or more amino acid mutations within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or the corresponding residues of another human germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein, one of ordinary skill in the art can easily generate a number of antibodies and antigen-binding fragments comprising 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 mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4 or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of one or more framework and / or CDR residues are mutated to one or more corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequences from which the original antibody is derived). In addition, the antibodies disclosed herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, wherein certain single residues are mutated to the corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are retained or mutated to the corresponding residues of different germline sequences. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, such as improved binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., KD), improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.

[0215] 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% of a tumor , 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 the tumor cells do not express the target antigen. In the following embodiments, less than 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% or more of a tumor In some embodiments, the target antigen is not expressed in 5% to 10% of the tumor cells. In some embodiments, the target antigen is not expressed in 5% to 10% of the tumor cells. In some embodiments, the target antigen is not expressed in 5% to 10% of the tumor cells.

[0216] The measurement of the expression or level of tumor antigens 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 within a tumor that do not express the target antigen" may refer to surface protein expression of the antigen. For example, tumor antigens can be identified by directly interrogating the tumor immune peptide group; that is, all endogenous peptides presented by MHC molecules on the cell surface. In this method, after the peptides are extracted from the tumor cells, they are eluted from the complex with the MHC molecules and then analyzed by liquid chromatography combined with tandem mass spectrometry (LC-MS / MS). The MS spectrum can be compared with a customized database generated by combining sequencing data of the patient's tumor with a reference protein sequence. In some embodiments, the quantification of tumor antigens uses a platform called SureQuant-IsoMHC, which uses a series of pMHC isotopologues and internal standard-triggered targeted mass spectrometry to generate an embedded multi-point calibration curve to determine the endogenous pMHC concentration. For more 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 US A. 2021 Sep 14; 118(37), incorporated herein by reference in its entirety. In addition, T cell receptor (TCR) mimetic antibodies can be used to estimate antigen copy number. Alternatively, measurement of target antigen (e.g., any target antigen disclosed herein) expression can be accomplished by measuring nucleic acid expression (e.g., RNA, such as mRNA expression) in tumor cells. Nucleic acid expression can be achieved by nucleic acid amplification and related techniques.

[0217] CD3 multispecific antibody

[0218] In some embodiments, the CD28 multispecific antibodies disclosed herein can be administered in conjunction with a CD3 antibody (eg, a CD3 multispecific antibody).

[0219] In some embodiments, the CD3 multispecific antibody is any one of the CD3 multispecific antibodies listed in Table 5.

[0220] Table 5: CD3 multispecific antibodies

[0221]

[0222]

[0223] 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 WO 2014 / 047231 or WO 2017 / 053856, a bispecific anti-CD20 x anti-CD3 antibody disclosed in WO 2014 / 047231, a bispecific anti-PSMA x anti-CD3 antibody disclosed in WO 2017 / 023761, a bispecific anti-MUC16 x anti-CD3 antibody disclosed in WO 2018 / 067331 or WO 2018 / 058003, a bispecific anti-STEAP2 x anti-CD3 antibody disclosed in WO 2018 / 058001, or a bispecific anti-BCMA x anti-CD3 antibody disclosed in WO 2020 / 018820, each of which is incorporated herein by reference.

[0224] Additional exemplary CD3 multispecific antibodies that can be used in the compositions and methods disclosed herein include, but are not limited to, for example, the bispecific CD3xCD123 antibodies disclosed in U.S. Patent No. 10,787,521 B2, U.S. Patent Application Publication Nos. 2018 / 0222987A1 and US2019 / 0241657A1, and International Application Publication Nos. WO 2016 / 036937A1, WO 2017 / 210443A1, WO 2019 / 050521A1, WO 2019 / 210147A1, WO 2019 / 232528A1, and WO 2020 / 092404A1; the bispecific CD3xSTEAP2 antibodies disclosed in International Application Publication Nos. WO 2018 / 058001A1; WO 2014 / 047231A1, WO 2015 / 143079A1, WO 2016 / 081490A1, WO 2017 / 112775A1, WO 2017 / 210485A1, WO 2018 / 114748A1, WO 2018 / 093821A8, WO 2018 / 223004A1, WO 2018 / 188612A1, WO 2019 / 155008A1, WO 2019 / 228406A1, WO 2020 / 088608A1, WO 2020 / 156405A1 and U.S. Patent Application Publication Nos. US2020 / 0199231A1 and US2020 / 0172627A1; International Application Publication No. WO The bispecific CD3xSSTR 2 antibody disclosed in 2018 / 005706A1; the bispecific CD3xCD38 antibody disclosed in international application numbers WO 2015 / 149077A1 and WO 2020 / 018556A1 and U.S. patent application publication numbers US2018 / 0305465A1 and US2020 / 0102403A1; the bispecific CD3xSTEAP1 antibody disclosed in Olivier Nolan-Stevaux (2020) Abstract of the Proceedings of the 2020 Annual Meeting of the American Association for Cancer Research; the bispecific CD3x5T4 antibody disclosed in international application publication number WO 2013 / 041687A1, U.S. patent application publication numbers US2017 / 0342160A1, US20200277397A1; the bispecific CD3x5T4 antibody disclosed in international application publication number WO The bispecific CD3xENPP3 antibody described in 2020 / 180726A1; the bispecific CD3xMUC16 antibody disclosed in International Application Publication Nos. WO 2018 / 067331A9 and WO 2019 / 246356A1;International application publication numbers WO 2013 / 072406A1, WO 2014 / 140248A1, WO 2016 / 166629A1, WO 2017 / 031104A1, WO 2017 / 134134A1, WO 2017 / 095267A1, WO 2019 / 220369A3, WO 2019 / 075359A1, WO 2019 / 226761A1, WO 2020 / 025596A1, WO 2020 / 191346A1, WO 2020018820A1, U.S. Patent Application Publication Nos. US2013 / 0273055A1, and US2019 / 0263920A1; International Application Publication Nos. WO 2012 / 055961A1, WO 2016 / 048938A1, WO 2017 / 087603A1, WO 2017 / 096368A1, WO 2018 / 188612A1, WO 2019 / 237081A1, WO 2020 / 048525A1, WO 2020 / 135335A1, U.S. Patent Application Publication Nos. US2016 / 0326249A1, US2020 / 0283523A1, US2019 / 0284279A1, U.S. Patent Nos. US 9,315,567B2, US 7,575,923B2, and US 7,635,472B2; the bispecific CD3xGPRC5D antibody disclosed in International Application Publication Nos. WO 2018 / 017786A3 and WO 2019 / 220369A3; the bispecific CD3xP SMA antibody disclosed in U.S. Patent Application Publication No. US2017 / 0320947A1; the trispecific CD3xCD28xCD38 antibody disclosed in U.S. Patent Application Publication No. US2020 / 0140552A1; or the international application publication No. WO Other CD3 multispecific antibodies disclosed in WO 2016 / 086189A2, WO 2020 / 088608A1, WO 2019191120A1 and WO 2016 / 105450A3, the contents of each of which are incorporated herein by reference in their entirety. ;

[0225] In some embodiments, the aforementioned multispecific (e.g., bispecific or trispecific) antigen binding molecules that specifically bind to CD3 and tumor antigens may include an anti-CD3 antigen binding molecule that binds to CD3 with a weak binding affinity such as exhibiting a KD greater than about 40 nM as measured by an in vitro affinity binding assay. The aforementioned bispecific antigen binding molecules may include anti-CD3 antigen binding molecules that bind to CD3 and exhibit an EC50 greater than about 100 nM as measured by a FACS titration assay. The aforementioned bispecific antigen binding molecules may include anti-CD3 antigen binding molecules that do not exhibit measurable or observable binding to CD3 as measured by an in vitro affinity binding assay or a FACS titration assay, but still retain the ability to activate human PBMC cells and / or induce cytotoxic activity against tumor antigen-expressing cell lines.

[0226] Treatment

[0227] Provided herein are methods and compositions for mediating killing of tumor cells in a tumor of a subject by 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, wherein the tumor cells do not express or are predicted not to express the target antigen. In some embodiments, at least a subpopulation of tumor cells in the tumor do not express the target antigen.

[0228] In some aspects, provided herein are methods and compositions for inducing killing of tumor cells in a subject and / or inducing T cell activation against tumor cells in a tumor, the method 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 one subpopulation of tumor cells in the tumor does not express the target antigen.

[0229] 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 subpopulation of tumor cells in the tumor do not express the target antigen.

[0230] In some aspects, provided herein are methods of selecting a subject for cancer therapy, the method comprising: i) determining that the subject contains a tumor containing 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 if expression of the target antigen is below the level of detection or below the signal-to-noise ratio, the tumor cells do not express the target antigen, thereby selecting the subject for cancer therapy.

[0231] 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 cell engagement antibody (BiTE), a dual affinity retargeting molecule (DART) and a tandem double antibody (TandAb). A bispecific T cell engager (BiTE) is a small fusion protein containing two antibody binding sites. DART is a bispecific adapter that uses a double antibody skeleton and adds a c-terminal disulfide bridge that improves stability. A tandem double antibody (TandAb) is a bispecific antibody fragment. TandAb is a tetravalent bispecific molecule with a 2+2 antigen binding valence, consisting of an Fv domain. TandAb is usually expressed in the form of a monomer subunit (single-chain Diabody, scDb) with four variable domains from two parent antibodies.

[0232] The methods provided herein can further comprise determining 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%, 4.1%, 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%, 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%, 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%) of the tumor cells do not express the target antigen (e.g., the target antigen, the first target antigen, or the second target antigen). .

[0233] The methods provided herein can further comprise determining at least a subset (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% In some embodiments, at least 50%-60% of the tumor cells do not express the target antigen (e.g., the target antigen, the first target antigen or the second target antigen).

[0234] One of ordinary skill in the art will appreciate that if a portion of cells in the tumor microenvironment do express or are predicted to express the target antigen, the present method can be used for subjects whose tumors do not express the target antigen (e.g., 100% of the tumors do not express or are predicted not to express the target antigen).

[0235] In some aspects, provided herein are methods of treating cancer in a subject having a tumor, inducing or mediating killing of tumor cells in a subject's tumor, and / or inducing T cell activation against tumor cells in a subject's tumor, 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 a 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 a CD3 protein, wherein the first target antigen and the second target antigen are not the same 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.

[0236] 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 cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb).

[0237] Therefore, in certain embodiments, the agents disclosed herein can be used alone or in combination with another type of therapeutic agent. For example, different therapeutic agents can be administered in the form of the same preparation or a separate preparation, simultaneously or sequentially. In certain embodiments, different therapeutic agents can be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about one week of each other. Therefore, the subject receiving such treatment can benefit from the combined effects of different therapeutic agents.

[0238] In certain embodiments, provided herein are methods comprising administering to a subject a multispecific antigen-binding molecule at a dosing frequency of about four times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or less frequently, as long as a therapeutic response is achieved.

[0239] In certain embodiments, a composition is provided herein, e.g., a pharmaceutical composition, containing at least one agent described herein and a pharmaceutically acceptable carrier. In one embodiment, the composition comprises a combination of multiple (e.g., two or more, three or more, four or more, or five or more) agents described herein.

[0240] In some embodiments, the pharmaceutical composition is delivered locally or systemically. In some embodiments, the pharmaceutical composition can be administered locally to a tumor or tumor microenvironment present in a subject. In some embodiments, the agent or pharmaceutical composition is administered together with a second cancer therapeutic agent.

[0241] The agents described herein can be used in combination with any other cancer therapy (including immunotherapy). Additional cancer treatment methods 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 produce to prevent or downregulate immune responses. Examples of immune checkpoint proteins include 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, butyrophilins, A2aR and combinations thereof. Immune checkpoint inhibitors can be cemiplimab (REGN2810), nivolumab (BM S-936558, MDX-1106, ONO-4538), pembrolizumab (MK-3475, SCH 900475), atezolizumab (MPDL3280A, RG7446, RO5541267), durvalumab (MEDI4736, MEDI-4736), avelumab (MSB0010718C), ipilimumab (BMS-734016, IBI310, MDX-010), SHR1210, sintilimab (IBI308), spartalizumab (PDR001), tislelizumab (BGB-A317), pidilizumab, BCD-100, toripalimab (JS001), BAY 1905254, ASP 8374, PF-06801591, AMP-224, AB122, AK105, AMG 404, 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, KN035, MDX-1105, MSB2311, SHR-1316, TG-1501, ZKA B001, INBRX-105, MCLA-145, KN046, M7824, LY3415244, INCB086550, CA-170, CX-072, ADU-1604, AGEN1181, AGEN1884, MK-1308, REGN4659, XmAb22841, ATOR-1015, PSB205, MGD019, AK104, XmAb20717, BMS-986249, tremelimumab, BMS-986258, BGB-A425, INCAGN02390, Sym023, JNJ 61610588, BI 754111, LAG525, MK-4280, REGN3767, Sym022, TSR-033, relatlimab, JTX-2011, MGD009, BMS-986207, OMP-313M32, MK-7684, or TSR-022.

[0242] Other cancer immunotherapies include adoptive immunotherapy, such as autologous or allogeneic T cell therapy or autologous or allogeneic CAR T cell therapy. Adoptive immunotherapy is a method of treating a patient's immune response to a tumor or cancer cell. The method involves removing immune cells from an individual, forming effector cells in vitro, expanding cells to a clinically relevant number, and re-injecting cells into the patient. The method provided herein includes co-administering an agent disclosed herein and expressing an allogeneic or autologous CTL of a T cell receptor specifically binding to a peptide (e.g., a cancer peptide or a subject-specific peptide) presented on a class I MHC. In some embodiments, CTL is from a cell bank or from a subject to whom CTL is being administered. In some embodiments, MHC is class I MHC. In some embodiments, class II MHC has an alpha chain polypeptide, which is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, the class II MHC has a beta 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 bank or cell bank prior to administration to a subject.

[0243] Other cancer therapies may be 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 T cell therapy, and any other adoptive therapy, such as therapy or macrophage therapy using natural killer cells. Cell therapy may include unmodified cells, such as traditional TIL therapy, or genetically modified cells. The methods included herein include any method known in the art for achieving cell targeting of tumor targets in cell therapy. For example, cell therapy may include cells comprising chimeric antigen receptors (CAR). Single-chain antibodies may be used, and CAR may also include costimulatory domains. CAR cell targets may be located on the membrane of target cells, and TCR modifications may use intracellular targets.

[0244] In one embodiment, cell therapy includes cells of a type selected from the group consisting of T cells, CD8+ cells, CD4+ cells, NK cells, δ-γT cells, regulatory T cells, and peripheral mononuclear blood cells. In another embodiment, TIL, T cells, CD8+ cells, CD4+ cells, NK cells, δ-γT cells, regulatory T cells, or peripheral blood mononuclear cells form cell therapy as disclosed herein. In a specific embodiment, cell therapy includes T cells. As used herein, "tumor infiltrating lymphocytes" or TILs refer to white blood cells that leave the bloodstream and migrate into tumors. Lymphocytes can be divided into three groups, including B cells, T cells, and natural killer cells. In another specific embodiment, cell therapy includes T cells modified with target-specific chimeric antigen receptors or, in particular, selected T cell receptors. As used herein, "T cells" include, but are not limited to, CD3+ cells (including CD4+ helper cells), CD8+ cytotoxic T cells, and γδT cells.

[0245] In some embodiments, T cells are contacted with antigen presenting cells (APCs), which present peptides specific to the cancer or tumor of the subject. In some embodiments, APCs are B cells, antigen presenting T cells, dendritic cells or artificial antigen presenting cells (e.g., aK562 cells). Dendritic cells for the process can be prepared by extracting PBMCs from patient samples and attaching them to plastics. In general, monocyte groups will adhere, and all other cells will be washed away. Then IL-4 and GM-CSF are used to differentiate the supporting group to produce dendritic cells derived from monocytes. These cells can be matured by adding IL-1β, IL-6, PGE-1 and TNF-α (important co-stimulatory molecules on the surface of up-regulated dendritic cells), and then transduced with one or more peptides provided herein. In some embodiments, APCs are artificial antigen presenting cells, such as aK562 cells. In some embodiments, 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 US 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.

[0246] Another exemplary adoptive immunotherapy scheme involves administering autologous tumor infiltrating lymphocytes (TIL). TIL cells have a strong killing power. TIL cells are effector cells that differentiate in vivo in solid tumors (see U.S. Patent No. 5,126,132, which describes a method for producing TIL cells for adoptive immunotherapy of cancer). TIL cells can be produced, for example, by taking a tumor sample from a patient, separating lymphocytes infiltrating into the tumor sample, growing these TIL cells in vitro in the presence of IL-2, and re-injecting these cells into the patient together with IL-2.

[0247] Other cancer therapies can be CAR-T cell therapy.Chimeric antigen receptor (CAR) is a molecule that combines the specificity of antibody-based tumor-associated surface antigens with the T cell receptor activation intracellular domain with specific anti-tumor cell immune activity (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 be fused with the intracellular domain providing T cell activation and co-stimulatory signals through single-chain Fv (scFv) antigen-specific extracellular regions to achieve primary activation independent of MHC. Second and third generation CARs also provide appropriate co-stimulatory signals through CD28 and / or CD137 (4-1BB) intracellular activation motifs, thereby enhancing cytokine secretion and anti-tumor activity in a variety of 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 the patient's autologous T cells to express CARs specific for tumor antigens, followed by ex vivo cell expansion and re-infusion back into the patient. CAR is a fusion protein of a selected single-chain fragment variant from a specific monoclonal antibody with one or more T cell receptor intracellular signaling domains. This genetic modification of T cells can be performed by viral-based gene transfer methods or non-viral methods, such as DNA-based transposons, CRISPR / Cas9 technology, or direct transfer of in vitro transcribed mRNA by electroporation.

[0248] Another cancer therapy may be natural killer cell therapy. Natural killer (NK) cells can recognize tumor cells as targets and therefore may be used 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). NK cell infusions have been used to treat patients with various forms 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). Available methods make it possible to obtain large numbers of human NK cells that exhibit higher anti-tumor capacity than unexpanded NK cells (see U.S. Pat. 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). Included herein are NK cells expanded from primary peripheral blood mononuclear cells (PBMCs). Also included herein are NK cells comprising chimeric antigen receptors or other modifications.

[0249] Another cancer therapy can be macrophage cell therapy. In some embodiments, cell therapy includes cytotoxic macrophages grown in vitro. Macrophages can recognize tumor cells as targets and can therefore be used for cancer immunotherapy (Andreesen R, Henneman n B, Krause SW. Adoptive immunotherapy of cancer using monocyt e-derived macrophages: rationale, current status, and perspectives. J Leukoc Biol. October 1998; 64 (4): 419-26). Macrophages are powerful immune effector cells, and their functional plasticity produces anti-tumor and pro-tumor functions in different environments. This plasticity has led to significant efforts to consume or repolarize tumor-associated macrophages. Alternatively, in some embodiments, macrophages are adoptively transferred following, for example, 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).

[0250] Also provided herein is a method for treating cancer in a subject, wherein a sample comprising T cells is obtained from the subject, cytotoxic T lymphocytes (CTLs) are separated from the sample, CTLs are amplified in vitro, and the amplified CTLs are administered to the subject in combination with at least one agent (e.g., any agent disclosed herein). Cytotoxic T cells may be tumor infiltrating lymphocytes. Amplifying CTLs may include contacting CTLs with antigen presenting cells (APCs) expressing cancer-specific or tumor-specific antigens to produce antigen-specific CTLs. In some embodiments, a sample comprising T cells or isolated CTLs is stimulated before being administered to a subject. The method may also include contacting CTLs with anti-CD3 monoclonal antibodies (OKT3) before being administered to a subject. In other embodiments, the method also includes contacting CTLs with human interleukin (IL)-2 before being administered to a subject.

[0251] Additional cancer therapies also include any known immune cell stimulators, such agents including, for example, those that induce proliferation, expansion or activation of such immune cells. Exemplary stimulation methods include administration of stimulatory cytokines such as IL-2, IL-12, IL-15, IL-18 and IL-21.

[0252] In some embodiments, the subject has received a chemotherapeutic agent prior to administration of the agent. The subject may be refractory to the chemotherapeutic agent. The subject may receive the chemotherapeutic agent and the agent of the additional cancer therapy disclosed herein sequentially or simultaneously. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide. TM); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine; polyacetylamines (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin, CC-1065 (including its adolesin derivatives); the synthetic analogs of adozelesin, carzelesin and bizelesin); scutellarin (particularly scutellarin 1 and scutellarin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, oxide hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin phili; dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicins; and new tumor suppressor protein chromophores and related chromophore enediyne antibiotic chromophores), aclacinomysins, actinomycins, authramycins, azaserine, bleomycin, cactinomycin, carabicin, carrninomycin, carzinophilin, chromomycins, actinomycin D, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin; TM) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, streptozotocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil ( 5-FU); folic acid analogs such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, cyclothiodine, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid, leucovorin, folate, doxycycline, cyclopentyl ether, doxycycline, oxadiaz ... acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids such as maytansine and maytansine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; PSK; TM ; razoxane; rhizoxin; sizofiran; spirogermanium; tricinosporic acid; triazoquinone; 2,2',2"-tricUorotriemylamine; trichothecenes (particularly T-2 toxin, verracurin A, baculozolin A, and anguidine); urethanes; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, for example, paclitaxel; TM , Bristol Meyers Squibb Oncology, Princeton, NJ) and docetaxel (Taxoteret TM , Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine (Gemzar TM ); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone; vincristine; vinorelbine (Navelbine TM ); mitoxantrone (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin; aminopterin; xeloda (xeloda); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. The definition of "chemotherapeutic agent" also includes anti-hormonal agents used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex TM), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston TM ); inhibitors of aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, TM ), exemestane, formestane, fadrozole, vorozole (Rivisor TM ), letrozole (Femara TM ) and anastrozole (Arimidex TM ); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprohde and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0253] As described in detail below, the pharmaceutical compositions and / or agents disclosed herein can be specifically 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 by subcutaneous, intramuscular, intravenous, intrathecal, intracerebral, or epidural injection in the form of, for example, a sterile solution or suspension or a sustained release formulation. The method of preparing a pharmaceutical formulation or composition includes the step of associating an agent described herein with a carrier and, optionally, one or more auxiliary ingredients. In general, the formulation is prepared by uniformly and intimately associating an agent described herein with a liquid carrier or a finely divided solid carrier, or both, and then shaping the product as desired.

[0254] The pharmaceutical compositions provided herein can be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in the formula book known to all pharmaceutical chemists (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA). These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles containing lipids (cations or anions) (such as LIPOFECTIN TM , Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0255] The dosage of the antigen binding molecule administered to a patient may vary depending on the patient's age and body size, target disease, condition, administration route, and the like.

[0256] 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, receptor-mediated endocytosis (see, e.g., 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 composition can be administered by any convenient route, such as by infusion or rapid injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered with other bioactive agents. It can be administered systemically or topically.

[0257] In some embodiments, the pharmaceutical compositions disclosed herein can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, relative to subcutaneous delivery, pen delivery devices have been easily applied to deliver pharmaceutical compositions as disclosed herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. After all the pharmaceutical composition in the cartridge has been applied 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 used again. In a disposable pen delivery device, there is no replaceable cartridge. In contrast, a disposable pen delivery device is pre-filled with a pharmaceutical composition held in a reservoir within the device. After the pharmaceutical composition in the reservoir is emptied, the entire device is discarded.

[0258] Many reusable pen and auto-injector delivery devices have been used to subcutaneously deliver the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM(NovoNordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM PEN (AbbottLabs, Abbott Park IL), to name a few.

[0259] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, a polymer material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In another embodiment, a controlled release system can be placed near the target of the composition, so only a portion of the systemic dose is required (see, e.g., Goodson, 1984, Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review conducted by Langer, 1990, Science 249: 1527-1533.

[0260] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods known to the public. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying the antibody described above or its salt in a conventional sterile aqueous medium or oily medium for injection. As aqueous media for injection, there are, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, etc., which can be used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50mol) adducts of hydrogenated castor oil)], etc. As oily media, there are, for example, sesame oil, soybean oil, etc., which have been used, which can be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. Therefore, the injection prepared is preferably filled in a suitable ampoule.

[0261] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in a dosage form in a unit dosage form suitable for meeting the dosage of the active ingredient. Such dosage forms of unit dosages include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0262] The agent described herein can be administered in multiple doses, such as an initial dose, a secondary dose, and a tertiary dose. The terms "initial dose", "secondary dose", and "tertiary dose" refer to the time sequence of administering the antigen binding molecules disclosed herein. Therefore, the "initial dose" is a dose (also referred to as a "baseline dose") administered at the beginning of the treatment regimen; "secondary dose" is a dose administered after the initial dose; and "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of therapeutic agents described herein, but may generally be different from each other in terms of frequency of administration. However, in certain embodiments, during the course of treatment, the amount of the antigen binding molecules contained in the initial, secondary, and / or tertiary doses is different from each other (e.g., up or down, as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of the treatment regimen, followed by less frequently administered subsequent doses (e.g., "maintenance doses").

[0263] In an exemplary embodiment disclosed herein, 1 to 26 (e.g., 1, 1 1 / 2 , 2, 2 1 / 2 ,3,3 1 / 2 , 4, 4 1 / 2 , 5, 5 1 / 2 ,6,6 1 / 2 ,7,7 1 / 2 ,8,8 1 / 2 , 9, 9 1 / 2 , 10, 10 1 / 2 , 11, 11 1 / 2 , 12, 12 1 / 2 , 13, 13 1 / 2 , 14, 14 1 / 2 , 15, 15 1 / 2 , 16, 161 / 2 , 17, 17 1 / 2 , 18, 18 1 / 2 , 19, 19 1 / 2 , 20, 20 1 / 2 , 21, 21 1 / 2 , 22, 22 1 / 2 , 23, 23 1 / 2 , 24, 24 1 / 2 , 25, 25 1 / 2 , 26, 26 1 / 2 Each secondary and / or tertiary dose is administered at least 2 weeks after administration of the therapeutic agent described herein. As used herein, the phrase "the immediately preceding dose" means, in a sequence of multiple administrations, the dose of the therapeutic agent described herein administered to the patient immediately prior to the administration of the next dose without an intervening dose in the administration sequence.

[0264] The method according to this aspect disclosed herein can include administering to the patient any number of secondary and / or tertiary doses of a 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., 2, 3, 4, 5, 6, 7, 8 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., 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

[0265] In embodiments involving multiple secondary doses, each secondary dose can be administered at the same frequency as the other secondary doses. For example, each secondary dose can be administered to the patient 1 to 2 weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose can be administered to the patient 2 to 4 weeks after the immediately preceding dose. Alternatively, the frequency of administering secondary and / or tertiary doses to the patient can be changed during the course of the treatment regimen. The frequency of administration can also be adjusted by the physician according to the needs of the individual patient after clinical examination during the course of the treatment.

[0266] Indications

[0267] In some embodiments, the methods described herein can be used to treat any cancer, including any cancerous or precancerous tumor. Cancers that can be treated by the methods and compositions provided herein include, but are not limited to, bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer. In addition, the cancer can be specifically of the following histological types, but is not limited to these: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct cancer; hepatocellular carcinoma; mixed hepatocellular carcinoma and bile duct cancer; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial colon polyps Sarcoid adenocarcinoma; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic cell carcinoma; oncocytic adenocarcinoma; basophilic granulocyte carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; cerumenous gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cyst adenocarcinoma; 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; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant blastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus Glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial disseminated melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor mixed tumor); Wilms' tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner's tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian thyroid tumor; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma;Ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectoderm; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; granulomatoid; small lymphocytic malignant lymphoma; Diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other specified non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0268] The composition or method can be used to treat cancer or tumors expressing CD20. The composition or method can be used to treat 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, cancer is follicular lymphoma. In some embodiments, cancer is diffuse large B cell lymphoma (DLBCL).

[0269] In some embodiments, cancer comprises a solid tumor. In some embodiments, the tumor is adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a hematogenous tumor, a brain / CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing's tumor, an eye tumor, a gallbladder tumor, a gastrointestinal tumor, a kidney tumor, a laryngeal or hypopharyngeal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penis tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymic tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms' tumor.

[0270] In certain embodiments, the tumor being treated is one that is predicted not to express a particular target antigen.The expression profiles of the exemplary target antigens (eg, exemplary TAAs) disclosed herein are known in the art and / or can be determined using the methods provided herein.

[0271] Expression profiles are known and can be found in, for example, the following databases: TANTIGEN 2.0 (available on the World Wide Web at projects.met-hilab.org / tadb / index.php; more details can be found in Zhang, G., Chitkushev, L., Olsen, LR et al. TAN TIGEN 2.0: a knowledge base of tumor T cellantigens and epitopes. BMC Bioinformatics 22, 40 (2021)); Cancer Epitope Database and Analysis Resource (CEDAR) (more details can be found in Z. et al., The Cancer Epitope Database and Analysis Resource: A Blueprint for the Esta blishment of a New Bioinformatics Resource for Use by the Cancer Immunology Community, Frontiers in Immunology, 12 (2021)); Human Protein Atlas (available on the World Wide Web at v17.proteinatlas.org / ); dbPe pNeo (available on the World Wide Web at biostatistics.online / dbPepNeo / search.php); and cBioPortal (available on the World Wide Web at cbioportal.org).

[0272] Figure 18-26 Exemplary expression profiles of certain exemplary target antigens are provided in.

[0273] In some embodiments, a tumor is predicted not to express a target antigen based on a known expression profile (e.g., an expression profile disclosed herein). In some embodiments, less than 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%, 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%, 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%, , 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%, 2 7%, 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 50% of the analyzed tumors express the target antigen, then the tumor type is predicted to not express the target antigen.

[0274] Primary tumors exhibit antigen expression patterns that vary within tumors and between different patients. Antigen expression differences between patients with the same histological tumor type may be very large. Therefore, the exemplary expression profiles disclosed herein are of reference value for tumor types that can be treated using the methods disclosed herein, but are not limited to cancers or tumor types that can be treated using the methods disclosed herein. In some embodiments, the treated tumor may be a tumor that has been tested and does not express the target antigen or contains a tumor cell subpopulation that does not express the target antigen, although the tumor antigen expression profile shows that the tumor type usually expresses the target antigen. In some embodiments, the target antigen is enriched in a specific tumor type, but the target antigen is not expressed in a tumor cell subpopulation in a specific tumor type of the patient. In some embodiments, the target antigen is usually expressed in a variety of tumor types, but the target antigen is not expressed in a tumor cell subpopulation of the patient.

[0275] Screening, diagnosis and prognostic testing

[0276] Also provided are screening, diagnostic and / or prognostic assays for identifying the level or quantifying the amount of a tumor-associated antigen on a cancer or tumor cell in a subject's cancer.

[0277] In some embodiments, at least one subpopulation of tumor cells in a tumor does not express the target antigen. The methods provided herein can further include determining at least a subpopulation (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% of the tumor cells in the tumor or in the tumor microenvironment. , 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%, 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%, 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%) of the tumor cells determined do not express the target antigen, and if the determined subpopulation / percentage does not express the target antigen, administering one or more agents disclosed herein to the subject. A person of ordinary skill in the art will appreciate that if a portion of cells in the tumor microenvironment do express the target antigen or are predicted to express the target antigen, the present method can be used for subjects whose tumors do not express the target antigen (e.g., 100% of the tumors do not express the target antigen or are predicted not to express the target antigen).

[0278] The methods provided herein can also include determining that at least the subpopulation of tumor cells in the tumor does not express the target antigen. In some embodiments, if the expression of the target antigen is below the detection level or below the signal-to-noise ratio, the tumor cells do not express the target antigen.

[0279] Provided herein is a method for screening a subject by measuring or calculating the amount or level of a tumor-associated antigen in a subject's cancer. Tumor-associated antigens can be measured by any method known in the art. For example, a biological sample can be collected from a patient. The sample can be obtained by any means known in the art. The sample can also 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 fluids present in a subject's body.

[0280] In some embodiments, the determined percentage of tumor cells expressing the target antigen can be determined by measuring the level or expression of the target antigen in a biological sample taken from a subject (e.g., a biopsy of a tumor or tumor microenvironment). In some embodiments, the percentage can then be extrapolated to quantify the predicted total percentage of tumor and / or tumor microenvironment cells expressing the target antigen in the subject.

[0281] The detection methods encompassed by the present disclosure can be used for in vitro and in vivo detection of mRNA, protein or genomic DNA of tumor-associated antigens or their biologically active fragments in biological samples. For example, in vitro techniques for detecting mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. In vitro techniques for detecting genomic DNA include Southern hybridization. In addition, in vivo techniques for detecting proteins include introducing labeled antibodies directed to the desired protein to be detected into the subject. For example, the antibody can be labeled with a radioactive marker, and the presence and location of the radioactive marker in the subject can be detected by standard imaging techniques.

[0282] The assays described herein may include measuring the level of tumor-associated antigens after separation from cells (e.g., after separation from a biopsy or biological sample). These may be performed in a cell-free format using known components of the gene expression of the tumor-associated antigens. Certain components of the assay may need to be fixed, and such embodiments may benefit from the use of well-known modifications for biomolecule fixation, such as the use of microtiter plates, beads, test tubes, microcentrifuge tubes in combination with derivatizable moieties, such as fusion protein domains, biotinylation, antibodies, etc.

[0283] In order to determine whether a subject suffers from a disease disclosed herein, whether there is a risk of developing such a disease, or whether it can benefit from the administration of an agent disclosed herein, a biological sample can be obtained from the subject, and the biological sample can be contacted with a compound or agent capable of detecting a tumor-associated antigen protein in the biological sample or a polynucleotide (e.g., mRNA or genomic DNA) encoding the tumor-associated antigen. The agent for detecting mRNA or genomic DNA may include a labeled nucleic acid probe capable of hybridizing with mRNA or genomic DNA. The nucleic acid probe may be, for example, a sequence complementary to a nucleic acid encoding a tumor-associated antigen, or a portion thereof, such as an oligonucleotide having a length of at least 15, 20, 25, 30, 25, 40, 45, 50, 100, 250 or 500 nucleotides, and is sufficient to specifically hybridize with the desired mRNA or genomic DNA under stringent conditions. Other suitable probes for diagnostic assays encompassed by the present disclosure are described herein.

[0284] 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 a protein, mRNA, or genomic DNA such that the presence of the desired protein, mRNA, or genomic DNA is detected in the biological sample; 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.

[0285] 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 may benefit from the use of well-known modifications for biomolecule immobilization, such as the use of microtiter plates, beads, test tubes, microcentrifuge tubes in combination with derivatizable moieties, such as fusion protein domains, biotinylation, antibodies, and the like.

[0286] Analysis of one or more nucleic acid regions of tumor-associated antigens in a subject can be used to predict whether the subject has or may benefit from the methods disclosed herein. For example, detecting a tumor-associated antigen in a portion of a cancer causes the cancer to be classified as a heterogeneous cancer, indicating that the subject will benefit from the methods disclosed herein. Similarly, analyzing the genomic copy number of a subject's tumor-associated antigen can be used to predict whether the subject will benefit from the methods disclosed herein. In some embodiments, the method encompassed by the present disclosure may be characterized by including the presence or absence of one or more polymorphic regions of a gene encoding a tumor-associated antigen in a cell sample from a subject.

[0287] In other detection methods, it is necessary to first amplify at least a portion of the nucleic acid detected in the biological sample. Amplification can be performed, for example, by PCR and / or LCR according to methods known in the art (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 produce the desired amount of amplified DNA.

[0288] Optional amplification methods include: self-sustaining 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), Q-β replicase (Lizardi, PM et al., 1988, Bio / Technology 6: 1197) and self-sustaining sequence replication (Guatelli et al., (1989) Proc. Nat. Acad. Sci. 87: 1874) and nucleic acid-based sequence 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. If the number of nucleic acid molecules is very small, these detection schemes are particularly useful for detecting such molecules.

[0289] In one embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence nucleic acids in a biological sample by comparing the sequence of the sample sequence to a 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). It is also contemplated that any of a variety of automated sequencing programs (Biotechniques (1995) 19:448) can be used when performing a subject assay, including sequencing by mass spectrometry (see, e.g., U.S. Patent No. 5,547,835 and International Patent Application Publication No. WO 94 / 16101, entitled "DNA Sequencing by Mass Spectrometry," by authors). U.S. Patent No. 5,547,835 and International Patent Application Publication No. WO 94 / 21822, entitled "DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation," by H. ), and U.S. Patent No. 5,605,798 and International Patent Application No. PCT / US96 / 03651, entitled “DNA Diagnostics Based on Mass Spectrometry,” by H. Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159). It will be apparent to those skilled in the art that for certain embodiments, it may be desirable to determine the occurrence of only one, two or three nucleic acid bases in a sequencing reaction. For example, A-track or the like may be performed, for example, where only one nucleotide is detected.

[0290] Antibodies against tumor-associated antigens can also be used for disease diagnosis and prognosis. In addition, such diagnostic methods can be used to detect abnormalities in the expression levels of such polypeptides, or abnormalities in the structure and / or tissue, cell or subcellular location of such polypeptides. Structural differences can include, for example, differences in the size, electronegativity or antigenicity of mutant polypeptides relative to normal polypeptides. Proteins from tissues or cell types to be analyzed can be easily detected or separated using techniques well known to those skilled in the art, including but not limited to Western blot analysis. For a detailed description of methods for performing Western blot analysis, see Sambrook et al., 1989, supra, Chapter 18. The protein detection and separation methods used herein can also be 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 are incorporated herein by reference in their entirety.

[0291] For example, this can be achieved by immunofluorescence techniques using fluorescently labeled antibodies (see below) in combination with optical microscopy, flow cytometry or fluorescence detection. In addition, antibodies (or fragments thereof) useful according to the present disclosure can be used histologically, such as in immunofluorescence or immunoelectron microscopy, for in situ detection of tumor-associated antigens. In situ detection can be achieved by taking out a histological sample from a subject and applying the labeled antibodies disclosed herein thereto. Antibodies (or fragments) can be applied by covering the labeled antibodies (or fragments) onto a biological sample. By using such a procedure, not only the presence of tumor-associated antigens can be determined, but also their distribution in the examined tissue can be determined. Those of ordinary skill in the art will readily recognize that a variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

[0292] Solid phase supports or carriers are usually used as supports 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. For the purposes covered by the present disclosure, the nature of the carrier can be soluble to a certain extent or insoluble. As long as the coupled molecule can bind antigens or antibodies, the support material can have virtually any possible structural configuration. Therefore, the support configuration can be spherical, such as beads, 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 a thin sheet, a test strip, etc. Supports include, but are not limited to, polystyrene beads. Those skilled in the art will appreciate many other carriers suitable for binding antibodies or antigens, or can determine these carriers by using routine experiments.

[0293] One method for labeling an antibody is to link it to an enzyme and use it 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., (ed.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme bound to the antibody will react with an appropriate substrate (e.g., a chromogenic substrate) in a manner to produce a chemical moiety that can be detected by spectrophotometric, fluorescent, or visual methods. Enzymes that can be used to detectably label antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Detection can be accomplished by colorimetry using a chromogenic substrate for the enzyme. Detection can also be performed by visually comparing the extent of the enzyme reaction of the substrate to a similarly prepared standard.

[0294] Any of a variety of other immunoassays may also be used for detection. For example, by radiolabeling antibodies or antibody fragments, it is possible to detect fingerprint gene wild-type or mutant peptides using radioimmunoassay (RIA) (see, e.g., 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 methods such as using a gamma counter or a scintillation counter or autoradiography.

[0295] Antibodies can also be labeled with fluorescent compounds. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected by fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine. Fluorescent emitting metals (such as 152 Antibodies can be detectably labeled with metals such as thiazolyl acetate (Eu) or other metals from the lanthanide series. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0296] The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antibody is then determined by detecting the presence of light generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, or oxalates.

[0297] Likewise, bioluminescent compounds can be used to label 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 purposes are luciferin, luciferase, and aequorin.

[0298] The methods described herein can be performed, for example, by utilizing a prepackaged diagnostic kit (such as the above-mentioned kit), the diagnostic kit comprising at least one probe or primer nucleic acid described herein, which can be conveniently used, for example, to determine whether a subject suffers from a disease associated with a particular allelic variant of interest or is at risk of developing the disease. The sample nucleic acid analyzed by any of the above-mentioned diagnostic and prognostic methods can be obtained from any cell type or tissue of the subject. For example, a body fluid (such as blood) of the subject can be obtained by known techniques (such as venipuncture). Alternatively, a dried sample (such as hair or skin) can be tested for nucleic acids. Fetal nucleic acid samples can be obtained from maternal blood, as described in International Patent Application No. WO91 / 07660 of Bianchi. Alternatively, amniotic fluid cells or chorionic villi can be obtained for prenatal testing.

[0299] Diagnostic procedures can also be performed directly on tissue sections (fixed and / or frozen) of subject tissue obtained from biopsy or resection in situ, thereby eliminating the need for nucleic acid purification. Nucleic acid reagents can be used as probes and / or primers for such in situ procedures (see, e.g., Nuovo, GJ, 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY).

[0300] In addition to methods that focus primarily on detecting a nucleic acid sequence, characteristics can also be assessed through such detection schemes. For example, fingerprints can be generated by utilizing differential display procedures, Northern analysis, and / or RT-PCR.

[0301] Also provided herein is a method for predicting whether a subject's tumor cells express a target antigen by a tissue or tumor expression profile of the target antigen. For example, a subject may be diagnosed with cancer, and if the known problem or tumor expression profile does not typically express the target antigen, the tumor cells in the subject's tumor are predicted not to express the target antigen. In addition, a subject may be diagnosed with cancer, and if the known problem or tumor expression profile shows less than 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%, 10 9%, 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% or less of the tumor cells in the subject's tumor are predicted to not express the target antigen.

[0302] The present disclosure also relates to novel agents identified by the above-mentioned screening assays. Thus, screening, diagnostic and / or prognostic assays are also provided for identifying antibodies specific for tumor-associated antigens that induce / mitigate killing of tumor cells that do not express tumor-associated antigens or activate T cells against tumor cells that do not express tumor-associated antigens.

[0303] In one embodiment, the disclosure provides assays for screening candidate or test compounds that are substrates for or interact with tumor associated antigens.

[0304] 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 determined. The ability of the test agent to perform the function in question can be determined by monitoring biomarkers described herein (e.g., biopsy, biomarker expression, physical assays, etc.).

[0305] The ability of the test agent to modulate the binding of the antibody to its target substrate can also be determined. Determining the binding ability of the test agent can be achieved, for example, by coupling the substrate to a radioisotope or enzyme label so that the binding of the substrate to the antibody can be determined by detecting the labeled substrate in the complex. The target substrate can also be coupled to a radioisotope or enzyme label to monitor the ability of the test agent to modulate the binding to the substrate in the complex. Determining the ability of the test agent to bind to the target protein can be achieved, for example, by coupling the agent to a radioisotope or enzyme label so that the binding can be determined by detecting the labeled agent in the complex. For example, such agents can be used 125 I. 35 S. 14 C or 3 The molecule may be labeled directly or indirectly with H, and the radioisotope detected by direct counting of radioactive emission or by scintillation counting. The agent may be further enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by measuring the conversion of an appropriate substrate to product.

[0306] In certain embodiments, the binding capacity of an agent is determined with or without labeling any interactants. For example, a microphysiometer can be used to detect interactions without labeling any components (McConnell, HM et al. (1992) Science 257: 1906-1912). As used herein, a "microphysiometer" (e.g., a cell sensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS).

[0307] In one aspect, a cell-based system as described herein can be used to identify agents for treating a cancer disclosed herein. For example, such a cell system can be exposed to an agent at a sufficient concentration and for a sufficient period of time to cause an improvement in disease symptoms in the exposed cells. After exposure, the cells can be examined to determine whether one or more disease phenotypes have changed to a more normal or wild-type-like phenotype.

[0308] In addition, animals 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 for identifying drugs, pharmaceuticals, therapies, and interventions that can effectively bind to tumor-associated antigens or other targets herein, such as to treat or prevent cancer disclosed herein.

[0309] example:

[0310] Example 1: CD22-targeting CD28 bispecific antibody enhances the anti-tumor efficacy of onitumumab in a refractory diffuse large B-cell lymphoma model

[0311] While many patients with diffuse large B-cell lymphoma (DLBCL) may achieve a complete response to first-line chemoimmunotherapy, patients with relapsed / refractory (r / r) disease typically have poor outcomes. Onitumab, a CD20xCD3 bispecific antibody that provides “signal 1” by activating the T-cell receptor / CD3 complex, has shown early, promising activity in patients with highly refractory DLBCL in a Phase I trial; however, not all patients achieve a complete response and many experience relapse, representing a large unmet medical need. Here, it was investigated whether the addition of a co-stimulatory “signal 2” by engaging the CD28 receptor on T cells could enhance the activity of onitumab. The data presented here demonstrate for the first time that REGN5837, a bispecific antibody that crosslinks CD22-expressing tumor cells to CD28-expressing T cells, enhances the activity of onitumab by enhancing T-cell activation and cytolytic function. In preclinical DLBCL tumor studies using animals reconstituted with the human immune system, REGN5837 promoted the antitumor activity of onitumab and induced intratumoral expansion of plastically reprogrammed T cells while avoiding a dysfunctional state. REGN5837 monotherapy showed activity and no toxicity in primate studies and enhanced T cell activation when given in combination with onitumab. In addition, analysis of non-Hodgkin lymphoma (NHL) clinical samples revealed that CD28 + CD8 + T cells were increased, demonstrating the presence of a population that can be targeted by the CD22xCD28 antibody. Together, these data suggest that REGN5837 enhances the antitumor activity of onitumumab in preclinical NHL models and that the combination of these two bispecific antibodies may provide a new chemotherapy-free approach for the treatment of r / r DLBCL.

[0312] One therapeutic strategy in the field of cancer immunotherapy is to redirect T cells to tumors using bispecific antibodies to enhance antitumor activity. This led to the approval of the CD19xCD3 T cell engager Blincyto for the treatment of acute lymphoblastic leukemia and the EpCAMxCD3 bispecific antibody Removab for the treatment of malignant ascites. These T cell redirecting therapeutics are designed to engage tumor antigens via one antigen binding fragment (Fab) arm and T cell activating receptors via the other Fab arm. Onitumumab is a CD20xCD3 bispecific antibody that effectively triggers T cell-mediated killing of CD20-expressing cells in preclinical tumor models. Clinical data from a Phase 1 trial showed promising activity and a manageable safety profile for this therapy in patients with highly refractory and relapsed B-NHL. However, since relapse is inevitable in heavily pretreated patients, there is still potential for improved efficacy in patients with aggressive lymphomas. For optimal T cell activation, T cells require the engagement of the T cell receptor complex (TCR), which provides "signal 1," and the engagement of co-stimulatory receptors, which provide "signal 2." Onitumab activates T cells by providing signal 1 by cross-linking CD3, while further enhancement of T cell effector function and activation can be mediated by the addition of co-stimulatory signals. In this study, we examined whether the addition of a CD22xCD28 bispecific antibody could enhance the anti-tumor activity of Onitumab.

[0313] REGN5837 or CD22xCD28 is a hinge-stabilized human IgG4-based antibody that is intended to enhance T cell responses against CD22-expressing tumors (i.e., NHL) by bridging CD22-expressing cells and CD28-expressing T cells. CD22 is a transmembrane protein that binds sialic acid and is present in normal B cells and malignant B cells (such as DLBCL tumors). Although the exact role of human CD22 is not fully understood, studies have shown that CD22 regulates signal transduction of B cell antigen receptors, B cell migration, and maintenance of peripheral B cell homeostasis and survival. By cross-linking CD22 on tumor cells with CD28 on T cells, key CD28 co-stimulatory signals can be conducted to enhance CD20xCD3-directed T cell activation. This study demonstrates that the anti-tumor activity of Onitumumab against DLBCL tumor cells can be effectively enhanced in vitro and in vivo by combining with REGN5837. Characterization of intratumoral T cell responses revealed that combined engagement of CD28 with onitumumab not only mediated curative tumor responses in preclinical models of DLBCL, but also expanded intratumoral T cells and induction of reprogrammable T cells. Therefore, these data support potential clinical studies of REGN5837 in combination with onitumumab in r / r DLBCL patients.

[0314] result

[0315] Intratumoral CD8 + CD28 expression was detected on T cells.

[0316] CD22 is a well-validated tumor target for the treatment of B-cell leukemias and lymphomas and has widespread but variable expression in DLBCL patient samples ( Figure 7A-7C ). In addition, it was observed that a large number of CD28 + Cells can be targeted with a CD22xCD28 bispecific antibody ( Fig. 8A and Figure 8B Analysis of baseline samples from a phase I dose-finding study of onitumumab (ClinicalTrials.gov identifier: NCT02290951) showed that CD28 expression was preferentially expressed in tumors with CD8 + On T cells, while on CD4 + The expression level on T cells is low ( Figure 1A and B). In addition, although limited in number, several pairs of patient samples were analyzed for the presence or absence of CD28 before and after onitumumab treatment. + CD8 + Five weeks after starting onitumab therapy, CD8 + CD28 + A specific increase in T cell density ( Figure 1C ), consistent with the persistence of an intratumoral T cell population that REGN5837 could target to enhance effector memory differentiation and further enhance antitumor activity. Although CD86 and, to a lesser extent, CD80 (both ligands for CD28) were also expressed in resected DLBCL patient samples, simultaneous expression of the co-inhibitory molecule CTLA4 was observed ( Fig. 8A and Figure 8B ), which has a higher affinity for CD80 and CD86 and can eliminate CD28 co-stimulatory activity. Therefore, the use of CD22xCD28 bispecific antibodies can achieve specific engagement of CD28 and potentially enhance the activity of onitumumab.

[0317] REGN5837 enhances onituzumab-mediated T cell activation and effector function in vitro.

[0318] To evaluate the ability of REGN5837 to enhance the cytolytic and T cell activation activities of onitumumab, 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 ( Fig. 9B ) and were titrated with doses of onitumab as a single agent (4.8 fM to 10 nM) or in the presence of fixed concentrations of REGN5837 (ranging from 77 pM to 10 uM). REGN5837 activity was determined in the presence of onitumab because, unlike the TeGenero class of CD28 superagonists, the CD28 bispecific antibody is unable to mediate costimulatory activity in the absence of signal 1 or TCR / CD3 complex engagement ( Fig. 9A Administration of REGN5837 co-stimulation enhanced the cytotoxicity of WSU-DLCL2 cells mediated by onitumumab ( Figure 2A ), upregulated CD4 + ( Figure 2B ) and CD8 + ( Figure 2D )T cells and induced CD4 + ( Figure 2C ) and CD8 + ( Figure 2E )T cell proliferation (Table 6).

[0319] Table 6: REGN5837 bispecific antibody enhances onituzumab-mediated T cell activation and cytotoxicity

[0320]

[0321] Analysis of supernatants from co-cultures revealed that REGN5837 also enhanced onituzumab-mediated cytokine release in a dose-dependent manner (Table 7). Figure 2F In addition, the ability of REGN5837 to enhance the activity of onitumumab was demonstrated by targeting B-cell acute lymphoblastic leukemia (NALM6) and Burkitt lymphoma (Raji CD80 / Cd86dko) cell lines ( Fig. 9C ).

[0322] Table 7: REGN5837 bispecific antibody enhances cytokine production induced by onituzumab treatment.

[0323]

[0324] Since CD22 is expressed in + and CD22 -The mixture of tumor cells in DLBCL patient samples is variable ( Figure 1A -C), we also evaluated whether REGN5837 enhanced T cell killing of CD22 + and CD22 - Purified T cells were incubated with CRISPR-edited CD22-deficient and CD22-wild-type WSU-DLCL2 cells and treated with a fixed concentration of onitumumab (5 pM) and a range of 4.63 x 10 -10 Up to 1.67x10 -8 REGN5837 stimulation of M. T cells with expression of CD22 + Incubation of target cells expressing CD22 with REGN5837 enhanced onitumumab-mediated tumor cell lysis; however, as expected, incubation of T cells with target cells that did not express CD22 did not result in enhanced cytotoxicity ( Figure 2G ). However, T cells with 20-80% CD22 + Mixed populations composed of targets were cultured together to induce CD22 in a REGN5837 dose-dependent manner. + and CD22 - Increased killing of target populations ( Figure 2G ) and increased CD4 and CD8 T cell activation ( Figure 2H ). Although at the highest REGN5837 concentration evaluated, CD22 - The degree of target cell lysis is related to CD22 + Target cells were different (when mixed at a 4:1 ratio, CD22 + The cell survival rate was 11%, while CD22 - The viability of cells was 21% compared with 60% without REGN5837 treatment, but even at the lowest CD22 + / CD22 - Cell ratio, CD22 + The presence of the target can also enhance the CD22 - Therefore, these data suggest that changes in CD22 expression on DLBCL tumor cells do not prevent REGN5837 from enhancing the in vitro cytotoxic activity of onitumumab.

[0325] REGN5837-mediated costimulation enhances the antitumor efficacy of onitumumab in a xenogeneic DLBCL tumor model.

[0326] The ability of REGN5837 to enhance the in vivo antitumor activity of onituzumab was evaluated in the WSU-DLCL2 tumor model, a xenogeneic DLBCL tumor in which onituzumab alone is insufficient to mediate tumor clearance, thereby mimicking patients who lack a complete response to CD3 bispecific antibody therapy. Dose titration of onituzumab showed that while the WSU-DLCL2 tumor model was initially responsive to CD20xCD3-mediated killing in vivo ( Fig. 10B and Fig. 10C ), but this treatment failed to mediate sustained tumor rejection, and all animals eventually died due to tumor burden despite treatment with high doses of onitumumab (10 mg / kg) ( Fig. 10B and 10D Therefore, we evaluated whether the addition of REGN5837 could further enhance the antitumor activity ( Figure 3A As expected, preventive treatment with REGN5837 alone had no effect on tumor growth ( Figure 3B , C). Although onitumumab monotherapy initially inhibited tumor growth ( Figure 3B , C left) and had a survival advantage compared with isotype control treated animals ( Figure 3C However, the combination of REGN5837 and onitumumab enhanced anti-tumor immunity, resulting in 86% (6 / 7) of the animals rejecting the tumor ( Figure 3B ) and significantly prolonged the overall survival of the animals compared with the monotherapy group ( Figure 3C right).

[0327] CD22xCD28-mediated co-stimulation enhances the anti-tumor efficacy of CD20xCD3 in preclinical models of B-cell malignancies

[0328] To investigate how the combination therapy enhances antitumor immunity, the modulation of intratumoral T cell responses by the different treatments was evaluated 26 days after implantation ( Fig.11A At this time, onitumumab treatment significantly reduced tumor mass, and combination therapy further suppressed tumor growth ( Fig. 11B ). High-dimensional dimensionality reduction analysis of intratumor immune subpopulations ( Figure 3D ) revealed the enrichment and depletion of certain populations in response to CD3 bispecific or combination therapy. Administration of onitumumab treatment preferentially amplified intratumoral CD4 + and CD8 + T cells, while reducing the percentage of WSU-DLCL2 tumor cells ( Fig. 11C Adding REGN5837 to onitumumab treatment further increased the frequency of T cells while reducing the frequency of tumor cells compared to monotherapy with the CD3 bispecific ( Fig. 11C ). The density of tumor and T cell subsets was counted, showing that onitumumab monotherapy significantly increased intratumoral CD4 +T The cell density ( Fig.11D ) and CD8 + The density of T cells ( Figure 3E Compared with onitumumab alone, the combination therapy further increased CD8 + The density of T cells was also reduced, while the number of WSU-DLCL2 cells per mg of tumor was reduced ( Figure 3E Examination of the induction of memory subsets revealed that treatment with onitumumab significantly promoted both effector and central memory CD8 T cells compared with isotype controls ( Figure 3F Left) and CD4 T cells ( Fig.11E Interestingly, the addition of REGN5837 significantly increased central and effector memory CD8 + The density of T cells ( Figure 3F Right), which is produced by CD8 + The density of T cell population increases ( Figure 3E Right). Combination treatment did not further alter the ratio of memory cells to naive cells.

[0329] Because of the strong synergistic effect observed when REGN5837 was combined with onitumumab for preventive treatment of WSU-DLCL2 tumors, it was decided to examine the combination efficacy in the context of delayed or curative treatment. Delaying study treatment by one week abolished the monotherapy activity of onitumumab ( Figure 4B , C), because tumor growth was no longer suppressed. However, the combination therapy still mediated a strong antitumor activity, significantly inhibiting tumor growth ( Figure 4C ), with an overall rejection rate of 40%, which represented a significant survival benefit (50% survival at 125 days after tumor implantation) compared to onituzumab alone (0% survival at 75 days). In addition, the efficacy of REGN5837 in enhancing the activity of onituzumab in a systemic tumor model of B-cell acute lymphoblastic leukemia (B-ALL) was evaluated. Immunodeficient animals were pre-implanted with human PBMCs and 12 days later, NALM-6B-ALL cells engineered to express luciferase were implanted intravenously for in vivo tracking using bioluminescent imaging (BLI). Delaying treatment until 8 days after implantation resulted in a non-significant trend toward a decrease in tumor burden in the onituzumab-treated group ( Figure 4E and Figure 4F However, the combination of REGN5837 and onitumumab induced a significant inhibition of tumor growth ( Figure 4F ).

[0330] Combination of REGN5837 with onitumumab enhances peripheral and intratumoral CD8 in human immune system reconstituted animals with DLBCL tumors + T cell response

[0331] To evaluate the in vivo efficacy of REGN5837 in a more physiologically relevant setting, WSU-DLCL2 tumors were implanted subcutaneously in the SIRPA h / h TPO h / m Rag2 - / - Iq - / - Mouse or human immune system (HIS) animal, the mouse or animal has been implanted with embryonic liver CD34 + Animals were randomized to assigned treatment groups based on fetal liver donor, frequency of human T cell engraftment, and sex. Monotherapy with 0.4 mpk of onitumumab failed to inhibit tumor growth, whereas monotherapy with CD3 bispecific antibody alone (0% survival at 64 days post-implantation) or isotype control (0% survival at 61 days post-implantation) ( Figure 5C Combination treatment with REGN5837 significantly inhibited tumor growth compared to (right) Figure 5C Left) and improved survival (80% survival 64 days after implantation). REGN5837 monotherapy also induced tumor rejection ( Figure 5B and Figure 5C ), because the basis of this antitumor response is the allogeneicity between donor T cells and WSU-DLCL2 tumor cells, which provides "signal 1". In vitro, REGN5837 enhanced T cell activation and effector function mediated by allogeneic signal 1 ( Fig. 12A and Fig. 12B ); therefore, this enhancement of antitumor activity in vivo could be attributed to co-stimulation of the alloreactive response.

[0332] To examine the possibility of peripheral T cell activation and enhanced cytokine release following combination treatment, serum cytokines were evaluated. Tumor-bearing mice with human immune systems were bled before and after dosing to evaluate the induction of lymphocyte subsets and serum cytokines. As expected, monotherapy treatment with REGN5837 showed no single-agent activity in peripheral blood, as there was no discernible T cell activation following treatment. However, monotherapy treatment with onitumumab effectively depleted circulating B cells ( Figure 5D Right) and increased CD8 + ( Figure 5D Left) T cells, a response also observed in preclinical and clinical studies in response to other CD3 bispecific antibodies. The combination of REGN5837 and onitumumab further boosted CD8 + ( Figure 5D Left) and CD4 + ( Fig.13A ) T cells. As expected, examination of serum cytokines after dosing showed that REGN5837 did not induce any serum cytokines, as there was no signal from the allogeneic tumor or from CD20xCD3 treatment 1. However, consistent with previous studies, onitumumab treatment was observed to induce serum cytokines ( Figure 5E and Fig. 13B ). Combination therapy induced significant cytokine release (TNFa, IL-2, IL-10) after the first dose compared with onitumumab monotherapy, with a trend toward increased IL-6. Overall, the increased but transient induction of serum cytokines and the expansion of T cells in peripheral blood are evidence of enhanced T cell activation induced by CD28 co-stimulation.

[0333] The combination of REGN5837 and onitumumab enhanced antitumor activity by increasing intratumoral T cell accumulation and biasing T cells toward a reprogrammable state and away from a dysfunctional phenotype.

[0334] To determine how REGN5837 enhances the antitumor activity of onitumab, immunoanalysis of intratumoral, splenic, and blood T cell responses was performed 30 days after implantation. At this time, the combination of onitumab and REGN5837 significantly inhibited tumor growth compared with animals treated with isotype and REGN5837 monotherapy, and showed a trend toward decreased tumor growth compared with onitumab monotherapy ( Fig. 13C All living cells from tumors, spleens, and blood were analyzed using FlowSOM (an unsupervised clustering algorithm) to identify distinct cell populations (metaclusters) and map these metaclusters to UMAP (Uniform Manifold Approximation and Projection for Dimensionality Reduction) maps ( Fig. 5F Left). Spleen ( Fig. 5F Right), tumor ( Fig. 5F Right) and blood ( Fig.14D )UMAP diagram showed that the relative proportions of different immune subsets in response to different treatments were significantly different. Fig.14A and Fig. 14C ) and blood( Fig. 14B and Fig.14E ), onitumumab monotherapy was sufficient to mediate significant depletion of B cells compared to isotype and REGN5837 monotherapy treatments. Importantly, the combination treatment depleted splenic B cells to a similar extent as onitumumab monotherapy, with spleen ( Fig.14A and C) and blood ( Fig. 14BMoreover, within the tumor, the combination of onitumumab and REGN5837 treatment resulted in a significant reduction in the frequency of tumor cells compared with onitumumab monotherapy ( Fig.13D Left), accompanied by intratumoral T cell expansion ( Fig.13D right).

[0335] Absolute counts of tumor cells revealed that tumor growth was significantly inhibited in response to onitumumab or combination treatment compared with isotype-treated animals ( Figure 5G Although there was no significant difference in tumor volume between combination therapy and onituzumab treatment ( Fig. 13C ) or absolute count of WSU-DLCL2 cells ( Figure 5G However, CD4 + and CD8 + T cells expanded significantly ( Figure 5G Examination of the induction of memory subsets showed that treatment with onitumumab or the combination therapy significantly promoted effector memory (EM) CD8 + ( Fig.13E ) and CD4 + ( Fig.13F ) T cells, of which more than 90% acquired the EM phenotype. Although there was a slight trend towards increased EM formation in response to combination treatment compared with onitumumab (CD8 + T cells: 96% vs. 91%; CD4 + T cells: (93% vs. 90%), but due to the overall increase in total T cells within the tumor, effector and central memory CD8 + ( Figure 13G ) and CD4 + ( Fig.13H )The density of T cells increased significantly.

[0336] To better understand how different treatments modulate intratumoral T cell phenotypes, FlowSOM was used to analyze intratumoral CD4 + and CD8 + T cell populations, and 11 metaclusters were identified ( Figure 5H Left) UMAP image shows that the combination therapy leads to intratumoral CD8 + Metacluster 1 was significantly enriched in T cells and metacluster 3 was suppressed, while CD4 + T cells are tilted toward meta-cluster 11 and away from meta-cluster 10 ( Figure 5H and Figure 5J ). Metacluster 1 represents reprogrammable (CD38 lo CD101 lo) phenotype or a reversibly dysfunctional state of CD8 + T cell populations, while metacluster 3 represents a highly proliferative but dysfunctional CD8 T cell population associated with lack of effector cytokine production and tumor progression (PD1 hi CD28 + Ki67 + )( Fig.5I ). Metacluster 11 is PD-1 + CD38 lo CD4 + T cell population, reportedly exhibited more tumor control than CD38 in metacluster 10 due to maintenance of stem cell properties and effector functions and prevention of metabolic exhaustion hi CD4 + T cells are better ( Fig.5I ). Thus, these data suggest that costimulation with a CD22xCD28 bispecific antibody in combination with onitumumab not only expands intratumoral T cells but also prevents the induction of dysfunctional T cells, thereby enhancing antitumor immunity.

[0337] Although REGN5837 monotherapy did not activate T cells in cynomolgus monkey studies, the CD28 bispecific antibody enhanced onituzumab-mediated T cell activation.

[0338] In order to evaluate the tolerance of REGN5837 alone or in combination with onitumab, a study was conducted in cynomolgus monkeys. Three monkeys in each group received a single intravenous slow push of REGN5837 alone (10mpk), onitumab alone (0.001, 0.01, 0.1 1mpk) or REGN5837 (1 or 10mpk) and then received onitumab (0.001, 0.01 or 0.1mpk). Throughout the study, the assessment of tolerance and pharmacological activity included body weight, clinical observation, veterinary physical examination (which includes the assessment of heart rate, temperature and respiratory rate), neuromuscular / musculoskeletal observation, clinical pathology (hematology, serum chemistry [including C-reactive protein], coagulation and urinalysis). In addition, 5 hours, 1 day, 4 days after administration and the 8th day after the completion of the study, blood samples were collected for cytokine analysis and immunophenotyping by flow cytometry. At autopsy, comprehensive macroscopic and microscopic evaluations were performed. Single or repeated intravenous administration of REGN5837 or onitumumab as monotherapy or combination therapy was not associated with any moribund state or early death. In addition, there were no adverse test article-related findings in clinical observations, including no signs of cytokine release syndrome (CRS) (e.g., vomiting or stool changes).

[0339] As previously described for other CD28-based co-stimulatory bispecific antibodies, treatment with REGN5837 as a single agent did not induce peripheral CD4 + or CD8 + T cell expansion or activation ( Figure 6B , C and Fig.15A -B), which is consistent with in vitro data demonstrating that CD28 bispecific antibodies do not induce T cell activation in the absence of signal 1 ( Fig. 9A In addition, REGN5837 monotherapy had no effect on peripheral B cell numbers ( Fig. 6A However, consistent with previously published data for CD3 bispecific antibodies, onitumumab induced an initial lymphocyte marginalization followed by the presentation of CD4 + ( Fig.15A , B) and CD8 + ( Figure 6B and Figure 6C )T cell expansion and activation trends, as indicated by ICOS upregulation and proliferation induction. T cell activation was accompanied by sustained depletion of peripheral B cells at all Onitumumab concentrations evaluated ( Fig. 6A ). Similar to onitumab alone, the addition of REGN5837 to onitumab effectively depleted peripheral B cells. Notably, the combination with 1 mpk of REGN5837 significantly expanded and activated peripheral CD8 + ( Figure 6B and 6C ) and CD4 + T( Fig.15A and 15B ) cells. While combination therapy with 10 mpk of REGN5837 also induced a trend toward enhanced onitumumab-mediated T cell activation, the addition of the co-stimulatory antibody at this concentration did not significantly enhance T cell activity to the same extent as REGN5837 administered at 1 mpk. This may be due to the single-arm binding of the CD28 bispecific molecule at high concentrations, resulting in a bell-shaped curve when examining T cell activation parameters.

[0340] Onitumumab monotherapy resulted in a dose-dependent increase in serum cytokines ( Fig.6D ), which correlates with previous observations from other tumor-targeting CD3 bispecific antibodies. The combination of onitumumab (0.1 mpk) and REGN5837 (1 mpk) significantly increased IL-2 production compared with placebo, and there was a trend toward increased serum IL-6, reflecting the increased T cell activation observed with the combination treatment ( Figure 6C and Fig. 15B). Notably, the combination of 0.1 or 0.01 mpk of onitumab and REGN5837 (1 mpk) did not induce IL-6 as strongly as 1 mpk of onitumab alone; however, the combination therapy expanded and activated CD4 + and CD8 + The extent of T cells was similar to that of 1 mpk of onitumab monotherapy ( Figure 6B and Fig.15A ). Overall, these results suggest that the addition of REGN5837 to onitumumab enhances T cell activation in cynomolgus monkeys by inducing increases in serum cytokines and expanding peripheral T cells.

[0341] in conclusion

[0342] Here we demonstrate that REGN5837 can enhance onitumab-mediated activity by promoting in vitro cytotoxicity against DLBCL and other NHL cell lines and by effectively enhancing antitumor activity against DLBCL tumors that could not be cleared by CD3 bispecific antibody treatment. While onitumab inhibited WSU-DLCL2 tumor growth as a single agent, only combination treatment with REGN5837 resulted in a curative response and improved overall survival in preclinical in vivo models. Furthermore, the combination of REGN5837 with onitumab not only maintained cytotoxicity against CD20-expressing cells, but also resulted in significant expansion of intratumoral T cells and enhanced induction of effector memory cells compared to onitumab monotherapy. Further characterization of the intratumoral immune compartment from HIS mice revealed that the addition of CD28 co-stimulation to onitumab resulted in a curative response in CD8 + T cell populations are biased towards a reprogrammable phenotype (CD38 lo CD101 lo ), or a plastic dysfunctional state that can be reversed under appropriate conditions, allowing the reactivated T cells to produce high levels of proinflammatory cytokines and mediate antitumor immunity. This expansion is accompanied by dysfunctional CD28 + Ki67 + CD8 + The reduction in induction of T cells, a hyperactivated cell population that has previously been described as lacking effector function, is amplified in melanoma and advanced NSCLC and is associated with resistance to PD-1 blockade therapy. Addition of REGN5837 also amplified PD-1. + CD38 lo CD4 + T cell populations reportedly have CD38 hiThese cells exhibited superior tumor control compared to T cells, which was due to the maintenance of effector function, promotion of T cell persistence, and metabolic reprogramming driven by enhanced glutaminolysis. Thus, these data suggest that REGN5837 can not only expand intratumoral T cells but also prevent the induction of dysfunctional T cells, thereby enhancing antitumor immunity.

[0343] Toxicology studies in cynomolgus monkeys and analysis of peripheral blood from HIS mice showed that despite the expression of CD22 on peripheral B cells, REGN5837 had low activity as a monotherapy and did not exhibit toxicity due to the lack of signal 1. Peripheral T cell activation and expansion were only observed when REGN5837 was administered in combination with onitumab, which mediates TCR aggregation. Notably, in the cynomolgus monkey study, the addition of REGN5837 to a low dose of onitumab was able to mediate peripheral T cell expansion at levels similar to 10-100-fold higher doses of CD3 bispecifics; however, this expansion was associated with lower induction of serum cytokines compared to high-dose monotherapy treatment with CD3 bispecifics in the cynomolgus monkey study. Therefore, treatment with either combination or high-dose CD3 bispecific antibodies was well tolerated in animals without any signs of cytokine release syndrome (CRS).

[0344] Onitumab is currently being evaluated in Phase 1 and Phase 2 clinical trials for the treatment of r / r B-cell NHL, where the patient population has received a large number of pre-treatments and has failed at least 2 previous lines of treatment. Results from early clinical trials of Onitumab showed encouraging activity for patients with refractory follicular lymphoma who received at least 5 mg of Onitumab weekly, with an ORR of 92.9% and a complete response rate (CR) of 75.0%. The ORR and CR rates of r / r DLBCL patients who had received a large number of pre-treatments, dosed at >=80 mg and had not received previous CAR-T therapy were 60%, while the ORR of patients refractory to CAR-T therapy was 33.3% and the CR rate was 23.8%. Overall, safety appears to be manageable and consistent with the CD20x CD3 bispecific antibody class. Despite the promising prospects, these data reveal that there is still room for improvement in the treatment of r / r DLBCL patients, especially in the setting after CAR-T therapy. Collectively, these data suggest that the novel CD22xCD28 bispecific antibody REGN5837 can enhance the antitumor activity of onitumab and provide support for studying REGN5837 together with onitumab in patients with difficult-to-treat aggressive lymphomas.

[0345] Example 2: Materials and methods of Example 1

[0346] Study Design

[0347] This study demonstrates that tumor-targeting CD28 bispecific antibodies can enhance onitumumab-mediated T cell activation to enhance antitumor activity. Control and experimental treatments were administered to age- and sex-matched mice. Sample size was chosen empirically to ensure adequate statistical power and to meet site standards for the techniques used in the study. All animals were randomized to different treatment groups based on tumor volume before treatment initiation, and the investigators were blinded to all treatment groups. The number of experimental replicates is indicated in the figure legends.

[0348] Cell lines

[0349] For WSU-DLCL2 studies, 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). TM Transfection system 100 μL kit (Invitrogen), by using TrueCut TM Cas9 protein v2 (Invitrogen) and high-scoring TrueGuide targeting human CD22 TM Synthetic sgRNA (Invitrogen guide RNA: CCGGTGCACCTCAATGACAG) was electroporated with Cas9 ribonucleoprotein (RNP) to generate CRISPR-edited CD22-deficient cell lines. CD22-deficient cells were bulk sorted 96 hours after electroporation.

[0350] For NALM6-luc tumor experiments, 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).

[0351] Animal studies

[0352] All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Regeneron Pharmaceuticals Institutional Animal Care and Use Committee (IACUC), and all animals were housed under pathogen-free conditions.

[0353] For NSG experiments, WSU-DLCL2 cells (3x10 6 cells) and compared them with 5x10 5 PBMCs (ReachBio) were mixed and then resuspended in a 1:1 mixture of PBS and GFR Matrigel (Corning). Female NSG mice (Jackson Laboratories) were injected subcutaneously in the right flank. For human immune system reconstitution experiments, WS U-DLCL2 cells (3x10 6 SIRPA cells) h / h TPO h / m Rag2 - / - Iq - / - Mice implanted with embryonic liver CD34 + Cells; Animals were separated so that the distribution of embryonic liver donors, frequency of human immune cell implantation, and sex of each treatment group were similar. Mice were randomly assigned to receive blind treatment of isotype controls (EGFRV3xCD3 or MUC16xCD28) or test articles (REGN5837, Onitumumab), which were administered by intraperitoneal injection at a specified concentration on the 1st, 8th, and 15th days after implantation as monotherapy or in combination for preventive treatment, and therapeutic treatment was performed on the 8th, 15th, and 22nd days. Tumor growth was monitored over time using X and Y diameter (vertical measurements of length and width) digital caliper (VWR) measurements. Tumor volume (X*Y*(X / 2) where X is the shorter diameter) was calculated. When the tumor reached the specified tumor endpoint (tumor diameter>20mm, or tumor ulceration), the mice were euthanized. The specified endpoint met IACUC standards.

[0354] For NALM6-luc tumor experiments, female immunodeficient NSG mice (Jackson Laboratories) were implanted with 4x10 6 5x10 PBMCs were injected intravenously into the successfully engrafted animals 12 days after PBMC engraftment. 6NALM6-luc cells. Mice were randomly assigned to receive blinded treatment with isotype controls (EGFRV3xCD3 or MUC16xCD28) or test articles (REGN5837, Onitumumab), which were administered as monotherapy or in combination at specified concentrations by intraperitoneal injection on days 8, 15, and 22 after implantation. Mice implanted with NALM6-luc were imaged twice a week using IVIS Spectrum (Perkin Elmer) after intraperitoneal injection of substrate luciferin (PerkinElmer). Bioluminescence (total flux) was quantified using Living Image software. The experiment ended when mice began to show signs of GVHD that met IACUC standards (weight loss>=20%).

[0355] Measurement of serum cytokines in mice

[0356] At the indicated time points, blood was collected from the submandibular vein into micro serum tubes (BD 365967). Cytokine concentrations were analyzed using the V-plex Human ProInflammatory-10Plex ​​kit according to the manufacturer's instructions (Meso Scale Diagnostics).

[0357] Flow cytometry

[0358] For immunophenotyping experiments, tumors, spleens and blood were collected on the specified date. Single cell suspensions were prepared and live / dead cell identification was performed using the Live / Dead Fixable Blue dead cell staining kit (Thermo Fisher Scientific). In order to quantify the number of cells in tissues, a fixed number of CountBright absolute counting beads (Thermo Fisher Scientific) were added to each sample before collection. Samples were obtained on Symphony (BD Biosciences) and analyzed using FlowJo (TreeStar) or OMIQ.

[0359] To automatically identify T cell clusters based on selected markers, OMIQ’s FlowSOM was run on samples acquired on Symphony (BD Biosciences). The same number of events was analyzed for each sample. The range of events was determined by the sample with the fewest cells. Intratumoral CD8 in Omiq.ai +FlowSOM clustering of T cells was run with default settings using selected parameters (activation / dysfunction and memory markers). To visualize the clusters identified by FlowSOM, the high-dimensional dimensionality reduction method UMAP was run on all samples in Omiq.ai. Samples from each treatment group were concatenated and the FlowSOM clusters were superimposed on the UMAP plot.

[0360] Wet plate coating assay

[0361] Antibodies diluted to 10 μg / ml in PBS were wet coated on polypropylene plates overnight. Diluted antibodies (100 μμl) were added to 96-well polypropylene assay plates in triplicate. The plates were stored overnight at 4°C to allow antibody adsorption. The plates were washed twice with PBS before being used for proliferation assays. PBMCs were isolated from leukocyte-rich peripheral blood (New York Blood Center) obtained from four healthy individual donors. PBMCs from each donor were resuspended in RPMI culture medium (Irvine Scientific) containing 10% human AB serum (GemCell) and penicillin / streptomycin / glutamine (respectively 100 units / ml, 100 μg / ml, 292 μg / ml, Gibco), and then added to 96-well assay plates at 100,000 cells / well, with a final volume of 200 μl / well. The assay plates were then incubated at 37°C+5% CO2 for 54 hours. At 54 hours, the 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 determined using the V-PLEX Proinflammatory Panel 1 Human kit according to the manufacturer's instructions. The mean and range of concentrations obtained from 4 donors (pg / mL) are plotted, with individual data points representing the mean concentration of each individual donor obtained from assays performed in triplicate wells.

[0362] To evaluate proliferation, 100 μl of 1 mCi / ml tritiated thymidine (Perkin Elmer) was added to each well and incubated at 37°C + 5% CO 2 The cells were incubated for 18 hours at 4 °C for 24 h. Each 96-well assay plate was harvested using a Filtermate Harvester (Perkin Elmer) and analyzed on a TopCount scintillation counter (Perkin Elmer). The amount of radioactivity was measured as counts per minute per well (CPM) and was proportional to the number of proliferating cells. The CPM values ​​were presented as four data points representing the average counts of each individual donor obtained from the assays performed in the three replicate wells.

[0363] In vitro T cell activation assay

[0364] 50mL SepMate will be used TM A tube of previously frozen human CD3 isolated from healthy donor leukocytes by density gradient centrifugation + T cells were thawed in stimulation medium (X-VIVO15 cell culture medium supplemented with 10% FBS, HEPES, NaPyr, NEAA, and 0.01 mM BME) containing 50 U / ml benzonase on the day of the assay and plated at 1x10 5 NALM6 cells were treated with 10 μg / mL mitomycin C in primary stimulation medium at a concentration of 10×10 6 cells / mL. At 37°C, 5% CO 2 After incubation for 1 h at 4 °C, the mitomycin C-treated cells were washed three times with D-PBS containing 2% FBS and then plated at 5 × 10 4 The final concentration of cells was added to the CD3 + T cells were added to the wells. An irrelevant hIgG1 mAb (100nM / well) was added to all wells to block Fc receptors. A fixed concentration of onituzumab or 500pM of a non-binding isotype control was added to the wells along with dose titrations of REGN5837 or a non-binding isotype control from 3.1pM to 200nM. The plates were incubated at 37°C and 5% CO2 for 72 hours, at which time 50μl of culture supernatant was collected. 5μl of this collected supernatant was tested according to the manufacturer's protocol for the human IL-2 (AL221F) AlphaLISA assay (Perkin Elmer). Measurements were obtained using an Envision multi-label plate reader (Perkin Elmer). A standard curve of known concentrations was generated to infer the concentration of IL-2 produced in the assay wells. To assess T cell proliferation, the culture medium was supplemented with a final concentration of 1.25μM [ 3 H] thymidine (Perkin Elmer), and the cells were incubated for 16 h at 37°C, 5% CO2. The plates were harvested using a Microbeta Filermat-96 cell harvester (Perkin Elmer), 30 μL MicroScint-20 (Perkin Elmer) was added, and the [ 3H] Thymidine incorporation. All serial dilutions were tested in triplicate. EC50 values ​​of the antibodies were determined by a four-parameter logistic equation on a 10-point dose-response curve using GraphPad Prism software.

[0365] In vitro cytotoxicity assay

[0366] Human PBMCs were thawed and cultured at 1x10 6 The density of cells / mL was inoculated in complete medium (RPMI cell culture medium supplemented with 10% FBS, penicillin-streptomycin-glutamine), and incubated overnight at 37°C to enrich lymphocytes by consuming adherent cells (such as macrophages, dendritic cells and some monocytes). The next day, PBMCs were harvested and labeled with 1 μM of Violet Cell Tracker fluorescent tracer dye. WSU-DLCL2 cells were labeled with 1 μM of fluorescent dye VybrantCFDA-SE. After labeling, 5,000 labeled target cells were inoculated in a round-bottom 96-well plate at a ratio of 1:5 with the labeled PBMCs. The serial dilutions of R5837 were combined with the serial dilutions of R1979 and added to the labeled target cells and effector cells, and the plates were then incubated at 37°C for 72 hours. After incubation, cells were washed and stained with LIVE / DEAD dye in PBS and then stained with a cocktail of fluorophore-labeled antibodies against CD2, CD4, CD8 and CD25 to analyze viable target cells, T cell activation and proliferation. Counting beads (20 μL per well) were added immediately before sample analysis on a BD Celesta flow cytometer.

[0367] Target cell killing was assessed by counting the number of live, CFDA-SE labeled target cells / well (normalized to the number of beads collected / well). Percent viability was normalized to the number of live target cells in control conditions (target cells in the presence of PBMCs alone). T cell activation was assessed by reporting CD25% on CD4+ and CD8+ T cells. T cell proliferation was assessed by reporting the percentage of cells with reduced MFI of the Violet Cell Tracker dye.

[0368] The supernatant of this assay is collected to analyze cytokine levels. According to the manufacturer's instructions, the concentration of IL 17a, IFNγ, TNFα, IL-10, IL-6, IL-4 and IL-2 is analyzed using flow cytometry bead array (Cytometric Bead Array, CBA) test kit. In brief, at room temperature, 1 / 15 dilution of supernatant is incubated 3 hours in 96-well assay plates together with standard samples and cytokine specific bead arrays. After incubation, the sample is washed twice, and then analyzed by flow cytometry on BD FACS CantoII. Cytokine levels are pushed inward by the MFI of the test kit standard, and are reported as pg / mL.

[0369] EC50 values ​​were determined by a four-parameter logistic equation on a 9-point dose-response curve using GraphPad Prism software. The maximum responses for percent cytotoxicity, T cell activation, proliferation, and cytokine release were considered plateau values ​​generated by Prism curve fitting. The fold change in EC50 was calculated as EC50. NoR5837 / EC50 [M]R5837 , and the fold change of maximum cytokine release was calculated as Max [M]R5837 / Max NoR5837 .

[0370] Chromogenic IHC

[0371] Chromogenic IHC assays were performed on formalin-fixed, paraffin-embedded DLBCL patient samples (Tristar) on the Ventana Discovery ULTRA platform. Antigen retrieval was performed using Tris-EDTA (pH 9) buffer, and slides were incubated with the primary antibodies listed below. The OptiView DAB detection system was used, and for some markers, an additional amplification step was required to visualize the signal. Slides were counterstained with Novolink hematoxylin (Leica Microsy stems, Inc.) and coverslips (Corning Coverglass, #1) were covered with Cytoseal 60 (Thermo Scientific / RichardAllen Scientific). Once the mounting medium was completely dry, slides were scanned at 40x on a Leica Aperio AT2 scanner. Use

[0372] Images were analyzed using Indica HALO software.

[0373] Table 8

[0374]

[0375] Multiplex IHC

[0376] Fully automated multiplex immunohistochemistry assays were performed on the Ventana Discovery ULTRA platform (Ventana Medical Systems, Tucson, AZ). Five consecutive rounds of application of primary and secondary antibody-horseradish peroxidase conjugated antibodies were performed. Heat denaturation was performed between each step to completely remove the bound primary and secondary antibodies, thereby eliminating downstream cross-reactions. This allows the use of primary antibodies produced in the same species. The fluorescent dyes used were carefully selected to ensure spectral separation and provide optimal staining. The combination and application order 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, applied in the following order and detected with the indicated fluorophores.

[0377] Table 9

[0378]

[0379]

[0380] After staining, the tissue was counterstained and the coverslip was sealed with Invitrogen Pro Long Gold Antifade Mountant containing NucBlue. Whole slide imaging was performed on a Zeiss Axioscan equipped with a Colibri light source and appropriate filters for visualizing these specific fluorophores. Quantitative image analysis was performed using the HALO Indica Labs Hyperplex module (Indica Labs, Albuquerque, NM). The number of positive cells for each immune subset and the density of the entire tumor area were measured.

[0381] Toxicology study in cynomolgus monkeys

[0382] The cynomolgus monkey study was conducted in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care, with Animal Welfare Assurance issued by the Office of Laboratory Animal Welfare and registered with the USDepartment of Agriculture and IACUC. The study was conducted at Altasciences Preclinical Seattle (formerly SNBL USA) using male cynomolgus monkeys (Macacafascicularis) (three animals per group). Subjects received a single dose of each test article by intravenous push (REGN5837) or intravenous infusion (onitumumab, infusion time of about 30 minutes). Combination therapy was administered sequentially by starting onitumumab infusion 5-10 minutes after REGN5837 administration. Evaluations of treatment-related effects included body weight measurements, clinical observations, veterinary physical examinations (including assessment of heart rate, temperature, and respiratory rate), neuromuscular / musculoskeletal observations, clinical pathology (hematology, collection of blood and / or tissue samples for cytokine analysis, immunophenotyping, histopathology, and toxicokinetic evaluation. For peripheral blood flow cytometry, blood was collected into potassium EDTA tubes; lysed; stained with CD3, CD4, CD8, CD14, CD16, CD20, CD28, and Ki67 (BD Biosciences) and CD278 (BioLegend); and then analyzed using a FACSCanto II flow cytometer. For cytokine analysis, blood was collected into serum separation tubes containing anticoagulant. Serum was separated by centrifugation at 1000 g to 2000 g for 10 to 15 minutes at 4°C and then analyzed using the MSD U-Plex platform (IL-1β, IL-2, IL-6, MCP-1, TNF-α, and IFN-γ).

[0383] Statistical analysis

[0384] Sample size was chosen empirically to ensure adequate statistical power and to meet the technical standards used in the study. Statistical significance was determined using one-way and two-way analysis of variance (ANOVA) and the Kaplan-Meier method with long-rank test to compare survival rates between groups. Graph generation and statistical analysis were performed using GraphPad Prism (version 8).

[0385] Example 3: REGN5837 enhances cytotoxicity mediated by human T cells activated by the CD20xCD3 bispecific antibody REGN1979 against B-cell lymphoma cells expressing or not expressing surface CD22.

[0386] T cells were isolated from freshly thawed PBMCs using the EasySep Human T Cell Isolation Kit and used immediately. WT Cells were labeled with 1 μM of the fluorescent dye Vybrant CFDA-SE. KO The cells were labeled with 1 μM fluorescent dye CellTrace Far Red. WT and WSU-DLCL2 / CD22 KO Target cells were seeded in round-bottom 96-well 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 different concentrations of REGN5837 (16.7nM, 2.77nM, 463pM), and the plates were incubated at 37°C for 72 hours.

[0387] After incubation, cells were washed and stained with LIVE / DEAD dye in PBS and then stained with a cocktail of fluorophore-labeled antibodies against CD4, CD8 and CD25 to analyze T cell activation. Counting beads (20 μL per well) were added immediately before sample analysis on a BD Celesta flow cytometer.

[0388] WSU-DLCL2 / CD22 was assessed by counting the number of live CFDA-SE labeled target cells / well (normalized to the number of collected beads / well). WT Killing. WSU-DLCL2 / CD22 was assessed by counting the number of live Far-Red labeled target cells / well (normalized to the number of beads collected / well). KO Killing. Percent viability was normalized to the number of live target cells in control conditions (target cells in the presence of effector cells only). T cell activation was assessed by reporting the MFI of CD25 on CD4+ and CD8+ T cells.

[0389] Results Overview:

[0390] The ability of REGN5837 to enhance cytotoxicity mediated by human T cells activated by the CD20xCD3 bispecific antibody REGN1979 against B-cell lymphoma cells expressing or not expressing surface CD22 was evaluated using flow cytometry. In addition, T cell activation was assessed, as measured by upregulation of CD25 on T cells.

[0391] 5pM REGN1979 activated and directed human T cells to kill WSU-DLCL2 / CD22 to a similar extent WT and WSU-DLCL2 / CD22 KO (Table 8). KO :CD22 WT Regardless of the ratio of cells, T cell activation was similar in the presence of only 5 pM REGN1979 (Table 11).

[0392] REGN5837 enhances REGN1979-mediated inhibition of WSU-DLCL2 / CD22 in a dose-dependent manner WT Cell killing; regardless of the presence of WSU-DLCL2 / CD22 in the culture KO , for WSU-DLCL2 / CD22 WT The cell killing was similar (Table 10).

[0393] In the presence of 20% or more WSU-DLCL2 / CD22 WT In cultured cells, REGN5837 enhanced REGN1979-mediated inhibition of WSU-DLCL2 / CD22 KO Cell killing. In the absence of any WSU-DLCL2 / CD22 WT In the case of WSU-DLCL2 / CD22 cells, REGN5837 did not enhance REGN1979-mediated KO Cell killing (Table 10).

[0394] In the presence of WSU-DLCL2 / CD22 WT In cultured cells, REGN5837 enhanced REGN1979-mediated T cell activation in a dose-dependent manner, with WSU-DLCL2 / CD22 WT In the absence of any WSU-DLCL2 / CD22 WT In the case of cells, REGN5837 did not enhance REGN1979-mediated T cell activation (Table 11).

[0395] In conclusion, REGN5837 costimulation enhanced REGN1979-mediated killing of target cells lacking CD22 expression as long as CD22-expressing cells were present in culture.

[0396] Table 10: WSU-DLCL2 / CD22 KO and WSU-DLCL2 / CD22 WT Survival rate%

[0397]

[0398] Table 11: T cell activation (CD25 upregulation)

[0399]

[0400]

[0401] Incorporated by Reference

[0402] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0403] Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences that reference accession numbers associated with entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web.

[0404] Equivalence Case

[0405] 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. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method for mediating killing of tumor cells in a tumor of a subject, 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, wherein the tumor cells do not express or are predicted not to express the target antigen.

2. A method for inducing killing of tumor cells in a subject's tumor, the method 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, wherein at least one subpopulation of the tumor cells does not express the target antigen.

3. A method for inducing T cell activation against tumor cells in a subject's tumor, the method 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, wherein at least one subpopulation of the tumor cells does not express the target antigen.

4. A method for treating cancer in a subject having a tumor, the method 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, wherein at least a subpopulation of tumor cells in the tumor does not express the target antigen.

5. The method of any one of claims 1-4, wherein the method further comprises determining that at least the subpopulation of tumor cells in the tumor does not express the target antigen, optionally wherein if the expression of the target antigen is below the detection level or below the signal-to-noise ratio, the tumor cells do not express the target antigen.

6. A method of treating cancer in a subject, comprising i) determining that the subject comprises a tumor comprising tumor cells that do not express the 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 CD28 protein.

7. A method of selecting a subject for cancer therapy, the method comprising: i) determining that the subject contains a tumor containing tumor cells that do not express the 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 CD28 protein, optionally wherein if expression of the target antigen is below the level of detection or below the signal-to-noise ratio, the tumor cells do not express the target antigen, thereby selecting the subject for cancer therapy.

8. The method of any one of claims 1 to 7, wherein the target antigen is: i) tumor-associated antigens (TAA), ii) an antigen associated with the tumor microenvironment of the tumor, optionally wherein the antigen associated with the tumor microenvironment is an antigen associated with 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) Immunizing antigens.

9. The method of claim 8, wherein the target antigen is a TTA selected from CD38, EGFR, CD22, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7.

10. The method of claim 9, wherein the target antigen is CD22.

11. The method of claim 8, wherein the target antigen is an antigen associated with a tumor stroma selected from the group consisting of PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin-EDB isoform.

12. The method of claim 8, wherein the target antigen is an antigen associated with the extracellular matrix of the tumor selected from the group consisting of connexin, versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) protein.

13. The method of claim 8, wherein the target antigen is an antigen associated with cancer-associated fibroblasts selected from 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.

14. The method of claim 8, wherein the target antigen is an antigen associated with blood vessels in the tumor microenvironment selected from DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1 and VEGFR2.

15. The method of claim 8, wherein the immunizing antigen is an antigen expressed on the surface of an immune cell, wherein the immune cell is selected from the group consisting of macrophages, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, natural killer cells, T cells, and B cells.

16. The method of claim 15, wherein the immune cells have infiltrated the tumor or the tumor microenvironment of the tumor.

17. The method of claim 8, wherein the target antigen is an immune antigen selected from any one of the immune antigens listed in Table 4.

18. The method of any one of claims 1 to 17, wherein the tumor comprises immune cells.

19. The method of claim 18, wherein the immune cells are B cells and / or cells expressing CD20.

20. The method of any one of claims 1 to 17, wherein the tumor cells that do not express the target antigen also do not express CD20.

21. The method of claim 8, wherein the target antigen is TTA and the tumor is a heterogeneous tumor that also contains tumor cells expressing the TAA.

22. The method of any of the preceding claims, wherein the tumor microenvironment of the tumor comprises cells expressing the target antigen.

23. The method of any of the preceding claims, wherein at least 20% of the tumor cells in the tumor do not express the target antigen.

24. The method of claim 23, wherein at least 30% of the tumor cells in the tumor do not express the target antigen.

25. The method of claim 23, wherein at least 40% of the tumor cells in the tumor do not express the target antigen.

26. The method of claim 23, wherein at least 50% of the tumor cells in the tumor do not express the target antigen.

27. The method of claim 23, wherein at least 60% of the tumor cells in the tumor do not express the target antigen.

28. The method of claim 23, wherein at least 70% of the tumor cells in the tumor do not express the target antigen.

29. The method of claim 23, wherein at least 80% of the cells in the tumor do not express the target antigen.

30. The method of claim 23, wherein at least 90% of the cells in the tumor do not express the target antigen.

31. The method of claim 23, wherein 100% of the cells in the tumor do not express the target antigen.

32. The method of any one of claims 1 to 21, wherein at least 10%-100% of the tumor cells in the tumor do not express the target antigen.

33. The method of claim 32, wherein at least 30%-100% of the tumor cells in the tumor do not express the target antigen.

34. The method of claim 32, wherein at least 50%-100% of the tumor cells in the tumor do not express the target antigen.

35. The method of claim 32, wherein at least 70%-100% of the tumor cells in the tumor do not express the target antigen.

36. The method of claim 32, wherein at least 90%-100% of the tumor cells in the tumor do not express the target antigen.

37. The method of any one of claims 1-36, wherein the multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T-cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), or a tandem diabody (TandAb).

38. The method of any one of claims 1 to 7, wherein the multispecific antigen-binding molecule is a bispecific antibody selected from the bispecific antibodies listed in Table 3.

39. The method of any one of claims 1 to 36, wherein the multispecific antigen-binding molecule is administered to the subject in combination with a second multispecific antigen-binding molecule having a first antigen-binding region specific for a second target antigen and a second antigen-binding region specific for a CD3 protein.

40. The method of claim 39, wherein the second multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), and a tandem diabody (TandAb).

41. The method of claim 39, wherein the second target antigen is selected from any one of the TAAs listed in Table 2.

42. The method of claim 39, wherein the second target antigen is CD20 protein.

43. The method of claim 39, wherein the second multispecific antigen-binding molecule is selected from any one of the multispecific antigen-binding molecules in Table 5.

44. The method of any one of claims 39 to 43, wherein the multispecific antigen-binding molecule exhibits a co-stimulatory effect when administered in combination with the second multispecific antigen-binding molecule.

45. The method of claim 44, wherein the co-stimulatory effect is one or more of: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in PBMCs, and increasing T cell-mediated cytotoxicity.

46. ​​The method of any one of claims 1 to 45, wherein the tumor cell is a tumor cell of a B-cell cancer.

47. The method of claim 46, wherein the B cell cancer 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 (Waldenstrom's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma).

48. The method of any one of claims 1 to 45, wherein the tumor is a solid tumor.

49. The method of claim 48, wherein the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a hematogenous tumor, a brain / CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing's tumor, an eye tumor, a gallbladder tumor, a gastrointestinal tumor, a kidney tumor, a laryngeal or hypopharyngeal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymic tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms' tumor.

50. The method of any one of claims 1 to 49, wherein the multispecific antigen-binding molecule is administered systemically, intravenously, subcutaneously, or intramuscularly.

51. The method of any of the preceding claims, wherein the multispecific antigen-binding molecule is administered to the subject in a pharmaceutically acceptable formulation.

52. The method of any of the preceding claims, wherein the method further comprises administering an additional anti-cancer agent.

53. The method of claim 52, wherein the additional anti-cancer agent is a chemotherapeutic agent, an immune checkpoint inhibitor, a tumor vaccine, or a cell therapy, optionally wherein the cell therapy comprises CAR-T cells, natural killer cells, or macrophages.

54. The method of claim 53, wherein the additional anti-cancer agent is an immune checkpoint inhibitor.

55. The method of claim 54, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, or an anti-LAG3 antibody.

56. The method of any one of claims 1 to 55, wherein the subject has a refractory cancer.

57. A method of treating cancer in a subject having a tumor, 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 a 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 a CD3 protein, Wherein the first target antigen and the second target antigen are not the same antigen.

58. A method of inducing tumor cell killing in a tumor in a 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 a 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 a CD3 protein, Wherein the first target antigen and the second target antigen are not the same antigen.

59. A method of inducing T cell activation against tumor cells in a tumor of a 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 a 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 a CD3 protein, Wherein the first target antigen and the second target antigen are not the same antigen.

60. The method of claims 57-59, further comprising determining that the tumor comprises at least a subpopulation of tumor cells that do not express the first target antigen.

61. The method of any one of claims 57 to 60, wherein at least a subpopulation of tumor cells in the tumor do not express the first target antigen.

62. The method of claim 61, wherein at least 10% of the tumor cells in the tumor do not express the first target antigen.

63. The method of claim 61, wherein at least 20% of the tumor cells in the tumor do not express the first target antigen.

64. The method of claim 61, wherein at least 30% of the tumor cells in the tumor do not express the first target antigen.

65. The method of claim 61, wherein at least 40% of the tumor cells in the tumor do not express the first target antigen.

66. The method of claim 61, wherein at least 50% of the tumor cells in the tumor do not express the first target antigen.

67. The method of claim 61, wherein at least 60% of the tumor cells in the tumor do not express the first target antigen.

68. The method of claim 61, wherein at least 70% of the tumor cells in the tumor do not express the first target antigen.

69. The method of claim 61, wherein at least 80% of the tumor cells in the tumor do not express the first target antigen.

70. The method of claim 61, wherein at least 90% of the tumor cells in the tumor do not express the first target antigen.

71. The method of claim 61, wherein 100% of the tumor cells in the tumor do not express the first target antigen.

72. The method of claim 61, wherein at least 10%-100% of the tumor cells in the tumor do not express the first target antigen.

73. The method of claim 61, wherein at least 30%-100% of the tumor cells in the tumor do not express the first target antigen.

74. The method of claim 61, wherein at least 50%-100% of the tumor cells in the tumor do not express the first target antigen.

75. The method of claim 61, wherein at least 70%-100% of the tumor cells in the tumor do not express the first target antigen.

76. The method of claim 61, wherein at least 90%-100% of the tumor cells in the tumor do not express the first target antigen.

77. The method of any one of claims 57 to 76, wherein the first target antigen is: i) tumor-associated antigens (TAA), ii) an antigen associated with the tumor microenvironment of the tumor, optionally wherein the antigen associated with the tumor microenvironment is an antigen associated with 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) Immunizing antigens.

78. The method of claim 77, wherein the first target antigen is a TTA selected from CD38, EGFR, CD22, MUC16, PSMA, CA9, FOLR1, HER2, and SLAMF7.

79. The method of claim 78, wherein the first target antigen is CD22.

80. The method of claim 77, wherein the first target antigen is an antigen associated with the tumor stroma selected from the group consisting of PSA, CEA, CA-125, CA-19, COL10, FAP, B7H3, LRRC15, and fibronectin-EDB isoform.

81. The method of claim 77, wherein the first target antigen is an antigen associated with the extracellular matrix of the tumor selected from connexin, versican (VACN), fibronectin, and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) proteins.

82. The method of claim 77, wherein the first target antigen is an antigen associated with cancer-associated fibroblasts selected from 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.

83. The method of claim 77, wherein the first target antigen is an antigen associated with blood vessels in the tumor microenvironment selected from DLK1, EphA2, HBB, NG2, NRP1, NRP2, PDGFRβ, PSMA, RGS5, TEM1, VEGFR1 and VEGFR2.

84. The method of claim 77, 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.

85. The method of claim 84, wherein the immune cells have infiltrated the tumor or the tumor microenvironment of the tumor.

86. The method of claim 84, wherein the first target antigen is an immune antigen selected from any one of the immune antigens listed in Table 4.

87. The method of any one of claims 57 to 76, wherein the first target antigen is selected from any one of the antigens listed in Table 2.

88. The method of any one of claims 57 to 87, wherein at least a subpopulation of tumor cells in the tumor do not express the second target antigen.

89. The method of any one of claims 57 to 88, wherein the tumor is a heterogeneous tumor comprising cells expressing the first target antigen and cells expressing the second target antigen.

90. The method of claim 89, wherein the tumor cells in the tumor do not express both the first target antigen and the second target antigen.

91. The method of any one of claims 57 to 90, wherein the first multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T-cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), or a tandem diabody (TandAb).

92. The method of any one of claims 57 to 90, wherein the first multispecific antigen-binding molecule is selected from the bispecific antibodies listed in Table 3.

93. The method of any one of claims 57 to 92, wherein the second target antigen is selected from any one of the antigens listed in Table 2.

94. The method of claim 93, wherein the second target antigen is CD20.

95. The method of any one of claims 57 to 92, wherein the second target antigen is an immuno-tumor antigen.

96. The method of claim 95, wherein the immuno-tumor antigen is CD22, CD20, CD72, CD19, CD21, CD24 or CD79.

97. The method of any one of claims 57 to 96, wherein the second multispecific antigen-binding molecule is a bispecific antibody or a bispecific antibody fragment, such as a bispecific T cell engaging antibody (BiTE), a dual affinity retargeting molecule (DART), or a tandem diabody (TandAb).

98. The method of any one of claims 57 to 97, wherein the second multispecific antigen-binding molecule is selected from the bispecific antibodies listed in Table 5.

99. The method of any one of claims 57 to 98, wherein the first multispecific antigen-binding molecule exhibits a co-stimulatory effect when administered in combination with the second multispecific antigen-binding molecule.

100. The method of claim 99, wherein the co-stimulatory effect is one or more of: activating T cells, inducing IL-2 release, inducing CD25+ upregulation in PBMCs, and increasing T cell-mediated cytotoxicity.

101. The method of any one of claims 57 to 100, wherein the tumor is from a B cell cancer.

102. The method of claim 101, wherein the B cell cancer 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 (Waldenstrom's macroglobulinemia), hairy cell leukemia, primary central nervous system (CNS) lymphoma, or primary intraocular lymphoma (ocular lymphoma).

103. The method of any one of claims 57 to 100, wherein the tumor is a solid tumor.

104. The method of claim 103, wherein the tumor is an adenocarcinoma, an adrenal tumor, an anal tumor, a bile duct tumor, a bladder tumor, a bone tumor, a hematogenous tumor, a brain / CNS tumor, a breast tumor, a cervical tumor, a colorectal tumor, an endometrial tumor, an esophageal tumor, an Ewing's tumor, an eye tumor, a gallbladder tumor, a gastrointestinal tumor, a kidney tumor, a laryngeal or hypopharyngeal tumor, a liver tumor, a lung tumor, a mesothelioma tumor, a multiple myeloma tumor, a muscle tumor, a nasopharyngeal tumor, a neuroblastoma, an oral tumor, an osteosarcoma, an ovarian tumor, a pancreatic tumor, a penile tumor, a pituitary tumor, a primary tumor, a prostate tumor, a retinoblastoma, a rhabdomyosarcoma, a salivary gland tumor, a soft tissue sarcoma, a melanoma, a metastatic tumor, a basal cell carcinoma, a Merkel cell tumor, a testicular tumor, a thymic tumor, a thyroid tumor, a uterine tumor, a vaginal tumor, a vulvar tumor, or a Wilms' tumor.

105. The method of any one of claims 57 to 104, wherein the first multispecific antigen-binding molecule is administered systemically, intravenously, subcutaneously, or intramuscularly.

106. The method of any one of claims 57 to 105, wherein the second multispecific antigen-binding molecule is administered systemically, intravenously, subcutaneously, or intramuscularly.

107. The method of any one of claims 57 to 106, wherein the first multispecific antigen-binding molecule and the second multispecific antigen-binding molecule are administered to the subject in a pharmaceutically acceptable formulation.

108. The method of any one of claims 57 to 107, wherein the method further comprises administering an additional anti-cancer agent.

109. The method of claim 108, wherein the additional anti-cancer agent is a chemotherapeutic agent, an immune checkpoint inhibitor, or a cell therapy, optionally wherein the cell therapy comprises CAR-T cells, natural killer cells, or macrophages.

110. The method of claim 109, wherein the additional anti-cancer agent is an immune checkpoint inhibitor.

111. The method of claim 110, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, or an anti-LAG3 antibody.

112. The method of any one of claims 1 to 111, wherein the subject has a refractory cancer.

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