Compositions and methods for treating cytokine release syndrome

Abstract

The present invention provides compositions and methods for treating cancer and suppressing cytokine release syndrome (CRS). The methods of the present invention comprise administering to a subject in need thereof a therapeutically effective amount of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist in combination with a therapeutically effective amount of a CD3 multispecific antigen-binding molecule.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 142,643, filed January 28, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Cytokine release syndrome (CRS) is a systemic inflammatory response that can be triggered by a variety of factors, including certain drugs. When symptoms associated with CRS occur within 6 hours after the start of a therapeutic infusion, they can be called infusion-related reactions (IRR).

[0003] T cell-activated cancer immunotherapy, including bispecific antibody therapy, is at particular risk for CRS (including IRR). In such therapies, CRS can be triggered by the massive release of IFN-γ by activated T cells or by the tumor cells themselves. Secreted IFN-γ induces activation of other immune cells, including macrophages, resulting in the production of excessive amounts of other cytokines, such as IL-6, TNF-α, and IL-10. In particular, IL-6 contributes to many of the major symptoms of CRS, including vascular leakage and activation of the complement and coagulation cascades that induce disseminated intravascular coagulation. IL-6 may also contribute to cardiomyopathy by promoting myocardial dysfunction (Non-Patent Document 1).

[0004] Addressing the significant harms of cancer immunotherapy, including CRS, is a clinical challenge. Mitigating CRS and / or IRR is important to ensure the safety of certain immunotherapy approaches, including the therapeutic use of bispecific antibodies targeting T cells. While low-grade CRS can generally be treated symptomatically with antihistamines, antipyretics, and fluid infusions, severe CRS may represent a life-threatening adverse event that requires prompt and aggressive treatment. Currently, specific anti-cytokine treatments, reductions in the dose of administered therapeutic drugs, and steroid premedication are used to reduce the incidence of severe CRS. For example, tocilizumab, an anti-IL-6 antibody, is used as an initial treatment for severe CRS in some circumstances. However, any of these currently available treatments may also reduce the therapeutic efficacy of immunotherapy in the treatment of cancer. Therefore, alternative strategies remain needed to reduce the potentially life-threatening effects of CRS without simultaneously adversely affecting the therapeutic efficacy of cancer immunotherapy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Shimabukaro-Vornhagen et al. (2018), Journal for Immunotherapy of Cancer, 6:1-14 Summary of the Invention

[0006] Provided herein are methods and compositions for the treatment and / or prevention of cytokine release syndrome (CRS), including the treatment and / or prevention of infusion-related reactions (IRR). As disclosed herein, administration of a CD40 antagonist (e.g., a CD40 blocking antibody) can reduce the release of cytokines associated with CRS without affecting the activation and cytotoxicity of T cells induced by administration of certain cancer immunotherapies (e.g., CD3 bispecific antibodies, CAR T cells). Thus, in certain aspects, the methods and compositions herein can reduce the potentially life-threatening effects of CRS without adversely affecting the therapeutic efficacy of T cell-activating cancer immunotherapies, including bispecific antibodies.

[0007] In some aspects, provided herein are methods of treating cancer and suppressing CRS (including IRR) in a subject, comprising co-administering to the subject: (a) a multispecific antigen binding molecule comprising a first antigen binding domain that specifically binds CD3 and a second antigen binding domain that specifically binds to a tumor antigen; and (b) a CD40 antagonist.

[0008] In some embodiments, the multispecific antigen binding molecule and the CD40 antagonist are administered simultaneously or sequentially, hi some embodiments, the CD40 antagonist is administered before the multispecific antigen binding molecule.

[0009] In some embodiments, the CD40 antagonist is an antibody or antigen-binding fragment thereof. In some embodiments, the CD40 antagonist antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, a composite antibody, a murine antibody, or a human antibody. In some embodiments, the CD40 antagonist antibody or antigen-binding fragment thereof is selected from an Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and a diabody fragment.

[0010] In some aspects, provided herein are methods of treating cancer and suppressing CRS (including IRR) in a subject, comprising co-administering to the subject: (a) a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen; and (b) a CAR-T cell that expresses a CD40 antagonist.

[0011] In some embodiments, the multispecific antigen-binding molecule and the CAR-T cells are administered simultaneously or sequentially, hi some embodiments, the CAR-T cells are administered before the multispecific antigen-binding molecule.

[0012] In some embodiments, the CAR-T cells secrete a CD40 antagonist. In some embodiments, the CD40 antagonist is an scFv or Fab. In some embodiments, the CAR-T cells express the CD40 antagonist upon activation.

[0013] The multispecific antigen-binding molecule may be a bispecific or trispecific antigen-binding molecule. In some embodiments, the trispecific antigen-binding molecule further comprises a third antigen-binding domain that specifically binds an additional T cell antigen or an additional tumor antigen. In some embodiments, the third antigen-binding domain specifically binds CD28. In some embodiments, the tumor antigen is selected from CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, and BCMA.

[0014] In some embodiments, the multispecific antigen-binding molecule comprises a multispecific antibody or antigen-binding fragment thereof. In some embodiments, the multispecific antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, a composite antibody, a murine antibody, or a human antibody. In some embodiments, the multispecific antigen-binding molecule is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

[0015] In some embodiments, the method activates T cells and / or enhances T cell cytotoxicity in the subject. In some embodiments, the method induces cancer cell death in the subject. In some embodiments, the method inhibits cytokine release syndrome. In some embodiments, cytokine release syndrome is inhibited as measured by maintaining C-reactive protein (CRP) levels below 7 mg / dL, IFN-γ below 75 pg / ml, and / or IL-10 below 60 pg / ml.

[0016] In some aspects, provided herein is a method of suppressing CRS (including IRR) caused by a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen in a subject, comprising administering to the subject a CAR-T cell that expresses a CD40 antagonist.

[0017] In some embodiments, the subject is a human. In some embodiments, the subject is a cancer patient. In some embodiments, the methods described herein further include identifying a subject susceptible to cytokine release syndrome or a subject in need of reduced cytokine release prior to administering to the subject a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist.

[0018] In some aspects, provided herein is a pharmaceutical composition comprising a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen, and a CD40 antagonist. In some embodiments, the pharmaceutical compositions described herein further comprise a pharma- ceutical acceptable carrier.

[0019] In some aspects, provided herein are methods of treating cancer and / or suppressing CRS (including IRR) in a subject, comprising administering to the subject a pharmaceutical composition described herein. In some aspects, provided herein are methods of treating cancer and / or suppressing CRS (including IRR) in a subject, comprising identifying a subject susceptible to cytokine release syndrome or in need of reduced cytokine release; and administering to the subject a pharmaceutical composition described herein.

[0020] In some aspects, provided herein are methods of treating cancer and / or suppressing CRS (including IRR) in a subject, comprising: (a) identifying a subject susceptible to cytokine release syndrome or in need of reduced cytokine release; and (b) co-administering to the subject: (1) a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen; and (2) a CD40 antagonist.

[0021] In some aspects, provided herein are methods of treating cancer and / or suppressing CRS (including IRR) in a subject, comprising: (a) identifying a subject susceptible to cytokine release syndrome or in need of reduced cytokine release; and (b) co-administering to the subject: (1) a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen; and (2) CAR-T cells that express a CD40 antagonist.

[0022] In some embodiments, subjects susceptible to CRS and / or subjects in need of reduced cytokine release are identified by detecting one or more biomarkers selected from the group consisting of fever, rash, respiratory symptoms, hypoxia, hypotension, cardiovascular insufficiency, neurotoxicity, liver dysfunction, renal dysfunction, coagulation, organ toxicity, tumor burden, cytokines, C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), blood urea nitrogen (BUN), alanine aminotransferase (ALT), creatinine (Cr), fibrinogen, prothrombin time (PT), partial thromboplastin time (PTT), eotaxin, and endothelial cell activation.

[0023] In some embodiments, the cytokine is one or more cytokines selected from the group consisting of sTNFR2, IP10, sIL1R2, sTNFR1, MIG, VEGF, sIL1R1, TNFα, IFNα, GCSF, sRAGE, IL1, IL2, IL4, IL5, IL10, IL12, IL13, IL18, IL1R1, IFNγ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, FLT-3L, fractalkine, and GM-CSF. In some embodiments, endothelial cell activation is detected by measuring serum levels of Ang-2 and / or von Willebrand factor. [Brief description of the drawings]

[0024] [Figure 1A] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1B] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1C] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1D] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1E] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1F] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1G] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1H] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 1I] We show that CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs. [Figure 2A] A 4-day assay with AML cell lines and PBMCs without added autologous B cells shows that CD40 blockade inhibited CD3 bispecific-mediated cytokine release. [Figure 2B] A 4-day assay with AML cell lines and PBMCs without added autologous B cells shows that CD40 blockade inhibited CD3 bispecific-mediated cytokine release. [Figure 2C] A 4-day assay with AML cell lines and PBMCs without added autologous B cells shows that CD40 blockade inhibited CD3 bispecific-mediated cytokine release. [Figure 2D] A 4-day assay with AML cell lines and PBMCs without added autologous B cells shows that CD40 blockade inhibited CD3 bispecific-mediated cytokine release. [Figure 2E] A 4-day assay with AML cell lines and PBMCs without added autologous B cells shows that CD40 blockade inhibited CD3 bispecific-mediated cytokine release. [Figure 3A] Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 3B] Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 3C] Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 3D]Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 3E] Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 3F] Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 3G] Four-day killing assays with prostate cell lines and PBMCs show that CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines without significantly affecting T cell activation and target killing. [Figure 4A-1] 1 shows that CD40 blockade reduced cytokines in PBMCs treated with CD20xCD3 bispecific (REGN1979). [Figure 4A-2] Same as above. [Figure 4B-1] 1 shows that CD40 blockade reduced cytokines in PBMCs treated with CD20xCD3 bispecific (REGN1979). [Figure 4B-2] Same as above. [Figure 4B-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] overview Provided herein are methods and compositions for the treatment and / or prevention of cytokine release syndrome (CRS), including the treatment and / or prevention of infusion-related reactions (IRR). As disclosed herein, administration of a CD40 antagonist (e.g., a CD40 blocking antibody) can reduce the release of cytokines associated with CRS without affecting the T cell activation and cytotoxicity induced by administration of certain cancer immunotherapies (e.g., CD3 bispecific antibodies, CAR T cells). Thus, in certain aspects, the methods and compositions herein can reduce the potentially life-threatening effects of CRS without adversely affecting the therapeutic efficacy of T cell-activating cancer immunotherapies, including bispecific antibodies.

[0026] Accordingly, in certain aspects, provided herein are methods of treating and / or preventing CRS and / or alleviating symptoms associated with CRS in a subject receiving cancer immunotherapy (e.g., with a multispecific antigen binding molecule comprising a first antigen binding domain that specifically binds CD3 and a second antigen binding domain that specifically binds to a tumor antigen) by administering to the subject a CD40 antagonist (e.g., an antagonist antibody that binds CD40) and / or CAR-T cells expressing a CD40 antagonist.

[0027] In some aspects, provided herein is a pharmaceutical composition comprising a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen; and a CD40 antagonist. In some embodiments, the pharmaceutical compositions described herein further comprise a pharma- ceutical acceptable carrier.

[0028] It is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention will be limited only by the appended claims.

[0029] All patents, applications and non-patent publications mentioned herein are hereby incorporated by reference in their entirety. definition For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about" when used in relation to a specific recited numerical value means that the value may vary by 1% or less from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0030] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0031] The term "administering" or "administration" as used herein means providing a pharmaceutical agent or composition to a subject, including, but not limited to, administration by a medical professional and self-administration. Such agents can contain, for example, CAR T cells provided herein.

[0032] 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 (herein referred to as V H Each light chain comprises a light chain variable region (abbreviated herein as V L and the light chain constant region. H Area and V L The regions 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 L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact 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.

[0033] As used herein, the terms "antigen-binding fragment" and "antigen-binding portion" of an antibody refer to one or more fragments of an antibody that retain the ability to bind to an antigen. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0034] "Cancer" broadly refers to the uncontrolled, abnormal proliferation of the host's own cells, leading to the invasion of surrounding tissues and potentially tissues distal to the initial site of abnormal cell proliferation in the host. Major classes include carcinomas, which are cancers of epithelial tissues (e.g., skin, squamous cells); sarcomas, which are cancers of connective tissues (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias, which are cancers of hematopoietic tissues (e.g., bone marrow tissue); lymphomas and myelomas, which are cancers of immune cells, and central nervous system cancers, including cancers of the brain and spinal cord tissue. "Cancer(s)" and "neoplasm(s)" are used interchangeably herein. As used herein, "cancer" refers to any type of cancer or neoplasm or malignant tumor, including leukemia, carcinoma, and sarcoma, whether primary or recurrent. Specific examples of cancers are carcinoma, sarcoma, myeloma, leukemia, lymphoma, and mixed tumors. Non-limiting examples of cancers are primary or recurrent cancers of the brain, melanoma, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, sarcoma, stomach, uterus, and medulloblastoma. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a metastatic cancer.

[0035] 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 generate a chimeric protein that exhibits specific anti-target cell immune activity. Generally, CARs consist of an extracellular single-chain antigen binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and when expressed in T cells, have the ability to redirect antigen recognition based on the specificity of a monoclonal antibody.

[0036] The phrase "co-administration" or "co-administering" as used herein refers to any administration form of two or more different therapeutic agents, such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., the two agents are effective in a subject simultaneously, which may include a synergistic effect of the two agents). For example, the different therapeutic agents can be administered simultaneously or sequentially in the same formulation, or in separate formulations. In certain embodiments, the different therapeutic agents can be administered within about 1 hour, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about one week of each other. Thus, subjects receiving such treatment can benefit from the combined effects of the different therapeutic agents.

[0037] A "costimulatory domain" or "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. The costimulatory domain may be a human costimulatory domain. Examples of 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.

[0038] "Costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including, but not limited to, proliferation activation, differentiation, etc. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind B7-H3.

[0039] A "costimulatory signal" refers to a signal that, in combination with a primary signal, causes T cell proliferation and / or up-regulation or down-regulation of key molecules. The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are those generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on an antigen.

[0040] The phrase "pharmacologically acceptable" as used herein refers to those agents, compounds, materials, compositions and / or dosage forms which are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0041] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent that encapsulates a material, that is involved in carrying or transporting a drug 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 injurious to the patient. Some examples of materials which can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible materials employed in pharmaceutical formulations.

[0042] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably. They refer to a natural or synthetic molecule, or any combination thereof, that contains one nucleotide or two or more nucleotides linked to the 5' end of another nucleotide by a phosphate group at the 3' position of one nucleotide. A polymeric form of nucleotides is not limited by length and can contain either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may have any three-dimensional structure and perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locuses) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, for example, by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides. A polynucleotide is not necessarily associated with a cell in which it is found in nature and / or is not operably linked to the polynucleotide with which it is naturally linked.

[0043] As used herein, a therapeutic agent that "prevents" a condition refers to a compound that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of the disorder or condition or reduces the severity of one or more symptoms in the treated sample relative to an untreated control sample.

[0044] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity at least about 95%, more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0045] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, for example, by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994), Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids 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: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992), Science, 256:1443-1445, which is incorporated herein by reference. A "reasonably conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0046] The term "specifically binds" or "specific binding" as used herein, when referring to a polypeptide, refers to a binding reaction that determines the presence of a protein or polypeptide or receptor in a heterogeneous population of proteins and other biologics. Thus, under specified conditions (e.g., immunoassay conditions in the case of antibodies), a particular ligand or antibody "specifically binds" to its particular "target" (e.g., an antibody specifically binds to an endothelial antigen) if it does not bind in any significant amount to other proteins present in the sample or to other proteins that the ligand or antibody may come into contact with in an organism. Generally, a first molecule that "specifically binds" to a second molecule will have about 10% or more binding affinity to that second molecule. 5 M -1 (For example, 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , and 10 12 M -1 For example, the ability of a PIG-specific CAR to bind a peptide presented on MHC (e.g., class I MHC or class II MHC); typically, the CAR specifically binds to that peptide / MHC with an affinity of at least about 10-4 M or less, KD, and binds to a given antigen / binding partner with an affinity (expressed as KD) that is at least 10-fold lower, at least 100-fold lower, or at least 1000-fold lower than the binding affinity for a non-specific, unrelated peptide / MHC complex (e.g., one that contains a BSA peptide or a casein peptide).

[0047] The term "subject" as used herein means a human or non-human animal selected for treatment or therapy. As used herein, the term "treatment" refers to a clinical intervention designed to modify the natural course of the individual being treated during the course of clinical pathology. The desired effects of treatment include slowing the rate of progression of a particular disease, disorder or condition, improving or alleviating pathological conditions, and improving remission or prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are reduced or eliminated.

[0048] Cytokine release syndrome (CRS) overview In certain aspects, provided herein are methods and compositions for treating and / or preventing cytokine release syndrome (CRS). Symptoms associated with CRS can include infusion-related reactions (IRR) if they occur within 6 hours after the start of a therapeutic infusion. Thus, in certain embodiments, the methods and compositions provided herein can be useful for treating and / or preventing CRS and / or CRS symptoms, including, but not limited to, IRR.

[0049] CRS is a potentially life-threatening cytokine-associated toxicity that can occur as a result of cancer immunotherapy, such as cancer antibody therapy (e.g., bispecific antibodies) and / or T cell immunotherapy (e.g., CAR T cells). CRS is caused by high levels of immune activation when large numbers of lymphocytes and / or myeloid cells release inflammatory cytokines upon activation. The severity of CRS and the timing of onset of symptoms can vary depending on the magnitude of immune cell activation, the type of treatment administered, and / or the extent of tumor burden in the subject. In the case of T cell therapy for cancer, onset of symptoms usually occurs several days to weeks after administration of T cell therapy, e.g., when T cell proliferation in vivo peaks. See, e.g., Lee et al. (2014), Blood. 124:188-195.

[0050] Symptoms of CRS can include neurotoxicity, disseminated intravascular coagulation, cardiac dysfunction, adult respiratory distress syndrome, renal failure, and / or hepatic failure. For example, symptoms of CRS can include fever with or without chills, malaise, malaise, myalgia, vomiting, headache, nausea, anorexia, arthralgia, diarrhea, rash, hypoxemia, tachypnea, hypotension, widening pulse pressure, decreased potential cardiac output (late), increased cardiac output (early), azotemia, hypofibrinogenemia with or without hemorrhage, elevated D-dimer, hyperbilirubinemia, transaminitis, confusion, delirium, altered mental status, hallucinations, tremors, seizures, changes in gait, word finding difficulty, frank aphasia, or dysmetria.

[0051] CRS grading In certain aspects, the methods and compositions provided herein can be used to treat and / or prevent CRS of any severity, hi certain embodiments, the methods and compositions provided herein are used to treat and / or prevent severe CRS.

[0052] Management of CRS may follow grade- and risk-adapted strategies for monitoring and treatment. Several CRS grading systems have been developed and are disclosed, for example, in Porter et al. (2018), J. Hematol. Oncol. 11:35, Lee et al. (2014), Blood, 124:188-195, Neelapu et al. (2018), Nat. Rev. Clin. Oncol. 15:47-62, Neelapu et al. (2018), Nat. Rev. Clin. Oncol. 15:218, Teachey et al. (2016), Cancer Discov. 6:664-679, and U.S. Patent Application Publication No. 2019 / 0336504, each of which is incorporated herein by reference in its entirety. Any of these CRS grading systems may be used to evaluate, diagnose, stratify, or identify subjects for the methods provided herein.

[0053] In some embodiments, CRS can be graded with severity from 1 to 5 as follows: Grades 1 to 3 are not severe CRS; Grades 4 to 5 are severe CRS; For Grade 1, only symptomatic treatment (e.g., nausea, fever, fatigue, myalgia, malaise, headache) is required and symptoms are not life-threatening; For Grade 2 CRS, symptoms require and generally respond to moderate interventions; Subjects with Grade 2 CRS develop hypotension that responds to either fluid infusion or a single low dose of vasopressor; or develop mild respiratory symptoms that respond to Grade 2 organ toxicity or low-flow oxygen (less than 40% oxygen). Subjects with Grade 3 CRS generally cannot reverse hypotension with fluid therapy or a single low dose of vasopressor. These subjects generally require more than low flow oxygen and have grade 3 organ toxicity (e.g., renal or cardiac insufficiency or coagulopathy) and / or grade 4 transaminitis. Grade 3 CRS subjects require more aggressive interventions, e.g., 40% or more oxygen, high doses of vasopressor(s), and / or multiple vasopressors. Grade 4 CRS subjects suffer from immediately life-threatening symptoms, including grade 4 organ toxicity or the need for mechanical ventilation. Grade 4 CRS subjects usually do not have transaminitis. In grade 5 CRS subjects, the toxicity causes death. For example, criteria for grading CRS are disclosed in U.S. Patent Application Publication No. 2019 / 0336504, which is incorporated herein by reference in its entirety.

[0054] In certain embodiments, the methods and / or compositions provided herein treat and / or prevent grade 5 CRS. In certain embodiments, the methods and / or compositions provided herein treat and / or prevent grade 4 CRS. In certain embodiments, the methods and / or compositions provided herein treat and / or prevent grade 3 CRS. In certain embodiments, the methods and / or compositions provided herein treat and / or prevent grade 2 CRS. In certain embodiments, the methods and / or compositions provided herein treat and / or prevent grade 1 CRS.

[0055] Identifying subjects at risk for CRS In certain embodiments, the methods provided herein include treating a subject susceptible to CRS and / or a subject in need of reduced cytokine release, hi some embodiments, the methods provided herein include identifying a subject susceptible to CRS and / or a subject in need of reduced cytokine release.

[0056] In some embodiments, one or more biomarkers are used to assess (e.g., diagnose or identify) a subject susceptible to CRS or in need of reduced cytokine release. In some embodiments, the one or more biomarkers are selected from fever, rash, respiratory symptoms, hypoxia, hypotension, cardiovascular insufficiency, neurotoxicity, hepatic insufficiency, renal insufficiency, coagulation, organ toxicity, tumor burden, cytokines, eotaxin, C-reactive protein (CRP), ferritin, creatinine, and endothelial cell activation, and the like.

[0057] In some embodiments, exemplary cytokines include, but are not limited to, sTNFR2, IP10, sIL1R2, sTNFR1, MIG, VEGF, sIL1R1, TNFα, IFNα, GCSF, sRAGE, IL1, IL2, IL4, IL5, IL10, IL12, IL13, IL18, IL1R1, IFNγ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, FLT-3L, fractalkine, and GM-CSF. In some embodiments, one or more (e.g., two or more or three or more) of the cytokines sTNFR2, IP10, sIL1R2, sTNFR1, MIG, VEGF, sIL1R1, TNFα, IFNα, GCSF, sRAGE, IL1, IL2, IL4, IL5, IL10, IL12, IL13, IL18, IL1R1, IFNγ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, FLT-3L, fractalkine, and GM-CSF are used to evaluate (e.g., diagnose or identify) a subject susceptible to CRS or a subject in need of reduced cytokine release.

[0058] Exemplary biomarkers used to assess (e.g., predict) CRS severity can also include disease burden assessment, e.g., the extent of disease (e.g., cancer) in a subject. For example, disease burden assessment can be performed by determining the level of disease (e.g., cancer) in a biological sample from a subject. For example, high disease burden is indicated by the presence of at least 25% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90% or more) cancer cells in a biological sample obtained from a subject (e.g., as determined by morphology of an aspirate or biopsy, flow assay of an aspirate or biopsy, and / or MRD). In some embodiments, high disease burden is indicated by the presence of at least 50% cancer cells in a biological sample obtained from a subject. For example, a low disease burden is indicated by the presence of less than 25% (e.g., 24%, 23%, 22%, 21%, 20%, 15%, 10%, 5% or less) cancer cells in a biological sample obtained from a subject (e.g., as determined by morphology of an aspirate or biopsy, flow assay of an aspirate or biopsy, and / or MRD). In some embodiments, a low disease burden is indicated by the presence of less than 0.1%, 1%, 5%, 10%, 15%, 20%, or 25% cancer cells in a biological sample obtained from a subject.

[0059] In some embodiments, one or more cytokines are used in conjunction with a disease burden assessment to assess (eg, diagnose or identify) a subject who is susceptible to CRS or who is in need of reduced cytokine release.

[0060] Another exemplary biomarker used to evaluate (e.g., diagnose or identify) subjects susceptible to CRS or subjects in need of reduced cytokine release includes C-reactive protein (CRP) level or activity. In embodiments, subjects at low risk of severe CRS are identified as having a CRP level of less than 7 mg / dL (e.g., 7, 6.8, 6, 5, 4, 3, 2, 1 mg / dL or less). In some embodiments, subjects at high risk of severe CRS are identified as having a higher level of CRP in a sample (e.g., blood sample) compared to subjects at low risk of severe CRS or compared to the level or activity of a control. In some embodiments, the higher level or activity is at least 2-fold higher (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 500, 1000-fold or more) compared to subjects at low risk of severe CRS or compared to the level or activity of a control.

[0061] In some embodiments, one or more biomarkers described herein are used to predict the risk or severity of CRS in a subject early after administration of a CD40 antagonist and / or CD3 multispecific antibody described herein. In some embodiments, one or more biomarkers described herein are used to predict the risk or severity of CRS in a subject within 2 weeks, e.g., within 1 week or less, after administration of a CD40 antagonist and / or CD3 multispecific antibody described herein. In embodiments, the biomarkers described herein are used to predict the risk or severity of CRS in a subject within 10 days (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 day or less after administration) of a CD40 antagonist and / or CD3 multispecific antibody described herein. In embodiments, the biomarkers described herein are used to predict the risk or severity of CRS in a subject within 1-10 days (e.g., within 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 day) after administration of a CD40 antagonist and / or CD3 multispecific antibody described herein. In embodiments, the biomarkers described herein are used to predict the risk or severity of CRS in a subject before the subject experiences one or more symptoms of grade 2, 3, 4, or 5 CRS (e.g., before the subject experiences one or more symptoms of grade 3, 4, or 5 CRS, or grade 4 or 5 CRS).

[0062] In some embodiments, elevated or decreased levels of one or more of the cytokines described herein, e.g., sTNFR2, IP10, sIL1R2, sTNFR1, VEGF, sIL1R1, TNFα, IFNα, GCSF, sRAGE, IL1, IL2, IL4, IL5, IL10, IL12, IL13, IL18, IL1R1, IFNγ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, FLT-3L, fractalkine, and GM-CSF, relative to control levels, indicate that the subject is susceptible to CRS or in need of reduced cytokine release, or is at risk of developing severe CRS. The control level may be a reference level, a baseline level, a level from a healthy subject, a level from a subject having a disorder or condition other than CRS (e.g., cancer), a level from a subject prior to administration of a CD3 multispecific antibody and / or CD40 antagonist described herein, or a level from a subject with a particular grade of CRS.

[0063] In some embodiments, an increase of at least a two-fold (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000-fold or more) relative to a control level (e.g., a baseline level) of one or more of the cytokines described herein (e.g., one or more of IFNγ, IL6, IL10, sgp130, IL18, TNFα, IL8, IP10, MCP1, MIG, MIP1β, and sIL6R) indicates that the subject is susceptible to CRS, is in need of a reduction in cytokine release, or is at risk of developing severe CRS.

[0064] In some embodiments, a decrease of at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) in one or more of the cytokines described herein (e.g., one or more of IL1R1, MIP1α, and IL13) compared to a baseline level indicates that the subject is susceptible to CRS, or in need of a decrease in cytokine release, or at risk for developing severe CRS. In some embodiments, the baseline level is a value that is independent of the baseline level of the cytokine in the subject. In some embodiments, the baseline level is a baseline cytokine value, or a baseline cytokine value by disease burden.

[0065] In some embodiments, an increase, e.g., at least a two-fold (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000-fold or more) relative to control levels of one or more of the cytokines described herein (e.g., IFNγ, IL6, IL10, sgp130, IL18, TNFα, IL8, IP10, MCP1, MIG, MIP1β, and sIL6R), e.g., when measured within 1-10 days (e.g., within 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 day) after administration of a CD3 multispecific antibody described herein, indicates that the subject is susceptible to CRS, or in need of reduced cytokine release, or at risk for developing severe CRS.

[0066] In some embodiments, a CRP level of less than 7 mg / dL (e.g., 7, 6.8, 6, 5, 4, 3, 2, 1 mg / dL or less), e.g., when measured within 1-10 days (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 day) after administration of a CD3 multispecific antibody described herein, indicates that the subject is at low risk of developing severe CRS.

[0067] In embodiments, a CRP level of 6 mg / dL or greater (e.g., 6, 6.8, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 mg / dL or greater), e.g., when measured within 1-10 days (e.g., within 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 day) after administration of a CD3 multispecific antibody described herein, indicates that the subject is at high risk for developing severe CRS.

[0068] In certain embodiments, the present disclosure provides a method of monitoring CRS (e.g., monitoring a patient with CRS0, CRS1, CSR2, or CRS3), or a method of monitoring the development of severe CRS, comprising assessing one or more CRS biomarkers herein. The method can include measuring one or more biomarkers at multiple time points, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more time points. In certain embodiments, the present disclosure provides a method of managing CRS, comprising assessing a subject at risk of developing CRS (e.g., severe CRS), and optionally administering a treatment for CRS, e.g., a treatment described herein.

[0069] Identifying subjects with CRS In certain embodiments, the methods provided herein include treating a subject with CRS. In some embodiments, the methods provided herein include determining whether a subject has CRS (e.g., severe CRS). The methods include, for example, obtaining a CRS risk status for the subject in response to a cancer immunotherapy, e.g., a CD3 multispecific antibody, and the CRS risk status can be determined by measuring the level of sTNFR2, IP10, sIL1R2, sTNFR1, MIG, VEGF, sIL1R1, TNFα, IFNα, GCSF, sRAGE, IL1, IL2, IL4, IL5, IL10, IL12, IL13, IL18, IL1R1, IFNγ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, FLT-3L, fractal cytoplasmic resorption, or cytoplasmic resorption in a sample (e.g., a blood sample). The method includes measuring one or more levels or activities of one or more cytokines (e.g., 3, 4, 5, 10, 15, 20, or more) selected from: erythrocyte sedimentation factor (ESD), erythrocyte colony-stimulating factor (ACS), and GM-CSF, or a laboratory test (e.g., an analyte) selected from: C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), blood urea nitrogen (BUN), alanine aminotransferase (ALT), creatinine (Cr), fibrinogen, prothrombin time (PT), partial thromboplastin time (PTT), or a combination thereof.

[0070] In some embodiments, a ferritin level of at least about 23,500, 25,000, 30,000, 40,000, 50,000, 70,000, 80,000, 90,000, 100,000, 150,000, 200,000, or 250,000 ng / ml, and optionally up to about 299,000 or 412,000 ng / ml, is indicative of CRS (e.g., severe CRS). In some embodiments, a ferritin level of less than about 23,500, 20,000, 18,000, 16,000, 14,000, 12,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 ng / ml, and optionally greater than about 280 ng / ml, indicates that the subject does not have CRS (e.g., severe CRS).

[0071] In some embodiments, an LDH level of at least about 1,700, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, or 20,000 U / L, and optionally up to about 24,000 U / L, indicates CRS (e.g., severe CRS). In some embodiments, an LDH level of less than about 1,700, 1,500, 1,400, 1,300, 1,200, 1,100, 1,000, 900, 800, 700, 600, 500, 400, 300, or 200 U / L, and optionally more than about 159 U / L, indicates that the subject does not have CRS (e.g., severe CRS).

[0072] In some embodiments, a CRP level of at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 mg / dl, and optionally up to about 38 mg / dl, indicates CRS (severe CRS). In some embodiments, a CRP level of less than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mg / dl, and optionally more than about 0.7 mg / dl, indicates that the subject does not have CRS (e.g., severe CRS).

[0073] In some embodiments, an ALT level of at least about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 980, 900, 950, or 1000 U / L, and optionally up to 1300 U / L, indicates CRS (e.g., severe CRS). In some embodiments, an ALT level of less than about 100, 90, 80, 70, 60, 50, 40, or 30 U / L, and optionally more than about 25 U / L, indicates that the subject does not have CRS (e.g., severe CRS).

[0074] In some embodiments, AST levels of at least about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 980, 900, 950, 1000 U / L, and optionally up to 1500 U / L, indicate CRS (e.g., severe CRS). In some embodiments, AST levels of less than about 150, 140, 130, 120, 100, 90, 80, 70, 60, 50, 40, or 30 U / L, and optionally greater than about 15 U / L, indicate that the subject does not have CRS (e.g., severe CRS).

[0075] In some embodiments, a BUN level of at least about 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 mg / dl, and optionally up to about 210 mg / dl, indicates CRS (e.g., severe CRS). In some embodiments, a BUN level of less than about 18, 17, 16, 15, 14, 13, 12, 11, or 10 mg / dl, and optionally more than about 5 mg / dl, indicates that the subject does not have CRS (e.g., severe CRS).

[0076] In some embodiments, fibrinogen levels less than about 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 mg / dl, and optionally greater than about 20 mg / dl, indicate CRS (e.g., severe CRS). In some embodiments, fibrinogen levels of at least about 150, 160, 170, 180, 190, 200, or 210 mg / dl, and optionally up to about 230 mg / dl, indicate that the subject does not have CRS (e.g., severe CRS).

[0077] In some embodiments, a PT level of at least about 17, 18, 19, 20, 21, or 22 seconds, and optionally up to about 24 seconds, indicates CRS (e.g., severe CRS). In some embodiments, a PT level of less than about 17, 16, 15, or 14 seconds, and optionally more than about 12 seconds, indicates that the subject does not have CRS (e.g., severe CRS).

[0078] In some embodiments, a PTT level of at least about 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, or 85 seconds, and optionally up to about 95 seconds, indicates CRS (e.g., severe CRS). In some embodiments, a PTT level of less than about 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, or 27 seconds, and optionally more than about 25 seconds, indicates that the subject does not have CRS (e.g., severe CRS).

[0079] In some embodiments, patients with severe CRS have IFN-γ>75 pg / ml and IL-10>60 pg / ml. In some embodiments, patients with severe CRS have IFN-γ levels of 40, 50, 60, 70, or 75 pg / ml or greater, IL-10 levels of 30, 40, 50, or 60 pg / ml or greater, or any combination thereof.

[0080] Additional biomarkers for assessing subjects having or at risk for CRS are disclosed in U.S. Patent Application Publication Nos. 2019 / 0336504 and 2018 / 0252727, each of which is incorporated by reference in its entirety.

[0081] therapeutic antibodies overview In certain aspects, the methods and compositions provided herein concern the use of therapeutic antibodies (eg, antagonistic anti-CD40 antibodies and / or bispecific CD3 binding antibodies).

[0082] As mentioned above, the term "antibody" as used herein encompasses both complete antibody molecules and antigen-binding fragments of complete antibody molecules. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of antibodies or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0083] An antigen-binding fragment of an antibody will usually contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated with V H For antigen-binding fragments containing domains, V H Domain and V LThe domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L , or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a dimer of monomeric V H Domain or V L It may contain domains.

[0084] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3 and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention may be linked to each other and / or to one or more monomeric V H or V L The domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently associated (e.g., by disulfide bond(s)).

[0085] Similar to intact antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies usually contain at least two different variable domains, where each variable domain can specifically bind to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in conjunction with antigen-binding fragments of antibodies of the invention using routine techniques available in the art. In certain embodiments provided herein, at least one variable domain of the multispecific antibody can specifically bind to CD3.

[0086] In some embodiments, the antibodies provided herein may function via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). "Complement-dependent cytotoxicity" (CDC) refers to the lysis of antigen-expressing cells by the antibodies of the present invention in the presence of complement. "Antibody-dependent cell-mediated cytotoxicity" (ADCC) refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, thereby causing lysis of the target cells. CDC and ADCC can be measured using assays that are well known and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998), Proc. Natl. Acad. Sci. (USA), 95:652-656). The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity, and therefore, the isotype of the antibody may be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0087] In certain embodiments provided herein, the anti-CD40 antagonist antibodies or CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The humanized antibodies of the invention may contain, for example, in the CDRs and particularly in CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0088] The antibodies provided herein may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibodies" as used herein is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described further below), antibodies isolated from recombinant combinatorial human antibody libraries (described further below), antibodies isolated from animals (e.g., mice) introduced with human immunoglobulin genes (see, e.g., Taylor et al. (1992), Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced or isolated by 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 animals transgenic for human Ig sequences are used), so that the V H Area and V L The amino acid sequence of the region is H and V L While derived from and related to sequences of, they may not naturally occur within the human antibody germline repertoire in vivo.

[0089] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa, in which the dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, resulting in a molecule of approximately 75-80 kDa (half antibody) composed of covalently linked light and heavy chains. These forms have been very difficult to separate, even after affinity purification.

[0090] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level normally observed using a human IgG1 hinge (Angal et al. (1993), Molecular Immunology, 30:105). The present invention provides a method for the production of a hinge, C, C-terminal region, or C-terminal region of an antibody that may be desirable to improve the yield of a desired antibody form in production. H 2 or C H The present invention encompasses antibodies with one or more mutations in three regions.

[0091] The antibody of the present invention may be an isolated antibody. "Isolated antibody" as used herein means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0092] In certain embodiments, the methods and compositions provided herein include a one-arm antibody that binds to CD40. As used herein, "one-arm antibody" refers to an antigen-binding molecule that comprises a single antibody heavy chain and a single antibody light chain.

[0093] Sequence variants In some embodiments, the anti-CD40 antagonist antibodies and / or CD3 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 and light chain variable domains compared to the corresponding germline sequences from which the antibody is derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations or combinations thereof. H and / or V LIn one embodiment, all of the framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only those mutated residues found within the first 8 amino acids of FR1, or only those mutated residues found within the last 8 amino acids of FR4, or only those mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residues in certain germline sequences while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in the different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may exhibit improved binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., K D The antibodies and antigen-binding fragments obtained by this general method are within the scope of the present invention.

[0094] In some embodiments, the anti-CD40 antagonist antibodies or CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, in certain embodiments, the anti-CD40 antagonist antibodies or CD3 multispecific (e.g., bispecific or trispecific) antibodies provided herein have HCVR, LCVR, and / or CDR amino acid sequences with, e.g., no more than 10, no more than 8, no more than 6, no more than 4, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0095] Fc variants According to certain embodiments provided herein, there are provided antibodies and multispecific antigen-binding molecules comprising an Fc domain that comprises one or more mutations that enhance or reduce binding of the antibody to the FcRn receptor at, for example, acidic pH compared to neutral pH. For example, the present invention provides antibodies and multispecific antigen-binding molecules comprising an Fc domain that comprises one or more mutations that enhance or reduce binding of the antibody to the FcRn receptor at, for example, acidic pH compared to neutral pH. H 2nd area or C HIncluded are antibodies containing mutations in the 3 region, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations may result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include the 428L (e.g., M428L) and 434S (e.g., N434S) modifications; the 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; the 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; the 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; the 250Q and 428L modifications (e.g., T250Q and M428L); and the 307 and / or 308 modifications (e.g., 308F or 308P).

[0096] For example, the present invention includes CD3 multispecific antigen binding molecules (e.g., anti-CD3 / anti-MUC16 bispecific antibodies, anti-BCMA×anti-CD3 antibodies, or anti-CD3 / anti-CD20 bispecific antibodies) that comprise an Fc domain that comprises one or more pairs or groups of mutations selected from the group consisting of 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations, as well as other mutations in antibody variable domains disclosed herein, are contemplated within the scope of the present invention.

[0097] biological equivalent Provided herein are antigen binding molecules having amino acid sequences that differ from those of the exemplary molecules disclosed herein but that retain the ability to bind the same antigen or antigens. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit essentially the same biological activity as the biological activity of the bispecific antigen binding molecules described.

[0098] The present invention includes antigen-binding molecules that are bioequivalent to any of the exemplary antigen-binding molecules shown herein. Two antigen-binding proteins or antibodies are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in their rate and extent of absorption when administered in single or multiple doses at the same molar dose under similar experimental conditions. Some antigen-binding proteins would be considered equivalents or pharmaceutical substitutes if they are absorbed to a similar extent but not at the same rate, but such differences in absorption rate may be considered bioequivalent because they are intentional and reflected in the package insert, are not essential to achieve effective body drug concentrations, such as in chronic use, and are considered medically insignificant for the particular pharmaceutical product studied.

[0099] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. In one embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biologic without a predicted increased risk of adverse effects, including clinically significant changes in immunogenicity, or a decrease in efficacy, compared to continuing treatment without such a switch.

[0100] In one embodiment, two antigen binding proteins are biologically equivalent if they both act through a common mechanism or mechanisms of action for the condition or conditions for which they are used, to the extent such mechanisms are known.

[0101] Bioequivalence may be demonstrated by in vivo and in vitro methods. Criteria for assessing bioequivalence include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of the antibody or its metabolites as a function of time in blood, plasma, serum, or other body fluids; (b) in vitro studies that correlate with and are reasonably predictive of in vivo bioavailability data in humans; (c) in vivo studies in humans or other mammals that measure the relevant acute pharmacological effect of the antibody (or its target) as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

[0102] Bioequivalent variants of the exemplary bispecific antigen-binding molecules shown herein may be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. In other contexts, bioequivalent antigen-binding proteins may include variants of the exemplary bispecific antigen-binding molecules shown herein, including amino acid changes that modify the glycosylation characteristics of the molecule, for example, mutations that remove or eliminate glycosylation.

[0103] Antibody binding The term "binding" as used herein, in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to a given antigen, e.g., a cell surface protein or fragment thereof, typically refers to an interaction or association between at least two entities or molecular structures, such as the interaction of an antibody and an antigen.

[0104] For example, binding affinities are typically around 10 as determined by surface plasmon resonance (SPR) techniques, e.g., on a BIAcore 3000 instrument using an antigen as the ligand and an antibody, Ig, antibody binding fragment, or Fc-containing protein as the analyte (or antiligand). -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 K below M D 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 competitive binding and SPR (Benedict, CA, J. Immunol. Methods. 1997, 201(2):223-31; Geuijen, CA, et al. J. Immunol. Methods. 2005, 302(1-2):68-77).

[0105] Thus, the antibodies or antigen-binding proteins provided herein have a K that is at least 10-fold lower than their affinity for binding to a non-specific antigen (e.g., BSA, casein). D According to the present invention, the K value is 10-fold lower than that of a non-specific antigen. D An antibody affinity corresponding to a value may be considered to be undetectable binding, but such an antibody may be paired with a second antigen-binding arm to create a bispecific antibody of the invention.

[0106] "K D The term "M" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. D There is an inverse relationship between binding affinity and K D The smaller the value, the higher the affinity, i.e., the stronger. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction, and thus a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction and thus a larger K D In some situations, the binding affinity (or K D ) relative to the binding affinity of that molecule (e.g., an antibody) for another interacting partner molecule (e.g., antigen Y) indicates a larger K D value (lower or weaker affinity) to smaller K D The binding affinity may be expressed as a binding ratio determined by dividing by the value (higher or stronger affinity), for example, 5-fold or 10-fold greater binding affinity, as the case may be.

[0107] "k dThe term "(sec-1 or 1 / s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody binding fragment. The value is k off Also called value.

[0108] "k a The term "(M-1 x sec-1 or 1 / M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody binding fragment. "K A The term "(M-1 or 1 / M)" refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is k a k d It is obtained by dividing by.

[0109] "EC50" or "EC 50 The term "half-maximal effective concentration" refers to the concentration of antibody that induces a response halfway between the baseline and maximum after a specified exposure time. EC 50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed. In certain embodiments, the EC 50 The EC value is equal to the concentration of an antibody of the invention that binds half-maximally to cells expressing CD3 or a tumor-associated antigen (e.g., CD123, STEAP2, CD20, PSMA, SSTR2, CD38, STEAP1, 5T4, ENPP3, MUC16, or BCMA) as determined, for example, by a FACS binding assay. Thus, the EC 50 , or half-maximal effective concentration, a decrease or weakening of binding is observed.

[0110] In one embodiment, the reduction in binding of a CD3 multispecific antibody is determined by EC2 that allows binding to half the maximum amount of target cells. 50 It can be defined as an increase in antibody concentration. In another embodiment, EC 50The values ​​represent the concentrations of CD3 multispecific antibodies of the invention that induce half-maximal depletion of target cells by T cell cytotoxicity. Thus, an increase in cytotoxicity (e.g., T cell-mediated tumor cell killing) is associated with an increase in EC 50 , or a decrease in half of the maximum effective concentration is observed.

[0111] pH-dependent binding In some embodiments, the invention includes antibodies and multispecific antigen-binding molecules with pH-dependent binding properties. For example, the CD3 multispecific antibodies of the invention may exhibit reduced binding to CD3 at acidic pH compared to neutral pH. Alternatively, the CD3 multispecific antibodies of the invention may exhibit enhanced binding to CD3 at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​below about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or lower. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. The expression "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0112] In some cases, "reduced binding at acidic pH compared to neutral pH" refers to the K D The K value of an antibody that binds to its antigen at neutral pH D For example, a CD3 multispecific antibody or antigen-binding fragment thereof may be expressed in terms of a ratio to a value of about 3.0 or greater (or vice versa). D If a ratio is shown, it may be considered for purposes of the present invention to indicate "reduced binding to CD3 at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K DThe ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or more.

[0113] Antibodies with pH-dependent binding properties may be obtained, for example, by screening antibody populations for decreased (or increased) binding to a particular antigen at acidic pH compared to neutral pH. Furthermore, antibodies with pH-dependent properties may be obtained by modification of the antigen-binding domain at the amino acid level. For example, an antibody with decreased antigen binding at acidic pH compared to neutral pH may be obtained by replacing one or more amino acids in the antigen-binding domain (e.g., within the CDRs) with histidine residues.

[0114] Preparation of antigen-binding domains and construction of multispecific molecules Antigen-binding domains specific for a particular antigen can be prepared by any antibody generation technique known in the art. Once obtained, two different antigen-binding domains specific for two different antigens (e.g., CD3 and a human tumor antigen (e.g., MUC16, BCMA, CD20, etc.)) can be appropriately positioned with respect to each other to create a bispecific antigen-binding molecule of the invention using routine methods. In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the multispecific antigen-binding molecule of the invention are derived from a chimeric antibody, a humanized antibody, or a fully human antibody. Methods for producing such antibodies are well known in the art. For example, one or more of the heavy and / or light chains of the bispecific antigen-binding molecule of the invention can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or other human antibody generation techniques), a high affinity chimeric antibody with human variable regions and mouse constant regions against a particular antigen (e.g., CD3 or a human tumor antigen (e.g., MUC16, BCMA, CD20, etc.)) is first isolated. Antibodies are characterized and selected for desirable properties including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate fully human heavy and / or light chains that can be incorporated into the bispecific antigen binding molecules of the invention.

[0115] Genetically engineered animals may be used to generate human bispecific antigen-binding molecules. For example, genetically engineered mice that cannot rearrange and express endogenous mouse immunoglobulin light chain variable sequences can be used, where the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to mouse kappa constant genes at the endogenous mouse kappa locus. Such genetically engineered mice can be used to generate fully human bispecific antigen-binding molecules that include two different heavy chains associated with the same light chain that includes a variable domain derived from one of two different human light chain variable region gene segments. (See, for example, US2011 / 0195454). Fully human refers to an antibody, or an antigen-binding fragment thereof, or an immunoglobulin domain that includes an amino acid sequence that is encoded by DNA derived from a human sequence throughout the entire length of each polypeptide of the antibody, or an antigen-binding fragment thereof, or an immunoglobulin domain. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other examples, the fully human protein or protein sequence includes a chimeric sequence in which each component sequence is derived from a human sequence. Without being bound by any theory, chimeric proteins or sequences are generally designed to minimize the generation of immunogenic epitopes at the junctions of the component sequences, e.g., compared to regions or domains of any wild-type human immunoglobulin.

[0116] CD40 antagonists In certain aspects, provided herein are methods of treating cancer and / or inhibiting cytokine release by administering to a subject (e.g., a subject in need thereof) a CD40 antagonist.

[0117] The term "CD40 antagonist" refers to any agent that inhibits or blocks CD40-mediated effects. It may be a small molecule, an antibody, an antisense oligonucleotide, siRNA, shRNA, sgRNA, a peptide, etc. In some embodiments, the CD40 antagonist is an antibody (e.g., an antigen-binding antibody fragment) that binds to CD40. An "antagonistic" effect of an anti-CD40 antibody refers to an effect that inhibits the binding of CD40 expressed on the surface of a cell, such as a B cell, a tumor cell, or a dendritic cell, to its ligand, or an effect that neutralizes one or more effects of the CD40 ligand on a CD40-expressing cell. An "antagonistic antibody" refers to an antibody that has such an effect. An example of an effect on a CD40-expressing cell includes suppressing B cell proliferation or suppressing antibody production.

[0118] In some embodiments, the CD40 antagonist antibody (e.g., antigen-binding antibody fragment) targets the CD40 receptor and interferes with CD40 signaling, particularly the CD40 signaling pathway mediated by the interaction of CD40 with CD40 ligand (CD40L). The term "CD40 antigen," "CD40 cell surface antigen," "CD40 receptor," or "CD40" refers to a transmembrane glycoprotein that belongs to the tumor necrosis factor (TNF) receptor family (see, e.g., U.S. Pat. Nos. 5,674,492 and 4,708,871; Stamenkovic et al. (1989), EMBO, 8:1403; Clark (1990), Tissue Antigens, 36:33; Barclay et al. (1997), The Leucocyte Antigen Facts Book, (2d ed.; Academic Press, San Diego)). At least six isoforms of human CD40 have been identified that are encoded by alternatively spliced ​​transcript variants of this gene (NM_001250.6 and NP_001241.1; NM_001302753.2 and NP_001289682.1; NM_001322421.2 and NP_001309350.1; NM_001322422.2 and NP_001309351.1; NM_001362758.2 and NP_001349687.1; and NM_152854.4 and NP_690593.1). For purposes of the present invention, the terms "CD40 antigen," "CD40 cell surface antigen," "CD40 receptor," or "CD40" encompass all isoforms of CD40.

[0119] The CD40 antigen can be presented on the surface of a variety of cell types, as described elsewhere herein. The terms "surface-presented" and "surface-expressed" refer to when all or a portion of the CD40 antigen is exposed to the exterior of the cell. The presented or expressed CD40 antigen may be fully or partially glycosylated.

[0120] By "antagonistic activity" it is intended that a substance functions as an antagonist. An antagonist of CD40 prevents or reduces the induction of any of the responses induced by binding of the CD40 receptor to an agonistic ligand, particularly CD40L. An antagonist may reduce the induction of any one or more of the responses to agonist binding by 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%, and most preferably 90%, 95%, 99%, or 100%. Methods for measuring the CD40 ligand binding specificity and antagonist activity of anti-CD40 therapeutics, e.g., anti-CD40 antibodies, are known in the art and include, but are not limited to, standard competitive binding assays, assays for monitoring immunoglobulin secretion by B cells, B cell proliferation assays, Banchereau-like B cell proliferation assays, T cell helper assays for antibody production, costimulation of B cell proliferation assays, and assays for upregulation of B cell activation markers. See, e.g., assays such as those disclosed in WO00 / 75348 and U.S. Patent No. 6,087,329, which are incorporated herein by reference. See also provisional and assigned U.S. patent applications 60 / 517,337, 60 / 525,579, and 60 / 565,710, each entitled "ANTAGONIST ANTI-CD40 MONOCLONAL ANTIBODIES AND METHODS OF USE THEREOF," filed November 4, 2003, November 26, 2003, and April 27, 2004, respectively, and International Patent Application No. PCT / US2004 / 037152, entitled "ANTAGONIST ANTI-CD40 MONOCLONAL ANTIBODIES AND METHODS OF USE THEREOF," filed November 4, 2004, and published as WO2005 / 044854; the contents of each are incorporated herein by reference in their entirety.

[0121] Any assay known in the art can be used to determine whether an anti-CD40 antibody acts as an antagonist of one or more B cell responses. In some embodiments, the anti-CD40 antibody acts as an antagonist of at least one B cell response selected from the group consisting of B cell proliferation, B cell differentiation, antibody production, cell-cell adhesion, B cell memory generation, isotype switching, upregulation of cell surface expression of MHC class II and CD80 / 86, and secretion of proinflammatory cytokines such as IL-8, IL-12, and TNF. Of particular interest are antagonist anti-CD40 antibodies that do not exhibit significant agonist activity with respect to B cell proliferation when bound to the human CD40 antigen on the surface of human B cells.

[0122] The term "CD40 ligand" includes any peptide, polypeptide, or protein capable of binding to and activating one or more CD40 signaling pathways. Thus, "CD40 ligand" includes, but is not limited to, full-length CD40 ligand protein and variants and fragments thereof that retain sufficient activity to perform the function of binding to CD40-expressing cells and stimulating CD40 signaling on CD40-expressing cells. Modifications to native CD40 ligand, e.g., human CD40 ligand (CD40L; also known as CD154), include, but are not limited to, substitutions, deletions, truncations, extensions, fusion proteins, fragments, peptidomimetics, and the like. In some embodiments of the invention, assays for evaluating the biological activity of antagonist anti-CD40 antibodies include the use of soluble CD40L, e.g., soluble recombinant human CD40L (Alexis Corporation, Bingham, Nottinghamshire, UK), to stimulate CD40 signaling on CD40-expressing cells.

[0123] "CD40L-mediated CD40 signaling" refers to any of the biological activities resulting from the interaction of the cell surface receptor CD40 with a CD40 ligand. Examples of CD40 signaling are signals that lead to the proliferation and survival of CD40-expressing cells, as well as signals that lead to the stimulation of one or more CD40 signaling pathways in CD40-expressing cells. A CD40 "signal transduction pathway" or "signal transduction pathway" is intended to mean at least one biochemical reaction, or a group of biochemical reactions, that result from the interaction of the CD40 receptor with a CD40 ligand, e.g., CD40L, and that, when transmitted through the signal transduction pathway, produces a signal that leads to the activation of one or more downstream molecules in a signal transduction cascade. A signal transduction pathway involves a number of signal transduction molecules that transmit a signal from the CD40 receptor on the cell surface, across the cell's plasma membrane, through one or more of a series of signal transduction molecules, through the cell's cytoplasm, and potentially to the cell's nucleus. CD40 signaling pathways include, for example, the AKT signaling pathway, which leads to activation of AKT and ultimately to activation of NF-κB via the NF-κB signaling pathway; and the mitogen-activated protein kinase (MAPK) signaling pathway, including the MEK / ERK signaling pathway and the MEK / p38 signaling pathway, which lead to activation of ERK and p38, respectively. The balance between activation and blockade of these signaling pathways favors either cell survival or apoptosis.

[0124] In some embodiments, the CD40 antagonist antibody is a monoclonal antibody. In certain embodiments, the CD40 antagonist antibody, or antigen-binding fragment thereof, is a chimeric antibody, a humanized antibody, a composite antibody, a murine antibody, or a human antibody. In some embodiments, the CD40 antagonist antibody, or antigen-binding fragment thereof, is selected from an Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and a diabody fragment.

[0125] CD40 antagonist antibodies provided herein include, for example, iscalimab (also known as CFZ533), disclosed by Kahaly et al. (2019), J. Endocr. Sco. 3:doi.org / 10.1210 / js.2019-OR19-6, Fisher et al. (2017), Arthritis Rheumatol. 69:1784, Farkash et al. (2019), Am. J. Transplant. 19:632, and International Patent Application Publication No. WO2012 / 075111A1; ravagalimab (also known as ABBV-323), disclosed in International Patent Application Publication No. WO2016 / 196314A1; BI655064, disclosed by Visvannathan et al. (2016), Arthritis Rheumatol. 68:1588); (2018), Biopharm. Drug Dispos. 39:245-255, Harland et al. (2017), Am. J. Transplant. 17:159-171, and U.S. Patent No. 8,716,451 B2; ch5D12, disclosed in Kasran et al. (2005), Aliment. Pharmacol. Ther. 22:111-122; Bensinger et al. (2012), British J. Haematology, 159:58-66, Byrd et al. lucatumumab (also known as HCD122 or CHIR-12.12), disclosed in International Patent Application No. PCT / US2004 / 037152; CHIR-5.9, disclosed in International Patent Application No. PCT / US2004 / 037152; 201A3, disclosed in Perper et al. (2019), J. Immunol. 203:58-75; KPL-404, disclosed in clinical trial NCT04497662 sponsored by Kiniksa Pharmaceuticals, Ltd.; Bankert et al. (2015), J. Immunol.194:4319-4327 and International Patent Application Publication No. WO2001 / 024823A1; and BIIB063 disclosed in Musselli et al. (2017), 2017 ACR / ARHP Annual Meeting Abstracts, the contents of each of which are incorporated herein by reference in their entirety.

[0126] Additional CD40 antagonist antibodies useful in certain embodiments of the methods and compositions provided herein are described, for example, in International Patent Application Publication Nos. WO02 / 11763A1, WO02 / 28481A9, WO03 / 045978A3, WO03 / 029296A1, WO03 / 028809A1, WO2005 / 044854, WO2006 / 073443A3, WO2007 / 124299A8, WO2011 / 123489A3, WO2016 / 196,314A1, WO2017 / 040566A1, WO2017 / 060242A No. 1, WO2018 / 217976A1, WO2019 / 156565A1, WO2020 / 144605A1, WO2020 / 106620A1, and WO2020 / 006347A1, U.S. Patent Application Publication Nos. US2020 / 0291123A1, US2017 / 0158771A1, and US2008 / 0057070A1, or U.S. Patent Nos. US9,125,893B2, US8,669,352B2, and US9,598,494B2, the contents of each of which are incorporated herein by reference in their entirety.

[0127] The anti-CD40 antagonist antibodies disclosed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LIn one embodiment, all of the framework and / or CDR residues within the domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residues in certain germline sequences while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in the different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may exhibit improved binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., K D The antibodies and antigen-binding fragments obtained by this general method are within the scope of the present invention.

[0128] Also provided herein are anti-CD40 antagonist antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes anti-CD40 antagonist antibodies or CD3 multispecific (e.g., bispecific or trispecific) antibodies having HCVR, LCVR, and / or CDR amino acid sequences with no more than 10, no more than 8, no more than 6, no more than 4, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0129] CD40 antagonist-expressing CAR-T cells In certain aspects, the methods and compositions provided herein relate to immune effector cells (e.g., CAR-T cells) that have been engineered to express a CD40 antagonist (e.g., a CD40 antagonist provided herein). In some embodiments, the CAR-T cells secrete the CD40 antagonist. In certain embodiments, the CD40 antagonist is an scFv or Fab. In some embodiments, the CAR-T cells express the CD40 antagonist upon activation. Methods for generating CAR-T cells that secrete antibodies or antigen-binding fragments thereof are disclosed, for example, in Choi et al. (2019), Nature Biotechnology, 37:1049-1058, which is incorporated herein by reference in its entirety.

[0130] CAR-T cells are T cells engineered to express a chimeric antigen receptor (CAR) polypeptide. A CAR is a receptor that comprises a targeting moiety associated with one or more signaling and / or costimulatory domains in a single fusion molecule. In certain embodiments, the binding moiety of the CAR comprises an antigen-binding domain of a single-chain antibody (scFv), which comprises variable fragments of the light and heavy chains of a monoclonal antibody linked by a flexible linker. In certain embodiments, the binding moiety further comprises a transmembrane domain and a hinge domain of a monoclonal antibody. In certain embodiments, the binding domain and / or extracellular domain of the CAR provided herein provides the CAR with the ability to bind to a target antigen of interest (e.g., a tumor antigen).

[0131] The "signal transduction domain" or "signal transduction domain" of a CAR as used herein is involved in intracellular signal transduction following binding of the extracellular ligand binding domain to a target, resulting in immune cell activation and immune response. In other words, the signal transduction domain is involved in the activation of at least one of the normal effector functions of the immune cell expressing the CAR. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "signal transduction domain" refers to a portion of a protein that transduces an effector function signal and instructs the cell to perform a specialized function. Examples of signal transduction domains for use in CARs can be cytoplasmic sequences of T cell receptors and co-receptors that cooperate to initiate signal transduction after binding to an antigen receptor, as well as derivatives or variants of these sequences, and synthetic sequences with the same functional capabilities. In some cases, the signal transduction domain includes two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can include signaling motifs known as ITAM immunoreceptor tyrosine-based activation motifs. ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Exemplary ITAMs include those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0132] In some embodiments, the cells are obtained from the subject to be treated (i.e., autologous), however, in certain embodiments, immune effector cell lines or donor effector cells (allogeneic) are used.

[0133] Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood drawn from a subject using any number of techniques known to those of skill in the art, such as Ficoll™ separation. For example, cells from an individual's circulating blood may be obtained by apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. Specific subpopulations of immune effector cells can be further isolated by techniques of positive or negative selection. For example, immune effector cells can be isolated using a combination of antibodies against surface markers specific to the positively selected cells, for example, by incubation with antibody-conjugated beads for a period of time sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of immune effector cell populations can be achieved by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.

[0134] The present disclosure provides a method of producing immune effector cells expressing a CAR and a CD40 antagonist as described herein. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from a subject, such as a subject having tumor cells expressing PIG and / or low density cancer antigens, such that the immune effector cells express one or more CARs and CD40 antagonists as described herein. In certain embodiments, immune effector cells are isolated from an individual and genetically engineered in vitro without further manipulation. Such cells can then be directly re-administered to the individual. In further embodiments, the immune effector cells are first activated and stimulated to proliferate in vitro before being genetically engineered to express a CAR and a CD40 antagonist. In this regard, the immune effector cells may be cultured before or after being genetically engineered (i.e., transduced or transfected to express a CAR or a CD40 antagonist as described herein).

[0135] Prior to in vitro or genetic manipulation of immune effector cells as described herein, a source of cells may be obtained from a subject. In particular, immune effector cells for use with the CAR and CD40 antagonists described herein include T cells. T cells may be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells may be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a buffer or medium appropriate for subsequent processing steps. In one embodiment, the cells are washed with PBS. In alternative embodiments, the wash solution lacks calcium, but may lack magnesium, or may lack many, but not all, divalent cations. As will be appreciated by those of skill in the art, the wash step may be accomplished by methods known to those of skill in the art, such as using a semi-automated flow-through centrifuge. After washing, the cells may be resuspended in a variety of biocompatible buffers, or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed by resuspending the cells directly in culture medium.

[0136] In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes, for example, by centrifugation through a Ficoll-Paque™ gradient. Specific subpopulations of T cells, such as CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or cell selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the cocktail of monoclonal antibodies typically includes antibodies against CD14, CD20, CD1b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may be used to isolate the cell population of interest.

[0137] PBMCs may be used directly for genetic engineering with CARs and CD40 antagonists using methods as described herein. In certain embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in certain embodiments, both cytotoxic and helper T lymphocytes may be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic engineering and / or expansion. CD8+ cells may be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells. In embodiments, memory T cells are present in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, expression of phenotypic markers of central memory TCMs includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and is negative for granzyme B. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and are positive for granzyme B and perforin. In some embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

[0138] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+ CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L positive and CD45RO positive. In some embodiments, effector CD4+ cells are CD62L negative and CD45RO negative.

[0139] Immune effector cells such as T cells can be genetically engineered after isolation using known methods, or immune effector cells can be activated and expanded (or differentiated in the case of precursor cells) in vitro before being genetically engineered. In another embodiment, immune effector cells such as T cells are genetically engineered with a chimeric antigen receptor as described herein (e.g., transduced with a viral vector containing a nucleic acid encoding a CAR or CD40 antagonist) and then activated and expanded in vitro. Methods for activating and expanding T cells are known in the art and are described, for example, in U.S. Patent Nos. 6,905,874; 6,867,041; 6,797,514; and WO2012079000. In general, such methods include contacting PBMCs or isolated T cells in a culture medium containing an appropriate cytokine, such as IL-2 (e.g., recombinant human IL-2), with stimulatory and costimulatory agents, such as anti-CD3 and anti-CD28 antibodies, typically attached to beads or other surfaces. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as "surrogate" antigen presenting cells (APCs). In other embodiments, T cells may be activated, stimulated, and expanded with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Patent Nos. 6,040,177; 5,827,642; and WO2012129514.

[0140] In some embodiments, immune effector cells include any white blood cell involved in the body's defense against infection and foreign substances. For example, immune effector cells can include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells can include T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0141] Helper T cells (T H CD4 T cells (CD4 T cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore + Also known as T cells, helper T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or aid in an active immune response. These cells are classified as T helper cells, which secrete a variety of cytokines to promote different types of immune responses. H 1. T H 2. T H 3. T H 17, T H 9, or T FH They can be differentiated into one of several subtypes, including:

[0142] Cytotoxic T cells (T C CTLs (cytotoxic T-lymphoblastic leukemia cells) destroy virus-infected and tumor cells and are involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore CD8 + Also known as T cells, these cells recognize their targets by binding to antigens associated with MHC class I molecules, which are present on the surface of all nucleated cells. They recognize their targets through IL-10, adenosine, and other molecules secreted by regulatory T cells, and through CD8 +Cells can be inactivated and put into an anergic state, thereby preventing autoimmune diseases.

[0143] Memory T cells are a subset of antigen-specific T cells that persist for a long time after an infection has subsided. They rapidly expand into large numbers of effector T cells upon reexposure to the cognate antigen, providing the immune system with a "memory" of past infections. Memory cells are CD4 + or CD8 + Memory T cells typically express the cell surface protein CD45RO.

[0144] Regulatory T cells (T reg CD4 T cells, formerly known as suppressor T cells, are important in maintaining immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. + T reg The cells were cultured with naturally occurring T reg Cells and adaptive T reg Two major classes of cells have been described.

[0145] Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) act as a bridge between the adaptive and innate immune systems. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d.

[0146] In some embodiments, the T cells are CD4 + In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T cells are enriched for one or more subsets based on cell surface expression. + Contains T lymphocytes.

[0147] Natural killer (NK) cells express CD56, which can kill virus-infected or transformed cells. +CD3 - These are large granular lymphocytes that constitute an important cell subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma, 2012, 53:1666-1676). + Unlike T lymphocytes, NK cells can initiate cytotoxicity against tumor cells and eradicate MHC-I negative cells without the need for prior sensitization (Narni-Mancinelli E, et al. Int. Immunol. 2011, 23:427-431). NK cells are safer effector cells because they may avoid the potentially fatal complications of cytokine storm (Morgan RA, et al. Mol. Ther. 2010, 18:843-851), tumor lysis syndrome (Porter DL, et al. N. Engl. J. Med. 2011, 365:725-733) and targeted extratumoral effects. CD3 multispecific antigen binding molecule In certain embodiments, the methods and compositions provided herein relate to CD3 antigen binding molecules (i.e., antigen binding molecules that comprise at least one antigen binding domain that binds to CD3). In certain embodiments, the CD3 multispecific antigen binding molecules provided herein further comprise an antigen binding domain that binds to a cancer antigen (i.e., an antigen expressed on a cancer cell). In certain embodiments, the CD3 multispecific antigen binding molecules provided herein further comprise an antigen binding domain that binds to a costimulatory receptor (e.g., CD28).

[0148] As used herein, the term "multispecific antigen-binding molecule" refers to a protein, polypeptide or molecular complex comprising at least a first antigen-binding domain and a second antigen-binding domain. In some embodiments, each antigen-binding domain in a multispecific antigen-binding molecule may comprise at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding domain specifically binds a first antigen (e.g., CD3), and the second antigen-binding domain specifically binds a second, different antigen (e.g., a tumor antigen).

[0149] In some embodiments, the CD3 multispecific antigen-binding molecule is a CD3 multispecific antibody. The CD3 multispecific antibodies provided herein may be, for example, bispecific or trispecific. A multispecific antibody may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains 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 CD3 bispecific antibodies provided herein can be linked to or co-expressed with another functional molecule, for example, another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent binding or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody with a second or additional binding specificity.

[0150] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR) and consisting of a homodimer or heterodimer formed from the association of two of the four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acid sequence set forth in SEQ ID NO: 116 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; human CD3-delta comprises the amino acid sequence set forth in SEQ ID NO: 117 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; human CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 118 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety; and CD3-gamma comprises the amino acid sequence set forth in SEQ ID NO: 119 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. All references herein to proteins, polypeptides and protein fragments are intended to refer to the human form of the respective protein, polypeptide or protein fragment, unless expressly specified as being from a non-human species. Thus, the term "CD3" refers to human CD3, unless specified that it is derived from a non-human species, e.g., "mouse CD3", "monkey CD3", etc.

[0151] As used herein, "antibodies that bind CD3" or "anti-CD3 antibodies" include antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The antibodies and antigen-binding fragments of the invention may bind soluble CD3 and / or cell surface expressed CD3. Soluble CD3 includes native CD3 proteins as well as recombinant CD3 protein variants that lack a transmembrane domain or are not associated with a cell membrane, such as, for example, monomeric and dimeric CD3 constructs.

[0152] As used herein, the phrase "cell surface expressed CD3" refers to one or more CD3 protein(s) expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to the antigen-binding portion of an antibody. Cell surface expressed CD3 includes CD3 protein contained within the context of a functional T cell receptor in the cell membrane. Cell surface expressed CD3 includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). Cell surface expressed CD3 also includes CD3 chains expressed by themselves on the cell surface without other types of CD3 chains (e.g., CD3-epsilon, CD3-delta, or CD3-gamma). Cell surface expressed CD3 can include or consist of CD3 protein expressed on the surface of a cell that normally expresses CD3 protein. Alternatively, CD3 expressed on the cell surface can comprise or consist of CD3 protein expressed on the surface of a cell that does not normally express human CD3 on its surface, but has been artificially engineered to express CD3 on its surface.

[0153] In some embodiments, the invention includes bispecific antibodies in which one immunoglobulin arm binds CD3 and the other immunoglobulin arm is specific for a cancer antigen (also referred to herein as a tumor antigen or "TAA"). In some embodiments, the invention includes trispecific antibodies in which a first immunoglobulin arm binds CD3, a second immunoglobulin arm is specific for a tumor antigen, and a third immunoglobulin arm binds an additional T cell antigen (e.g., CD28) or an additional tumor antigen.

[0154] In some embodiments, the CD3 binding arm may comprise any of the amino acid sequences of HCVR / LCVR or CDRs disclosed in WO2014 / 047231 or WO2017 / 053856. In certain embodiments, the CD3 binding arm binds human CD3 and induces human T cell activation. In certain embodiments, the CD3 binding arm binds weakly to human CD3 and induces human T cell activation. In other embodiments, the CD3 binding arm binds weakly to human CD3 and induces killing of tumor-associated antigen-expressing cells in association with a bispecific or multispecific antibody. In other embodiments, the CD3 binding arm binds or weakly associates with human and cynomolgus (monkey) CD3, but the binding interaction is not detectable by in vitro assays known in the art.

[0155] In certain embodiments, a multispecific antibody or antigen-binding fragment for use in the invention comprises an antigen-binding arm that binds to CD28, ICOS, HVEM, CD27, 4-1BB, OX40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1, or TIM2 to induce T cell activation.

[0156] In certain embodiments, the CD3 multispecific antigen binding molecule comprises an antigen binding domain specific for a cancer antigen. In certain embodiments, the cancer antigen is selected from the group consisting of AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCMA, BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD20, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin 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, glypican-3, GnTV, gp100 / Pmel17, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), LAGE-1, LDLR-fucosyltransferase, and HL-1. fusion protein, lentinol, 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, mellow, midkine, MMP-2, MMP-7, MUC1, MUC5AC, MUC16, mucin, MUM-1, MUM-2, MUM-3, Myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, Se Selected from cerunin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, STEAP2, survivin, SYT-SSX1 or -SSX2 fusion proteins, TAG-1, TAG-2, telomerase, TGF-beta RII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, and XAGE-1b / GAGED2a.

[0157] In some embodiments, cancer antigens include ADAM17, BCMA, CA-IX, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD56, CD70, 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. In some embodiments, the tumor antigen may be CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, or BCMA.

[0158] In some embodiments, the cancer antigen is CD20, MUC16, BCMA, PSMA, or STEAP2. CD20 is a non-glycosylated phosphoprotein expressed on the cell membrane of mature B cells. CD20 is considered a B cell tumor-associated antigen because it is expressed in more than 95% of B cell non-Hodgkin's lymphomas (NHL) and other B cell malignancies, but is absent from precursor B cells, dendritic cells, and plasma cells. Human CD20 protein has the amino acid sequence shown in SEQ ID NO:5 of U.S. Patent Application Publication No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0159] MUC16 refers to mucin 16. MUC16 is a single transmembrane domain highly glycosylated integral membrane glycoprotein that is highly expressed in ovarian cancer. The amino acid sequence of human MUC16 is shown in SEQ ID NO: 1899 of US Patent Application Publication No. US2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety.

[0160] BCMA refers to B cell maturation antigen. BCMA (also known as TNFRSF17 and CD269) is a cell surface protein expressed on malignant plasma cells and plays a central role in regulating B cell maturation and differentiation into immunoglobulin-producing plasma cells. The amino acid sequence of human BCMA is shown in SEQ ID NO: 115 of U.S. Patent Application Publication No. US2020 / 0024356, the contents of which are incorporated herein by reference in their entirety. It can also be found in GenBank Accession No. NP_001183.2.

[0161] PSMA refers to prostate-specific membrane antigen, also known as folate hydrolase 1 (FOLH1). PSMA is an endogenous non-decidual 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 contents of which are incorporated herein by reference in their entirety.

[0162] STEAP2 refers to six-transmembrane epithelial antigen of prostate 2. STEAP2 is a six-transmembrane integral protein that is highly expressed in prostate epithelial cells and is a cell surface marker for prostate cancer. STEAP2 is a 490 amino acid protein encoded by the STEAP2 gene located in chromosomal region 7q21 in humans. The amino acid sequence of human STEAP2 is shown in SEQ ID NO:9 of U.S. Patent Application Publication No. US2020 / 0129617, the contents of which are incorporated herein by reference in their entirety.

[0163] In some embodiments the CD3 multispecific antibody may be a bispecific CD3×CD19 antibody, a bispecific CD3×GPRC5D antibody, a bispecific CD3×CD123 antibody, a bispecific CD3×STEAP2 antibody, a bispecific CD3×CD20 antibody, a bispecific CD3×SSTR2 antibody, a bispecific CD3×CD38 antibody, a bispecific CD3×STEAP1 antibody, a bispecific CD3×5T4 antibody, a bispecific CD3×ENPP3 antibody, a bispecific CD3×MUC16 antibody, a bispecific CD3×BCMA antibody, a bispecific CD3×PSMA antibody or a trispecific CD3×CD28×CD38 antibody.

[0164] In some embodiments, the invention includes an antibody having the amino acid sequence of the HCVR, LCVR and / or CDR of an antibody set forth herein, an anti-CD3 antibody disclosed in WO2014 / 047231 or WO2017 / 053856, a bispecific anti-CD20 x anti-CD3 antibody disclosed in WO2014 / 047231, a bispecific anti-PSMA x anti-CD3 antibody disclosed in WO2017 / 023761, a bispecific anti-MUC16 x anti-CD3 antibody disclosed in WO2018 / 067331, a bispecific anti-STEAP2 x anti-CD3 antibody disclosed in WO2018 / 058001, or a bispecific anti-BCMA x anti-CD3 antibody disclosed in WO2020 / 018820, each of which is incorporated herein by reference.

[0165] In certain embodiments, the multispecific antigen-binding molecule is a multispecific antibody or an antigen-binding fragment thereof. Each antigen-binding domain of the multispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "A1", and the CDRs of the second antigen-binding domain may be designated with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3. In the context of a trispecific antigen-binding molecule (e.g., a trispecific antibody) comprising a first, second, and third antigen-binding domain, the CDRs of the first antigen-binding domain may be designated with the prefix "A1", the CDRs of the second antigen-binding domain may be designated with the prefix "A2", and the CDRs of the third antigen-binding domain may be designated with the prefix "A3". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3; and the CDRs of the third antigen-binding domain may be referred to herein as A3-HCDR1, A3-HCDR2, and A3-HCDR3.

[0166] The bispecific antigen-binding molecule described above or herein may be a bispecific antibody. Optionally, the bispecific antibody comprises a human IgG heavy chain constant region. Optionally, the human IgG heavy chain constant region is of isotype IgG1. Optionally, the human IgG heavy chain constant region is of isotype IgG4. In various embodiments, the bispecific antibody comprises a chimeric hinge that reduces Fcγ receptor binding compared to a wild-type hinge of the same isotype.

[0167] The first and second antigen-binding domains may be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first and second antigen-binding domains may each be connected to a separate multimerization domain. The binding of one multimerization domain to another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerization domain" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerization domain of the same or similar structure or composition. For example, a multimerization domain may be a multimerization domain that is capable of associating with a second multimerization domain of the same or similar structure or composition. H A non-limiting example of a multimerizing component is the Fc portion of an immunoglobulin (C H 2-C H The Fc domain of IgG includes 3 domains, for example, the isotypes IgG1, IgG2, IgG3, and IgG4, as well as allotypes within each isotype group.

[0168] A bispecific antigen-binding molecule of the invention will usually comprise two multimerization domains, e.g., two Fc domains, each being an individual part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0169] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length 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 that include or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0170] Any bispecific antibody format or technique may be used to generate the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent bonding, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to generate a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab. 2 and bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0171] In the context of the bispecific antigen-binding molecules provided herein, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring form of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules that contain one or more modifications in the Fc domain that result in a modified Fc domain with an engineered binding interaction (e.g., enhanced or decreased) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a C H 2 or C H 3 region, where the modification increases the affinity of the Fc domain for FcRn in an acidic environment (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, for example, 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, the modifications include the 428L (e.g., M428L) and 434S (e.g., N434S) modifications; the 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; the 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; the 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; the 250Q and 428L modifications (e.g., T250Q and M428L); and the 307 and / or 308 modifications (e.g., 308F or 308P).

[0172] In certain embodiments, provided herein is a first C H 3 domain and second Ig C H a bispecific antigen-binding molecule comprising three domains, wherein a first and a second IgC HThe three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds protein A and the second Ig C H The 3 domain contains a mutation that reduces or abolishes Protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further include a Y96F modification (according to IMGT; Y436F according to the EU). See, e.g., U.S. Patent No. 8,586,713. H Further modifications that may be found within 3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I according to EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU).

[0173] In certain embodiments, the Fc domain may be a chimera that combines Fc sequences from more than one immunoglobulin isotype. For example, a chimeric Fc domain may be a chimeric Fc domain that combines Fc sequences from human IgG1, human IgG2, or human IgG4. H C derived from 2 regions H 2 sequences, and C derived from human IgG1, human IgG2 or human IgG4 HThe chimeric Fc domain may comprise some or all of the three sequences. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may comprise an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region combined with 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 of the antigen-binding molecules provided herein is, from N-terminus to C-terminus: [IgG4 C H Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules provided herein comprises, from the N-terminus to the C-terminus: [IgG1 C H IgG1 CH3]-[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 of the invention are described in U.S. Patent Application Publication No. 2014 / 0243504, published August 28, 2014, and incorporated herein in its entirety. Chimeric Fc domains having these general structural arrangements and variants thereof can alter Fc receptor binding, which in turn affects Fc effector function.

[0174] The CD3 multispecific (e.g., bispecific or trispecific) antibodies disclosed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LIn one embodiment, all of the framework and / or CDR residues within the domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residues in certain germline sequences while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in the different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations may exhibit, for example, improved binding specificity, increased binding (e.g., as measured by cell binding titration or FACS binding) or binding affinity (e.g., K D The antibodies and antigen-binding fragments obtained by this general method are within the scope of the present invention.

[0175] Also provided herein are CD3 multispecific (e.g., bispecific or trispecific) antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the invention includes anti-CD40 antagonist antibodies or CD3 multispecific (e.g., bispecific or trispecific) antibodies that have HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0176] Exemplary CD3xMUC16 Antibodies In some embodiments, the methods and compositions provided herein include bispecific antibodies in which one immunoglobulin arm binds human CD3 and the other immunoglobulin arm is specific for human MUC16. The term "MUC16" as used herein refers to human MUC16 protein unless specified to be from a non-human species (e.g., "mouse MUC16", "monkey MUC16", etc.). Human MUC16 protein has the amino acid sequence set forth in SEQ ID NO: 1899 of U.S. Patent Application Publication No. US2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein, for example, as "anti-CD3 / anti-MUC16", or "anti-CD3xMUC16" or "CD3xMUC16" bispecific molecules, or other similar terms (e.g., anti-MUC16 / anti-CD3). Such bispecific antigen-binding molecules are constructed with a first antigen-binding arm that binds MUC16 and a second antigen-binding arm that binds CD3. The MUC16-binding arm can include any of the amino acid sequences of the HCVR / LCVR or CDRs shown in Table 1 herein. The CD3-binding arm can include any of the amino acid sequences of the HCVR / LCVR or CDRs shown in Tables 2-6 herein. The sequences in Tables 1-6 are disclosed in U.S. Patent Application Publication No. US2018 / 0118848A1, the contents of which are incorporated herein by reference in their entirety. The SEQ ID NOs in Tables 1-6 refer to the sequences in U.S. Patent Application Publication No. US2018 / 0118848A1.

[0177] Table 1 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-MUC16 antibodies of the invention.

[0178] [Table 1]

[0179] Table 2 provides amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-CD3 antibodies of the invention. Methods for making the anti-CD3 antibodies disclosed herein can also be found in U.S. Patent Application Publication No. 2014 / 0088295.

[0180] [Table 2-1]

[0181] [Table 2-2]

[0182] [Table 2-3]

[0183] Tables 3 and 4 provide amino acid sequence identifiers for the heavy chain variable regions (Table 3) and light chain variable regions (Table 4) of additional anti-CD3 HCVRs and LCVRs useful in the anti-MUC16 x anti-CD3 bispecific antibodies of the invention, and their corresponding CDRs.

[0184] [Table 3]

[0185] [Table 4]

[0186] Table 5 shows the amino acid sequence identifiers of the heavy chain variable regions and CDRs of the engineered anti-CD3 antibodies of the invention. The amino acid sequence identifiers of the light chain variable regions and CDRs are also identified in Table 6 below.

[0187] [Table 5]

[0188] [Table 6]

[0189] In certain exemplary embodiments, a first antigen-binding domain that specifically binds human CD3 comprises a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 1730, 1762, 1778, 1786, and 1866, and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) from a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 26.

[0190] In certain exemplary embodiments, the first antigen-binding domain that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2 and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2 and A1-LCDR3), where A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788, and 1868; HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, 1782, 1790, and 1870; A1-HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1736, 1768, 1784, 1792, and 1872; A1-LCDR1 comprises the amino acid sequence of SEQ ID NO: 28; A1-LCDR2 comprises the amino acid sequence of SEQ ID NO: 30; and A1-LCDR3 comprises the amino acid sequence of SEQ ID NO: 32.

[0191] In certain exemplary embodiments, the first antigen-binding domain that specifically binds human CD3 comprises the heavy and light chain CDRs of a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1730 / 26, 1762 / 26, 1778 / 26, 1786 / 26, and 1866 / 26.

[0192] In certain exemplary embodiments, a first antigen-binding domain that specifically binds human CD3 comprises three heavy chain complementarity determining regions (A1-HCDR1, A1-HCDR2, and A1-HCDR3) and three light chain complementarity determining regions (A1-LCDR1, A1-LCDR2, and A1-LCDR3), and a second antigen-binding domain that specifically binds human MUC16 comprises three heavy chain complementarity determining regions (A2-HCDR1, A2-HCDR2, and A2-HCDR3) and three light chain complementarity determining regions (A2-LCDR1, A2-LCDR2, and A2-LCDR3), where A1-HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1732, 1764, 1780, 1788, and 1868; A1-HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1734, 1766, A1-HCDR1 comprises the amino acid sequence of SEQ ID NO:28; A1-LCDR2 comprises the amino acid sequence of SEQ ID NO:30; and A1-LCDR3 comprises the amino acid sequence of SEQ ID NO:32, and A2-HCDR1 comprises the amino acid sequence of SEQ ID NO:20; A2-HCDR2 comprises the amino acid sequence of SEQ ID NO:22; A2-HCDR3 comprises the amino acid sequence of SEQ ID NO:24; A2-LCDR1 comprises the amino acid sequence of SEQ ID NO:28; A2-LCDR2 comprises the amino acid sequence of SEQ ID NO:30; and A2-LCDR3 comprises the amino acid sequence of SEQ ID NO:32.

[0193] Further bispecific anti-MUC16 x anti-CD3 antibodies are disclosed, for example, in WO2018 / 067331, which is incorporated herein by reference. Exemplary CD3×BCMA Antibodies In some embodiments, the methods and compositions provided herein include bispecific antibodies in which one arm of the immunoglobulin binds human CD3 and the other arm of the immunoglobulin is specific for human BCMA. The term "BCMA" as used herein refers to human BCMA protein unless specified to be from a non-human species (e.g., "mouse BCMA", "monkey BCMA", etc.). Human BCMA protein has the amino acid sequence set forth in SEQ ID NO: 115 of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein, for example, as "anti-BCMA x anti-CD3" or "anti-CD3 / anti-BCMA", or "anti-CD3 x BCMA" or "CD3 x BCMA" bispecific molecules, or other similar terms (e.g., anti-BCMA / anti-CD3). The BCMA binding arm may comprise any of the amino acid sequences of the HCVR / LCVR or CDRs set forth in Table 7 herein. The CD3 binding arm can comprise any of the amino acid sequences of the HCVR / LCVR or CDRs shown in Table 8 herein, or an anti-CD3 antibody disclosed in WO2014 / 047231 or WO2017 / 053856. The sequences in Tables 7 and 8 are disclosed in U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety. The SEQ ID NOs in Tables 7 and 8 refer to the sequences in U.S. Patent Application Publication No. US2020 / 0024356A1.

[0194] Table 7 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-BCMA antibodies of the invention.

[0195] [Table 7]

[0196] Table 8 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-CD3 antibodies. Other anti-CD3 antibodies for use in preparing bispecific antibodies according to the present invention can be found, for example, in WO2014 / 047231.

[0197] [Table 8]

[0198] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises a first antigen binding domain comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 66; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the isolated bispecific antigen binding molecule comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 68, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 70, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 72. Optionally, the isolated bispecific antigen binding molecule comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 88. In some cases, the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 66 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82. The above SEQ ID NOs refer to sequences in U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0199] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises a second antigen binding domain comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98; and (b) three light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 82. Optionally, the second antigen binding domain comprises: (a) an HCDR1 comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100; (b) an HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102; and (c) an HCDR3 comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104. Optionally, the second antigen binding domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 84, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 86, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 88. In some cases, the second antigen-binding domain comprises: (a) an HCDR1, HCDR2, HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 92, 94, 96, respectively; an LCDR1, LCDR2, LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively; or (b) an HCDR1, HCDR2, HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 100, 102, 104, respectively; and an LCDR1, LCDR2, LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 84, 86, 88, respectively. In some cases, the second antigen-binding domain comprises: (a) an HCVR comprising the amino acid sequence of SEQ ID NO: 90 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82; or (b) an HCVR comprising the amino acid sequence of SEQ ID NO: 98 and an LCVR comprising the amino acid sequence of SEQ ID NO: 82. The above SEQ ID NOs refer to the sequences of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0200] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively; and (b) a second antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. Optionally, the isolated bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 66, and a LCVR comprising the amino acid sequence of SEQ ID NO: 82; and (b) a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 90, and a LCVR comprising the amino acid sequence of SEQ ID NO: 82.

[0201] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 68, 70, 72, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively; and (b) a second antigen binding domain comprising HCDR1, HCDR2, HCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 100, 102, 104, respectively, and LCDR1, LCDR2, LCDR3 domains comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88, respectively. Optionally, the isolated bispecific antigen binding molecule comprises: (a) a first antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 66, and a LCVR comprising the amino acid sequence of SEQ ID NO: 82; and (b) a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 98, and a LCVR comprising the amino acid sequence of SEQ ID NO: 82. The above sequence numbers refer to sequences from U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated by reference in their entirety.

[0202] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule comprises: (a) a first antigen binding domain that specifically binds human BCMA and comprises a CDR of an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 122, and 124, and comprises a CDR of an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 82, 123, and 125; and (b) a second antigen binding domain that specifically binds human CD3. In some cases, the first antigen-binding domain comprises CDRs derived from a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 122 / 123, 124 / 125, 2 / 82, 18 / 82, 34 / 82, 50 / 82, 66 / 82, 122 / 82, and 124 / 82. In some cases, the first antigen binding domain comprises a HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domain selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64, 68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88, 52-54-56-84-86-88, and 68-70-72-84-86-88, respectively. In some cases, the first antigen-binding domain comprises a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 122 / 123, 124 / 125, 2 / 82, 18 / 82, 34 / 82, 50 / 82, 66 / 82, 122 / 82, and 124 / 82. In some cases, the second antigen-binding domain comprises the CDRs of a pair of HCVR / LCVR amino acid sequences selected from the group consisting of SEQ ID NOs: 90 / 82 and 98 / 82. The above SEQ ID NOs refer to the sequences of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0203] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule competes for binding to BCMA or binds to the same epitope on BCMA as a reference antibody, where the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 90 / 82 or SEQ ID NO: 98 / 82. The above SEQ ID NOs refer to the sequences of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0204] In certain exemplary embodiments, the isolated anti-BCMA x anti-CD3 bispecific antigen binding molecule competes for binding to human CD3 or binds to the same epitope on human CD3 as a reference antibody, where the reference antibody comprises a first antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of SEQ ID NO: 66 / 82 and a second antigen binding domain comprising a HCVR / LCVR pair comprising the amino acid sequence of either SEQ ID NO: 90 / 82 or SEQ ID NO: 98 / 82. The above SEQ ID NOs refer to the sequences of U.S. Patent Application Publication No. US2020 / 0024356A1, the contents of which are incorporated herein by reference in their entirety.

[0205] Additional bispecific anti-BCMA x anti-CD3 antibodies are disclosed, for example, in WO2020 / 018820. CD3×CD20 antibody In some embodiments, provided herein are bispecific antibodies in which one arm of the immunoglobulin binds human CD3 and the other arm of the immunoglobulin is specific for human CD20. The term "CD20" as used herein refers to human CD20 protein unless specified to be derived from a non-human species (e.g., "mouse CD20", "monkey CD20", etc.). Human CD20 protein has the amino acid sequence shown in SEQ ID NO: 1369 of U.S. Patent No. 9,657,102B2, the contents of which are incorporated herein by reference in their entirety. Such molecules may be referred to herein, for example, as "anti-CD3 / anti-CD20", or "anti-CD3xCD20", or "CD3xCD20" bispecific molecules, or other similar terms.

[0206] In certain embodiments, the first antigen binding domain that specifically binds CD3 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1250, 1266, 1282, 1298, 1314, and 1329, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. All of the sequences disclosed in this section for antigen binding domains that specifically bind CD3 or CD20, and corresponding SEQ ID NOs, are derived from U.S. Patent No. US9,657,102B2, the contents of which are incorporated herein by reference in their entirety.

[0207] In certain embodiments, the first antigen-binding domain that specifically binds CD3 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322 and 1333, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0208] In certain embodiments, the first antigen-binding domain that specifically binds CD3 comprises a pair of HCVR and LCVR (HCVR / LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 1250 / 1258, 1266 / 1274, 1282 / 1290, 1298 / 1306, 1314 / 1322, and 1329 / 1333.

[0209] In certain embodiments, the first antigen-binding domain that specifically binds CD3 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1252, 1268, 1284, 1300, 1316 and 1330, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1256, 1272, 1288, 1304, 1320 and 1332, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; 1275, 1276, 1277, 1278, 1279, 1280, 1296, 1312, 1328 and 1336, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0210] In certain embodiments, the first antigen-binding domain that specifically binds CD3 comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1252-1254-1256-1260-1262-1264; 1268-1270-1272-1276-1278-1280; 1284-1286-1288-1292-1294-1296; 1300-1302-1304-1308-1310-1312; 1316-1318-1320-1324-1326-1328; and 1330-1331-1332-1334-1335-1336, respectively.

[0211] In certain embodiments, the second antigen-binding domain that specifically binds CD20 comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 1242, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0212] In certain embodiments, the second antigen-binding domain that specifically binds CD20 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1258, 1274, 1290, 1306, 1322, and 1333, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0213] In certain embodiments, the second antigen-binding domain that specifically binds CD20 comprises a pair of HCVR and LCVR (HCVR / LCVR) amino acid sequences selected from the group consisting of SEQ ID NOs: 1242 / 1258, 1242 / 1274, 1242 / 1290, 1242 / 1306, 1242 / 1322 and 1242 / 1333.

[0214] In certain embodiments, the second antigen-binding domain that specifically binds CD20 is selected from the group consisting of: a heavy chain CDR1 (HCDR1) domain having the amino acid sequence of SEQ ID NO: 1244, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR2 (HCDR2) domain having the amino acid sequence of SEQ ID NO: 1246, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a heavy chain CDR3 (HCDR3) domain having the amino acid sequence of SEQ ID NO: 1248, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1262, 1278, 1294, 1310, 1326 and 1335, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1264, 1280, 1296, 1312, 1328 and 1336, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0215] In certain embodiments, the second antigen binding domain that specifically binds to CD20 comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1244-1246-1248-1260-1262-1264; 1244-1246-1248-1276-1278-1280; 1244-1246-1248-1292-1294-1296; 1244-1246-1248-1308-1310-1312; 1244-1246-1248-1324-1326-1328; and 1244-1246-1248-1334-1335-1336, respectively.

[0216] Additional bispecific anti-CD20 / anti-CD3 antibodies are disclosed, for example, in US Pat. No. 9,657,102, which is incorporated by reference herein in its entirety. Other Exemplary CD3 Multispecific Antibodies Additional exemplary CD3 multispecific antibodies that can be used in the compositions and methods of the invention include those described, for example, in U.S. Pat. No. 10,787,521 B2, U.S. Patent Application Publication Nos. 2018 / 0222987 A1, and 2019 / 0241657 A1, and International Application Publication Nos. WO2016 / 036937 A1, WO2017 / 210443 A1, WO2019 / 050521 A1, WO2019 / 210147 A1, and / or U.S. Patent Application Publication Nos. 2019 / 0233621, ... , WO2019 / 232528A1, and WO2020 / 092404A1; the bispecific CD3×STEAP2 antibody disclosed in International Application Publication No. WO2018 / 058001A1; WO2014 / 047231A1, WO2015 / 143079A1, WO2016 / 081490A1, WO2017 / 112775A1, WO2017 / 210485A1, WO2 and the bispecific CD3xCD3 antibodies disclosed in U.S. Patent Application Publication Nos. US2020 / 0199231A1 and US2020 / 0172627A1. 20 antibodies; the bispecific CD3×SSTR2 antibody disclosed in International Application Publication No. WO2018 / 005706A1; the bispecific CD3×CD38 antibody disclosed in International Application Publication Nos. WO2015 / 149077A1 and WO2020 / 018556A1, and U.S. Patent Application Publication Nos. US2018 / 0305465A1 and US2020 / 0102403A1; the Olivier antibody disclosed in Proceedings of the 2020 American Association for Cancer Research Annual Meeting the bispecific CD3 x STEAP1 antibody disclosed in Nolan-Stevaux (2020) Abstract; the bispecific CD3 x 5T4 antibody disclosed in International Application Publication No. WO2013 / 041687A1, U.S. Patent Application Publication No. US2017 / 0342160A1, US20200277397A1; the bispecific CD3 x ENPP3 antibody as described in International Application Publication No. WO2020 / 180726A1;Bispecific CD3xMUC16 antibodies disclosed in International Application Publication Nos. WO2018 / 067331A9 and WO2019 / 246356A1; International Application Publication Nos. WO2013 / 072406A1, WO2014 / 140248A1, WO2016 / 166629A1, WO2017 / 031104A1, WO2017 / 134134A1, WO2017 / 095267A1, WO2019 / 220369A3, WO2019 / 075359A1, WO2019 / 226761A1, WO2020 / 02559 6A1, WO2020 / 191346A1, WO2020018820A1, U.S. Patent Application Publication Nos. US2013 / 0273055A1, US2019 / 0263920A1; the bispecific CD3×BCMA antibodies disclosed in International Application Publication Nos. WO2012 / 055961A1, WO2016 / 048938A1, WO2017 / 087603A1, WO2017 / 096368A1, WO2018 / 188612A1, WO2019 / 237081A1, WO2020 / 048525A1, WO2020 / 1 35335A1, the bispecific CD3×CD19 antibodies disclosed in U.S. Patent Application Publication Nos. US2016 / 0326249A1, US2020 / 0283523A1, US2019 / 0284279A1, U.S. Patent Nos. US9,315,567B2, US7,575,923B2, and US7,635,472B2; the bispecific CD3×GPRC5D antibodies disclosed in International Application Publication Nos. WO2018 / 017786A3 and WO2019 / 220369A3; and the bispecific CD3×GPRC5D antibodies disclosed in U.S. Patent Application Publication No. US2017 / 0320947 A1; the trispecific CD3xCD28xCD38 antibody disclosed in U.S. Patent Application Publication No. US2020 / 0140552A1; or other CD3 multispecific antibodies disclosed in International Application Publication Nos. WO2016 / 086189A2, WO2020 / 088608A1, WO2019191120A1, and WO2016 / 105450A3, the contents of each of which are incorporated herein by reference in their entirety;

[0217] In some embodiments, the multispecific (e.g., bispecific or trispecific) antigen-binding molecule that specifically binds CD3 and a tumor antigen has a K of greater than about 40 nM as measured by an in vitro affinity binding assay. D The bispecific antigen-binding molecule may comprise an anti-CD3 antigen-binding molecule that binds to CD3 with weak binding affinity such that it exhibits an EC50 of greater than about 100 nM as measured by a FACS titration assay. The bispecific antigen-binding molecule may comprise an anti-CD3 antigen-binding molecule that binds to CD3 and exhibits an EC50 of greater than about 100 nM as measured by a FACS titration assay. The bispecific antigen-binding molecule may comprise an anti-CD3 antigen-binding molecule that does not exhibit measurable or observable binding to CD3 as measured by an in vitro affinity binding assay or a FACS titration assay, but retains the ability to activate human PBMC cells and / or induce cytotoxic activity against tumor antigen-expressing cell lines.

[0218] Therapeutic Formulations and Administration In some embodiments, provided herein are pharmaceutical compositions comprising a CD40 antagonist (e.g., a CD40 antagonist antibody, or antigen-binding fragment thereof) as described herein. In some embodiments, provided herein are pharmaceutical compositions comprising a CD3 multispecific antigen-binding molecule as described herein. In some embodiments, provided herein are pharmaceutical compositions in which a CD3 multispecific antigen-binding molecule as described herein is co-formulated with a CD40 antagonist (e.g., a CD40 antagonist antibody, or antigen-binding fragment thereof) as described elsewhere herein.

[0219] The pharmaceutical compositions provided herein can be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. Many suitable formulations can be found in a formulary 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, lipids (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also, for example, Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998), J. Pharm. Sci. Technol. 52:238-311.

[0220] The dose of the antigen-binding molecule administered to a patient may vary depending on the age and physique of the patient, the target disease, condition, administration route, and the like. A variety of delivery systems, 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), are known and can be used to administer the pharmaceutical compositions provided herein. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local.

[0221] In some embodiments, the pharmaceutical compositions provided herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device can be easily applied in delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can be reused. A disposable pen delivery device does not have a replaceable cartridge. Rather, a disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition is emptied from the reservoir, the entire device is discarded.

[0222] Many reusable pen and autoinjector delivery devices are applicable for subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN ... Examples of disposable pen delivery devices applicable for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR Pen (Sanofi-aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA Pen (Abbott Labs, Abbott Park IL), to name a few.

[0223] In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material may be used: see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be placed in close proximity to the target of the composition, so that only a fraction of the systemic dose is required (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science, 249:1527-1533.

[0224] The injection may include dosage forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, etc. These injections may be prepared by known methods. For example, the injection may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or oily medium conventionally used for injections. The aqueous medium for injection may be, for example, an isotonic solution containing physiological saline, glucose, or other auxiliary agents, etc., which may be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)], etc. As the oily medium, for example, sesame oil, soybean oil, etc. are employed, which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled into a suitable ampoule.

[0225] Advantageously, the above-mentioned pharmaceutical composition for oral or parenteral use is prepared in a dosage form suitable for the dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0226] In some aspects, provided herein is a pharmaceutical composition comprising a CAR-T cell expressing a CD40 antagonist (e.g., a CD40 antagonist antibody, or an antigen-binding fragment thereof) as described herein.

[0227] In certain embodiments, the CAR-T cell population expressing the CD40 antagonist may be administered as a pharmaceutical composition, alone or in combination with a pharma- ceutical or physiologically acceptable carrier, diluent, excipient, and / or in combination with other components or cell populations. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions disclosed herein may be formulated for intravenous administration.

[0228] Administration of the CAR-T cells expressing the CD40 antagonist may be performed in any convenient manner, including injection, infusion, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In some embodiments, the disclosed pharmaceutical compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by iv injection. The compositions may be injected directly into a tumor or lymph node.

[0229] Methods for Treating Cancer and / or Inhibiting Cytokine Release Syndrome - Patent application In some aspects, the invention includes a method for treating cancer and suppressing cytokine release syndrome (CRS) in a subject. In some embodiments, the method may include administering to a subject in need thereof (e.g., simultaneously or sequentially) (1) a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist and (2) a multispecific (e.g., bispecific or trispecific) antigen binding molecule comprising at least a first antigen binding domain against CD3 and a second antigen binding domain against a tumor antigen.

[0230] In some embodiments, the method may comprise administering to a subject in need thereof a pharmaceutical composition comprising: (1) a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist; and (2) a multispecific (e.g., bispecific or trispecific) antigen binding molecule comprising at least a first antigen binding domain to CD3 and a second antigen binding domain to a tumor antigen. The therapeutic composition may comprise any of the CD40 antagonist antibodies and CD3 multispecific antigen binding molecules as disclosed herein, and a pharma- ceutically acceptable carrier or diluent.

[0231] In some embodiments, the present invention includes a method of suppressing cytokine release syndrome (CSR) in a subject or reducing cytokine release caused by a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen. The method according to this embodiment of the present invention includes administering to a subject in need thereof a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist.

[0232] In some embodiments, the methods further include diagnosing or identifying a subject susceptible to cytokine release syndrome or in need of reduced cytokine release.

[0233] As used herein, terms such as "treat," "treating," and the like, refer to temporarily or permanently alleviating a symptom or eliminating the cause of a symptom. For example, "treating cancer" can mean slowing or inhibiting tumor growth, reducing tumor cell mass or burden, promoting tumor regression, causing tumor shrinkage, necrosis, and / or disappearance, preventing tumor recurrence, and / or prolonging the survival of a subject.

[0234] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of cancer and / or CRS and / or having been diagnosed with cancer and / or CRS and in need of treatment thereof. In many embodiments, the term "subject" may be used interchangeably with the term "patient."

[0235] In some embodiments, cancers that may be treated by the methods and compositions provided herein include, but are not limited to, cancers originating from the cervix, anus, vagina, vulva, penis, base of tongue, larynx, tonsils, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, non-melanoma skin cancer (NMSC), cutaneous squamous cell carcinoma (SCC), stomach, testis, tongue, or uterus. Furthermore, cancer may specifically include the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; piloma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumors; carcinoid tumor, malignant; branchial alveolar adenocarcinoma; papillary adenocarcinoma. Cancer; chromophobe carcinoma; eosinophil carcinoma; eosinophil adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; noncapsular sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma; Cancer; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; with squamous metaplasia Adenocarcinoma;malignant thymoma;malignant ovarian stromal tumor;malignant meningioma;malignant granulosa cell tumor;malignant erythroblastoma;Sertoli cell carcinoma;malignant Leydig cell tumor;malignant lipid cell tumor;malignant paraganglioma;malignant extramammary paraganglioma;pheochromocytoma;glomerular sarcoma;malignant melanoma;amelanotic melanoma;superficial spreading 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;Müllerian mixed tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;malignant mesenchymoma;malignant Brenner tumor;malignant phyllodes tumor;synovial sarcoma;malignant mesothelioma;dysgerminoma;embryonal carcinoma;malignant teratoma;malignant ovarian thyroid tumor;choriocarcinoma;malignant 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 sarcoma;malignant ameloblastoma;ameloblastic fibrosarcoma;malignant pinealoma;chordoma;malignant glioma;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioma;primitive neuroectodermal;cerebellar sarcoma;ganglioneoblastoma;neuroblastoma;retinoblastoma;olfactory neuroblastoma;malignant meningioma;neurofibrosarcoma;malignant neurilemmoma;malignant granular cell tumor;malignant lymphoma;Hodgkin's disease;Hodgkin's lymphoma;paragranuloma;small The cancer may be, but is not limited to, lymphocytic lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic 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.

[0236] In some embodiments, cancers that may be treated by the methods and compositions provided herein express a tumor antigen targeted by a CD3 multispecific antigen binding molecule (e.g., tumors with expression of the tumor antigen in 20% or more of the tumor cells as measured by flow cytometry). In particular, the compositions and methods of the invention may be used to treat, prevent and / or ameliorate a disease or disorder associated with or mediated by, for example, expression of CD20, PSMA, MUC16, STEAP2 or BCMA, or activity or proliferation of CD20+, PSMA+, MUC16+, STEAP2+, or BCMA+ cells. The mechanism of action by which the therapeutic methods of the invention are achieved includes the killing of cells expressing such antigens in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or a combination of two or more of these mechanisms.

[0237] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the present invention is a bispecific anti-CD3 x anti-PSMA antibody. The compositions or methods are useful for treating cancers that express PSMA, including prostate cancer, kidney cancer, bladder cancer, colon cancer, and gastric cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0238] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-MUC16 antibody. The compositions or methods are useful for treating MUC16 expressing cancers, including ovarian cancer, breast cancer, pancreatic cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma mass forming type, adenocarcinoma of the cervix, and adenocarcinoma of the gastrointestinal tract. In some embodiments, the cancer is ovarian cancer.

[0239] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-STEAP2 antibody. The compositions or methods are useful for treating cancers expressing STEAP2, including prostate cancer, bladder cancer, cervical cancer, lung cancer, colon cancer, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, uterine cancer, and ovarian cancer. In some embodiments, the cancer is prostate cancer (e.g., castration-resistant prostate cancer).

[0240] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the invention is a bispecific anti-CD3 x anti-BCMA antibody. The compositions or methods are useful for treating multiple myeloma or BCMA expressing cancers including Waldenstrom's macroglobulinemia, Burkitt's lymphoma, and other B-cell or plasma cell cancers such as diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin's lymphoma. In some embodiments, the cancer is multiple myeloma.

[0241] In some embodiments, the CD3 multispecific antigen binding molecule used in the compositions or methods of the present invention is a bispecific anti-CD3 x anti-CD20 antibody. The compositions or methods are useful for treating cancers expressing CD20, including non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom's macroglobulinemia, primary mediastinal B-cell lymphoma, lymphoblastic lymphoma, or Burkitt's lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0242] In some embodiments, the methods of the invention are used in subjects (e.g., cancer patients) who have one or more symptoms or signs of cytokine release syndrome described herein, have been diagnosed with cytokine release syndrome, and / or are susceptible to cytokine release syndrome, or are in need of reduced cytokine release.

[0243] In certain embodiments, the methods of the invention are used in subjects being treated with a particular anti-cancer agent (e.g., cancer immunotherapy, CAR-T cell therapy, or a CD3 multispecific antigen-binding molecule such as those described herein).

[0244] In some embodiments, for any of the methods disclosed herein, the subject being treated or evaluated is a subject who is to be treated or has been treated with a cancer immunotherapy, e.g., a CD3 multispecific antigen binding molecule described herein.

[0245] In some embodiments, the subject is at risk (e.g., high risk) of developing CRS (e.g., severe CRS). In some embodiments, the subject is at low risk (e.g., no risk) of developing CRS (e.g., severe CRS). In some embodiments, the subject has CRS grade 0, CRS grade 1, CRS grade 2, or CRS grade 3. In some embodiments, the subject's risk of developing CRS (e.g., severe CRS) is determined using the assessment or prediction methods described herein.

[0246] In some embodiments, the methods provided herein treat, slow, or inhibit tumor growth or induce tumor cell death. In certain embodiments, the methods provided herein promote tumor regression. In certain embodiments, the methods provided herein reduce tumor cell burden or reduce tumor burden. In certain embodiments, the methods provided herein prevent tumor recurrence.

[0247] In some embodiments, the methods provided herein prevent, suppress, alleviate, or treat CRS, e.g., by alleviating at least one symptom or sign associated with CRS, reducing CRS severity, or reducing cytokine release, etc. In some embodiments, the methods provided herein prevent, suppress, alleviate, or treat CRS without adversely affecting the therapeutic benefits of the CD3 multispecific antigen binding molecules described herein.

[0248] In certain embodiments, the methods of the invention comprise administering to a subject in need thereof a therapeutically effective amount of a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist in combination with a therapeutically effective amount of a CD3 multispecific antigen-binding molecule, wherein administration of the CD40 antagonist or the CAR-T cell expressing a CD40 antagonist results in suppression of CRS (e.g., suppresses or reduces at least one symptom, sign, or biomarker of CRS as disclosed herein). In certain embodiments, CRS is suppressed by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to a subject administered the CD3 multispecific antigen-binding molecule as monotherapy.

[0249] In certain embodiments, administration of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist reduces cytokine release. In certain embodiments, the levels of a cytokine (e.g., TNFα, IL4, IL6, IL10, IL2, IFN-γ, IL-17A, IL13, or CD40L) are inhibited by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% compared to subjects administered a CD3 multispecific antigen binding molecule as monotherapy.

[0250] In certain embodiments, administration of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist in combination with a CD3 multispecific antigen-binding molecule does not significantly reduce the anti-tumor effect (e.g., inhibiting or slowing tumor growth, inducing tumor regression, preventing tumor recurrence, and / or prolonging survival, etc.) compared to a subject administered the CD3 multispecific antigen-binding molecule as monotherapy. For example, administration of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist in combination with a CD3 multispecific antigen-binding molecule may reduce the anti-tumor effect by less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% compared to a subject administered the CD3 multispecific antigen-binding molecule as monotherapy. In certain embodiments, administration of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist does not affect the anti-tumor efficacy of the CD3 multispecific antigen-binding molecule.

[0251] In certain embodiments, administration of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist in combination with a CD3 multispecific antigen-binding molecule does not significantly reduce T cell (e.g., CD8+ T cell) activation, proliferation, and / or cytotoxicity compared to a subject administered the CD3 multispecific antigen-binding molecule as monotherapy. For example, administration of a CD40 antagonist or CAR-T cells expressing a CD40 antagonist in combination with a CD3 multispecific antigen-binding molecule may reduce T cell (e.g., CD8+ T cell) activation, proliferation, and / or cytotoxicity by about 40%, about 30%, about 20%, about 15%, about 10%, about 5%, about 1%, about 0.5%, or less than about 0.1% compared to a subject administered the CD3 multispecific antigen-binding molecule as monotherapy. In certain embodiments, administration of a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist does not affect T cell (e.g., CD8+ T cell) activation, proliferation, and / or cytotoxicity induced by a CD3 multispecific antigen-binding molecule.

[0252] In certain embodiments, the disclosed CD40 antagonists, CAR-T cells expressing CD40 antagonists, and / or CD3 multispecific antigen binding molecules are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, an additional cancer or CRS treatment.

[0253] Exemplary CRS treatments include, for example, IL-6 inhibitors or IL-6 receptor (IL-6R) inhibitors (e.g., tocilizumab or siltuximab), anti-IFN-γ therapy, anti-sIL2Ra therapy, antipyretics such as acetaminophen, sgp130 blockers, vasoactive drugs, corticosteroids, immunosuppressants, and mechanical ventilation. Exemplary vasoactive drugs include, but are not limited to, angiotensin-11, endothelin-1, alpha adrenergic agonists, rostanoids, phosphodiesterase inhibitors, endothelin antagonists, vasoactive drugs (e.g., adrenaline, dobutamine, isoprenaline, ephedrine), hypertensive drugs (e.g., noradrenaline, vasopressin, metaraminol, vasopressin, methylene blue), inotropic dilators (e.g., milrinone, levosimendan), and dopamine. Exemplary hypertensive agents include, but are not limited to, norepinephrine, dopamine, phenylephrine, epinephrine, and vasopressin. Exemplary corticosteroids include, but are not limited to, dexamethasone, hydrocortisone, and methylprednisolone. Exemplary immunosuppressants include, but are not limited to, inhibitors of TNFα or inhibitors of IL-1. Additional exemplary treatments for CRS are disclosed in International Application WO2014011984, which is incorporated herein by reference.

[0254] The additional therapeutically active ingredient(s) may be administered immediately prior to, simultaneously with, or immediately following administration of the antigen-binding molecule of the present invention (for purposes of this disclosure, such a dosing regimen will be considered as administering the antigen-binding molecule "in combination" with the additional therapeutically active ingredient).

[0255] As stated above, the co-administration may be simultaneous, separate or sequential. In the case of simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0256] Treatment regimen In certain embodiments, provided herein are methods that include administering a CD40 antagonist (e.g., a CD40 antagonist antibody) or a CAR-T cell expressing a CD40 antagonist to a subject at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0257] In certain embodiments, provided herein are methods comprising administering a CD3 multispecific antigen binding molecule to a subject at a dosing frequency of about 4 times per week, twice per week, once per week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0258] In certain embodiments, the methods comprise administering a CD40 antagonist (e.g., a CD40 antagonist antibody) or a CAR-T cell expressing a CD40 antagonist in combination with a CD3 multispecific antigen-binding molecule at a dosing frequency of about 4 times per week, twice per week, once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 8 weeks, once per 12 weeks, or less frequently as long as a therapeutic response is achieved.

[0259] According to certain embodiments, multiple doses of a CD40 antagonist (e.g., a CD40 antagonist antibody) in combination with a CD3 multispecific antigen-binding molecule, or CAR-T cells expressing a CD40 antagonist, may be administered to a subject over a defined time course. The method according to this aspect of the invention may include sequentially administering to a subject multiple doses of a CD40 antagonist (e.g., a CD40 antagonist antibody) in combination with a CD3 multispecific antigen-binding molecule, or CAR-T cells expressing a CD40 antagonist. As used herein, "sequentially administering" means that each dose of the antigen-binding molecule is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The invention includes methods comprising sequentially administering to a patient a single initial dose of a CD40 antagonist (e.g., a CD40 antagonist antibody) or CAR-T cells expressing a CD40 antagonist, followed by one or more secondary doses of a CD40 antagonist (e.g., a CD40 antagonist antibody) or CAR-T cells expressing a CD40 antagonist, optionally followed by one or more tertiary doses of a CD40 antagonist (e.g., a CD40 antagonist antibody) or CAR-T cells expressing a CD40 antagonist. In certain embodiments, the invention further comprises sequentially administering to a patient a single initial dose of a CD3 multispecific antigen-binding molecule, followed by one or more secondary doses of a CD3 multispecific antigen-binding molecule, optionally followed by one or more tertiary doses of a CD3 multispecific antigen-binding molecule.

[0260] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the time sequence of administration of the antigen-binding molecule of the present invention. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also called a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary, and tertiary doses may all contain the same amount of therapeutic agent as described herein, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antigen-binding molecule contained in the initial, secondary, and / or tertiary doses varies from each other during the course of treatment (e.g., adjusted up or down as necessary). In certain embodiments, two or more doses (e.g., 2, 3, 4, or 5) are administered as "loading doses" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0261] In an exemplary embodiment of the invention, each secondary dose and / or tertiary dose is 1 to 26 (e.g., 1, 1 and 1 / 2, 2, 2 and 1 / 2, 3, 3 and 1 / 2, 4, 4 and 1 / 2, 5, 5 and 1 / 2, 6, 6 and 1 / 2, 7, 7 and 1 / 2, 8, 8 and 1 / 2, 9, 9 and 1 / 2, 10, 10 and 1 / 2, 11, 11 and 1 / 2, 12, 12 and 1 / 2, 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, 3 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 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 weeks, or more) weeks later. As used herein, the phrase "immediately preceding dose" refers to a dose of a therapeutic agent described herein that is administered to a patient prior to administration of the very next dose in the sequence, without an intervening dose, in a series of multiple doses.

[0262] The method according to this aspect of the invention may include administering any number of secondary and / or tertiary doses of a therapeutic agent described herein to the patient. 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.

[0263] In embodiments including multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the immediately preceding dose. Similarly, in embodiments including multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the immediately preceding dose. Alternatively, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may be adjusted by the physician during treatment depending on the needs of the individual patient following clinical testing. EXAMPLES

[0264] Example 1: CD40 blockade inhibited CD123×CD3-mediated cytokine release without affecting T cell activation in a 4-day assay with autologous B cell-enriched PBMCs.

[0265] Experimental setup The effect of CD40 blockade on cytokine release was assessed in assays with autologous B cell-enriched human PBMCs and the AML cell line MOLM13. B cells were isolated from human PBMCs using the EasySep Human B Cell Isolation Kit (StemCells cat#17954), labeled with CFSE, and plated with autologous PBMCs and MOLM13 cells at a ratio of (5:10:1) in complete medium (RPMI supplemented with 10% FBSm, 100 U / mL penicillin, 100 μg / ml streptomycin, 292 μg / ml L-glutamine). CD123×CD3(G) or one-arm CD3 control were added to the wells in a 10-fold dose titration starting at 1 μg / ml. Anti-CD40 antibody (IgG1 variant, REGN3794) was used at a constant concentration of 5 μg / ml. Cells were incubated in complete medium at 37°C for 4 days. Cytokines and T cell activation were assessed 4 days after setup. Cytokines were measured in supernatants using the LegendPlex human B cell panel (Biolegend catalog #740527). For assessment of T cell activation, cells were stained with directly conjugated antibodies against CD2, CD25, and live / dead staining was performed. T cell activation is reported as the percentage of live CD2+ cells expressing CD25.

[0266] result: Blockade of CD40 with anti-CD40 antibodies inhibited bispecific CD123xCD3 antibody-mediated cytokine release in 4-day in vitro assays with CD123+ tumor cells, human PBMCs, and enriched B cells without significantly affecting T cell activation (Figures 1A-1I).

[0267] Example 2: CD40 blockade inhibited CD3 bispecific-mediated cytokine release in a 4-day assay with AML cell lines and PBMCs without added autologous B cells, without significantly affecting T cell activation Experimental setup The effect of CD40 blockade on cytokine release was assessed in assays with human PBMC and the AML cell line THP-1. Human PBMC were labeled with CFSE and THP-1 cells were labeled with Violet Cell Trace. Labeled PBMC and THP-1 cells were plated at a ratio (10:1) in complete medium. CD123xCD3(G) or one-arm CD3 control were added to the wells in a 10-fold dose titration starting at 1 μg / ml. Anti-CD40 antibody (IgG1 variant, REGN3794) was used at a constant concentration of 5 μg / ml. Cells were incubated for 4 days at 37°C in complete medium. Cytokines and T cell activation were assessed 4 days after setup. Cytokines were measured in supernatants using the LegendPlex human B cell panel (Biolegend catalog #740527). For the assessment of T cell activation, cells were stained with directly conjugated antibodies against CD2, CD4, CD8, CD16, CD25 and live / dead staining was performed. T cell activation is reported as the percentage of viable CD2+CD16-CD8+ cells expressing CD25.

[0268] result: Blockade of CD40 with anti-CD40 antibodies inhibited CD3 bispecific-mediated cytokine release in 4-day in vitro assays with CD123+ tumor cells and human PBMCs (without additional B cells) without significantly affecting T cell activation (Figures 2A-2E). In general, more robust cytokine release and more significant blockade of this release by anti-CD40 antibodies was observed in assays with enriched B cells (Figures 1A-1H) compared to assays without them (Figures 2A-2E).

[0269] Example 3: CD40 blockade inhibited CD3 bispecific-mediated release of selected cytokines in a 4-day killing assay with prostate cell lines and PBMCs without significantly affecting T cell activation and target killing Experimental setup The effect of CD40 blockade on cytokine release was evaluated in a killing assay with human PBMC and the prostate cell line 22Rv1. Human PBMC were labeled with CFSE and 22Rv1 cells were labeled with Violet Cell Trace. Labeled PBMC and 22Rv1 cells were seeded in a ratio (20:1) in complete medium. Steap2xCD3(G) or one-arm CD3 control were added to the wells in a 10-fold dose titration starting at 1 μg / ml. Anti-CD40 antibody (IgG1 variant, REGN3794) was used at a constant concentration of 5 μg / ml. Cells were incubated in complete medium at 37°C for 4 days. At the end of the culture, cytokine release, surviving target cells, and T cell activation were analyzed by flow cytometry. Cytokines were measured in the supernatants using the LegendPlex human B cell panel (Biolegend catalog #740527). For assessment of T cell activation and target cell killing, cells were washed and stained with directly conjugated antibodies against CD2, CD4, CD8, CD16, and CD25, and live / dead staining was performed. T cell activation is reported as the percentage of live CD2+CD16-CD8+ cells expressing CD25. To assess 22Rv1 survival, gates were placed on the violet-labeled live cell population. Percentages of live cell population normalized to untreated samples are reported.

[0270] result: Blockade of CD40 with anti-CD40 antibodies inhibited CD3 bispecific-mediated cytokine release in 4-day in vitro killing assays with STEAP2+ tumor cells and human PBMCs without significantly affecting T cell activation and cytotoxicity (Figures 3A-3G). The extent of anti-CD40-mediated inhibition varied depending on the cytokine (Figures 3C-3G).

[0271] Example 4: Blockade of CD40 reduced cytokines in PBMCs treated with CD20xCD3 bispecific (REGN1979) Experimental setup The effect of CD40 blockade on cytokine release was assessed in killing assays with human PBMCs and the NHL cell line Ramos. Ramos cells were labeled with CFSE and mixed with human PBMCs in a ratio (1:10) in complete medium. CD20xCD3 (REGN1979) or one-arm CD3 control were added to the wells in a 10-fold dose titration starting at 10 μg / ml. Anti-CD40 antibody (IgG1 variant, REGN3794) was used at a constant concentration of 5 μg / ml. Cells were incubated in complete medium at 37°C for 4 days. At the end of the culture, cytokine release, viable target cells, and T cell activation were analyzed by flow cytometry. Cytokines were measured in the supernatants using the LegendPlex human B cell panel (Biolegend catalog #740527). For evaluation of T cell activation and target cell killing, cells were washed and stained with directly conjugated antibodies against CD2, CD4, CD8, CD16, CD25, and live / dead staining was performed. T cell activation is reported as the percentage of viable CD2+CD16-CD8+ cells expressing CD25. To assess Ramos viability, the absolute number of viable Ramos cells per well was calculated using CountBright beads.

[0272] result: Blockade of CD40 with anti-CD40 antibodies inhibited CD20xCD3 bispecific-mediated cytokine release in a 4-day in vitro killing assay with CD20+ tumor cells and human PBMCs without adversely affecting cytotoxicity (Figures 4A-4B). The combination of CD20xCD3(REGN1979) + anti-CD40 antibodies reduced T cell activation in one of two donors tested (approximately 35% reduction compared to CD20xCD3(REGN1979) alone) (Figure 4A).

[0273] Incorporation by Reference All publications, patents, patent applications, and sequence accession numbers referred to herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0274] Equivalent Numerous embodiments of the present invention have been described. Of course, it will be understood that various modifications may be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.

[0275] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A CD40 antagonist or a CAR-T cell expressing a CD40 antagonist for use in treating cancer and suppressing cytokine release syndrome in a subject, said use comprising: (a) a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to CD3 and a second antigen-binding domain that specifically binds to a tumor antigen; (b) the CD40 antagonist or a CAR-T cell expressing the CD40 antagonist; co-administering to said subject a CD40 antagonist or a CAR-T cell expressing a CD40 antagonist.

2. The CD40 antagonist or CD40 antagonist-expressing CAR-T cell of claim 1, wherein the multispecific antigen-binding molecule is a bispecific or trispecific antigen-binding molecule.

3. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist described in claim 2, wherein the multispecific antigen-binding molecule is a trispecific antigen-binding molecule, and the trispecific antigen-binding molecule further comprises a third antigen-binding domain that specifically binds an additional T cell antigen or an additional tumor antigen.

4. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist of claim 3, wherein the third antigen-binding domain specifically binds CD28.

5. The CD40 antagonist or CAR-T cell expressing a CD40 antagonist according to any one of claims 1 to 4, wherein the tumor antigen is selected from CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, and BCMA.

6. The CD40 antagonist or CD40 antagonist-expressing CAR-T cell of any one of claims 1 to 5, wherein the multispecific antigen-binding molecule comprises a multispecific antibody or an antigen-binding fragment thereof.

7. 7. The CD40 antagonist or CAR-T cell expressing a CD40 antagonist according to any one of claims 1 to 6, wherein the multispecific antigen-binding molecule is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

8. The CD40 antagonist or CAR-T cells expressing a CD40 antagonist described in any one of claims 1 to 7, wherein the use comprises administering to the subject the multispecific antigen-binding molecule and the CD40 antagonist in combination, and the CD40 antagonist is an antibody or an antigen-binding fragment thereof.

9. The CD40 antagonist or CAR-T cells expressing a CD40 antagonist described in any one of claims 1 to 7, wherein the use comprises administering to the subject in combination the multispecific antigen-binding molecule and CAR-T cells expressing the CD40 antagonist, and the CAR-T cells secrete the CD40 antagonist.

10. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist according to claim 9, wherein the CD40 antagonist is an scFv or a Fab.

11. The CD40 antagonist or CAR-T cell expressing a CD40 antagonist according to any one of claims 1 to 10, wherein the cytokine release syndrome is suppressed by maintaining C-reactive protein (CRP) levels below 7 mg / dL, IFN-γ below 75 pg / ml, and / or IL-10 below 60 pg / ml.

12. The CD40 antagonist or CAR-T cells expressing a CD40 antagonist described in any one of claims 1 to 11, wherein the use further comprises identifying a subject susceptible to cytokine release syndrome or in need of reduced cytokine release prior to administering to the subject in combination with the multispecific antigen-binding molecule and the CD40 antagonist or the CAR-T cells expressing the CD40 antagonist.

13. A CD40 antagonist or a CAR-T cell expressing a CD40 antagonist for use in suppressing cytokine release syndrome caused by a multispecific antigen-binding molecule, wherein the multispecific antigen-binding molecule comprises a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen.

14. The CD40 antagonist or CD40 antagonist-expressing CAR-T cell of claim 13, wherein the multispecific antigen-binding molecule is a bispecific or trispecific antigen-binding molecule.

15. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist described in claim 14, wherein the multispecific antigen-binding molecule is a trispecific antigen-binding molecule, and the trispecific antigen-binding molecule further comprises a third antigen-binding domain that specifically binds an additional T cell antigen or an additional tumor antigen.

16. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist of claim 15, wherein the third antigen-binding domain specifically binds CD28.

17. The CD40 antagonist or CAR-T cell expressing a CD40 antagonist according to any one of claims 13 to 16, wherein the tumor antigen is selected from CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, and BCMA.

18. The CD40 antagonist or CD40 antagonist-expressing CAR-T cell of any one of claims 13 to 17, wherein the multispecific antigen-binding molecule comprises a multispecific antibody or an antigen-binding fragment thereof.

19. 19. The CD40 antagonist or CAR-T cell expressing a CD40 antagonist according to any one of claims 13 to 18, wherein the multispecific antigen-binding molecule is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

20. A CD40 antagonist or a CAR-T cell expressing a CD40 antagonist described in any one of claims 13 to 19, wherein the CD40 antagonist is used in the manufacture of a medicine and the CD40 antagonist is an antibody or an antigen-binding fragment thereof.

21. A CD40 antagonist or a CAR-T cell expressing a CD40 antagonist described in any one of claims 13 to 19, wherein the CAR-T cell expressing the CD40 antagonist is used in the manufacture of a medicine, and the CAR-T cell secretes the CD40 antagonist.

22. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist of claim 21, wherein the CD40 antagonist is an scFv or a Fab.

23. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist according to any one of claims 13 to 22, wherein the use comprises: (a) identifying a subject susceptible to cytokine release syndrome or in need of reduced cytokine release; and (b) administering to the subject the CD40 antagonist or a CAR-T cell expressing the CD40 antagonist.

24. 1. A pharmaceutical composition comprising: (a) a multispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds CD3 and a second antigen-binding domain that specifically binds a tumor antigen; and (b) a CD40 antagonist.

25. 25. The pharmaceutical composition of claim 24, wherein the multispecific antigen-binding molecule is a bispecific or trispecific antigen-binding molecule.

26. The pharmaceutical composition of claim 25, wherein the multispecific antigen-binding molecule is a trispecific antigen-binding molecule, the trispecific antigen-binding molecule further comprising a third antigen-binding domain that specifically binds an additional T cell antigen or an additional tumor antigen.

27. The pharmaceutical composition of claim 26, wherein the third antigen-binding domain specifically binds CD28.

28. 28. The pharmaceutical composition of any one of claims 24 to 27, wherein the tumor antigen is selected from CD19, CD123, STEAP2, CD20, SSTR2, CD38, STEAP1, 5T4, ENPP3, PSMA, MUC16, GPRC5D, and BCMA.

29. The pharmaceutical composition of any one of claims 24 to 28, wherein the multispecific antigen-binding molecule comprises a multispecific antibody or an antigen-binding fragment thereof.

30. 30. The pharmaceutical composition of any one of claims 24 to 29, wherein the multispecific antigen-binding molecule is selected from a bispecific CD3xCD19 antibody, a bispecific CD3xGPRC5D antibody, a bispecific CD3xCD123 antibody, a bispecific CD3xSTEAP2 antibody, a bispecific CD3xCD20 antibody, a bispecific CD3xSSTR2 antibody, a bispecific CD3xCD38 antibody, a bispecific CD3xSTEAP1 antibody, a bispecific CD3x5T4 antibody, a bispecific CD3xENPP3 antibody, a bispecific CD3xMUC16 antibody, a bispecific CD3xBCMA antibody, a bispecific CD3xPSMA antibody, and a trispecific CD3xCD28xCD38 antibody.

31. The pharmaceutical composition of any one of claims 24 to 30, wherein the CD40 antagonist is an antibody or an antigen-binding fragment thereof.

32. The pharmaceutical composition of any one of claims 24 to 31, further comprising a pharma- ceutically acceptable carrier.

33. A pharmaceutical composition described in any one of claims 24 to 32 for use in treating cancer and suppressing cytokine release syndrome.

34. The use of claim 2, (a) identifying a subject susceptible to cytokine release syndrome or a subject in need of reduced cytokine release; (b) administering said pharmaceutical composition to said subject.

35. 35. The CD40 antagonist or CAR-T cell expressing a CD40 antagonist of claim 12 or 23, or the pharmaceutical composition of claim 34, wherein a subject susceptible to cytokine release syndrome and / or a subject in need of reduced cytokine release is identified by detecting one or more biomarkers selected from the group consisting of fever, rash, respiratory symptoms, hypoxia, hypotension, cardiovascular insufficiency, neurotoxicity, liver disease dysfunction, renal dysfunction, coagulation, organ toxicity, tumor burden, cytokines, C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH), aspartate aminotransferase (AST), blood urea nitrogen (BUN), alanine aminotransferase (ALT), creatinine (Cr), fibrinogen, prothrombin time (PT), partial thromboplastin time (PTT), eotaxin, and endothelial cell activation.

36. The CD40 antagonist or the CAR-T cell expressing a CD40 antagonist of claim 35, wherein the cytokine is one or more cytokines selected from the group consisting of sTNFR2, IP10, sIL1R2, sTNFR1, MIG, VEGF, sIL1R1, TNFα, IFNα, GCSF, sRAGE, IL1, IL2, IL4, IL5, IL10, IL12, IL13, IL18, IL1R1, IFNγ, IL6, IL8, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, FLT-3L, fractalkine, and GM-CSF.

37. The CD40 antagonist or a CAR-T cell expressing a CD40 antagonist or a pharmaceutical composition according to claim 35, wherein the endothelial cell activation is detected by measuring serum levels of Ang-2 and / or von Willebrand factor.

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