ANTI-TCR ANTIBODY MOLECULES AND USES THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 31, 2020, is named 53676-702_301_SL.txt and is 311,084 bytes in size.

BACKGROUND

Current molecules designed to activate and expand T cells encoding exogenous receptors (e.g., CAR T cells, T cells expressing an exogenous TCR) ex vivo for cancer immunotherapy typically target the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) alone or in combination with targeting the costimulatory receptor CD28. However, there are limitations to this approach. Previous studies have shown that use of these anti-CD3ε targeting molecules can produce T cells that when infused into a subject either produce or stimulate other cells to produce proinflammatory cytokines (e.g., IL-1, IL-6 and TNFα) associated with inflammatory conditions such cytokine release syndrome (CRS), macrophage activation syndrome, neurological toxicities, and tumor lysis syndrome. Thus, a current need exists to develop additional methods of activating and expanding T cells ex vivo, which do not pose these significant risks to patients.

SUMMARY

Disclosed herein are, inter alio, methods of expanding T cells ex vivo using antibodies directed to the variable chain of the beta subunit of TCR (TCRβV). In some embodiments, methods described herein result in less or no production of cytokines associated with cytokine release syndrome (CRS), e.g., IL-6, IL-1beta and TNF alpha; and enhanced and/or delayed production of IL-2 and IFNγ. In some embodiments, methods described herein limit the unwanted side-effects of CRS, e.g., CRS associated with anti-CD3e targeting.

According, in one aspect, provided herein are methods of expanding T cells ex vivo comprising contacting a plurality of T cells to a first agent, wherein the first agent comprises a first domain that specifically binds to a T cell receptor beta variable beta chain (TCRβV) region, thereby generating a first population of T cells.

In some embodiments, the first agent further comprises a second domain that binds to a protein expressed on the surface of a population of T cells in the plurality.

In some embodiments, the first agent is a bispecific antibody molecule.

In some embodiments, the second domain specifically binds to a T cell receptor variable beta chain (TCRβV) region.

In some embodiments, the second domain and the first domain specifically bind to different T cell receptor variable beta chain (TCRβV) regions.

In some embodiments, the second domain and the first domain specifically bind to TCRβVs belonging to different subfamilies or different members of the same TCRβV subfamily.

In some embodiments, the first domain specifically binds to a TCRβV region of a TCRβV belonging to a TCRβV6 subfamily, a TCRβV10 subfamily, a TCRβV12 subfamily, a TCRβV5 subfamily, a TCRβV7 subfamily, a TCRβV11 subfamily, a TCRβV14 subfamily, a TCRβV16 subfamily, a TCRβV18 subfamily, a TCRβV9 subfamily, a TCRβV13 subfamily, a TCRβV4 subfamily, a TCRβV3 subfamily, a TCRβV2 subfamily, a TCRβV15 subfamily, a TCRβV30 subfamily, a TCRβV19 subfamily, a TCRβV27 subfamily, a TCRβV28 subfamily, a TCRβV24 subfamily, a TCRβV20 subfamily, TCRβV25 subfamily, or a TCRβV29 subfamily, and the second domain specifically binds to a TCRβV region of a TCRβV belonging to a TCRβV6 subfamily, a TCRβV10 subfamily, a TCRβV12 subfamily, a TCRβV5 subfamily, a TCRβV7 subfamily, a TCRβV11 subfamily, a TCRβV14 subfamily, a TCRβV16 subfamily, a TCRβV18 subfamily, a TCRβV9 subfamily, a TCRβV13 subfamily, a TCRβV4 subfamily, a TCRβV3 subfamily, a TCRβV2 subfamily, a TCRβV15 subfamily, a TCRβV30 subfamily, a TCRβV19 subfamily, a TCRβV27 subfamily, a TCRβV28 subfamily, a TCRβV24 subfamily, a TCRβV20 subfamily, TCRβV25 subfamily, or a TCRβV29 subfamily.

In some embodiments, the first domain specifically binds to a TCRβV region of a TCRβV belonging to a TCRβV12 subfamily.

In some embodiments, the second domain and the first domain specifically bind to TCRβVs belonging to different subfamilies.

In some embodiments, the second domain and the first domain specifically bind to different members of the same TCRβV subfamily.

In some embodiments, the second domain specifically binds to an antibody molecule. In some embodiments, the antibody molecule is expressed by a population of T cells in the plurality. In some embodiments, the antibody molecule comprises a variable heavy chain and a variable light chain. In some embodiments, the antibody molecule is a scFv or a Fab.

In some embodiments, the second domain specifically binds to a light chain region of the antibody molecule. In some embodiments, the second domain specifically binds to a κ light chain region of an antibody molecule. In some embodiments, the second domain comprises protein L.

In some embodiments, the first domain comprises LC CDR1, LC CDR2, LC CDR, HC CDR1, HC CDR 2, and HC CDR 3 of an antibody described in Table 2, Table 3, Table 4 or Table 5. In some embodiments, the first domain comprises a VH and VL chain sequences of an antibody disclosed in Table 2, Table 3, Table 4, or Table 5.

In some embodiments, the first agent comprises LC CDR1, LC CDR2, LC CDR, HC CDR1, HC CDR 2, and HC CDR 3 of an antibody described in Table 2, Table 3, Table 4 or Table 5. In some embodiments, the first agent comprises a VH and VL chain sequences of an antibody disclosed in Table 2, Table 3, Table 4, or Table 5.

In some embodiments, said first agent specifically binds to at least two TCRβVs belonging to different subfamilies.

In some embodiments, said first agent specifically binds to at least three, four, five, or six TCRβVs belonging to different subfamilies.

In some embodiments, said first agent specifically binds to at least two different members of the same TCRβV subfamily.

In some embodiments, said first agent specifically binds to at least three, four, five, six, or seven different members of the same TCRβV subfamily.

In some embodiments, the method further comprises contacting the plurality of T cells with a second agent, wherein the second agent comprises a domain that specifically binds to a T cell receptor variable beta chain (TCRβV) region, wherein the first and the second agents specifically bind to different TCRβV regions.

In some embodiments, the first agent comprises a domain that specifically binds to a TCRβV region of a first TCRβV, and the second agent comprises a domain that specifically binds to a TCRβV region of a second TCRβV, wherein the first and the second TCRβVs belong to different TCRβV subfamilies or are different members of the same TCRβV subfamily.

In some embodiments, the first agent comprises a domain that specifically binds to a TCRβV region of a first TCRβV belonging to a TCRβV12 subfamily.

In some embodiments, the first and the second agent each specifically bind to a TCRβV belonging to a different subfamily.

In some embodiments, the first and the second agent each specifically bind to different members of the same TCRβV subfamily.

In some embodiments, the first population of T cells exhibit at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) of: a lower level of IL-1β expression, a lower level of IL-6 expression, a lower level of TNFα expression, a lower level of IFNγ expression, a lower level of IL-1β expression, a lower level of IL-17 expression, a higher level of IL-2 expression, or a higher level of IL-15 expression, relative to a comparable population of T cells that contacted with an agent comprising a domain that specifically binds CD3ε (e.g., an anti-CD3ε antibody).

In some embodiments, expression is measured by determining the level of the protein secreted from the population of T cells, as measured by an assay described herein.

In some embodiments, the level of IL-1β expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% less than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of IL-6 expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% less than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of IL-10 expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% less than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of IL-17 expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% less than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of IFN-γ expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% less than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of TNF-α expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% less than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of IL-15 expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% higher than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the level of IL-2 expression is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% greater than the level expressed by the comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε, as measured by an assay described herein.

In some embodiments, the number of T cells in the first population of T cells, it at least about 10 fold higher (e.g., at least 50, 100, 500, 1000, or 10000 fold higher) than the number of T cells in the plurality of T cells.

In some embodiments, the number of T cells in the first population of T cells that express CD45R, express CD95, and exhibit low or no detectable expression of CCR7 is higher compared to the number of T cells in a comparable population that express CD45R, express CD95, and exhibit low or no detectable expression of CCR7 contacted with an agent comprising a domain that specifically binds CD3ε (e.g., an anti-CD3ε antibody).

In some embodiments, the number of T cells in the first population that express CD45R, express CD95, and exhibit low or no detectable expression of CCR7 is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 500, or 1000 fold higher than the number of T cells in in a comparable population that express CD45R, express CD95, and exhibit low or no detectable expression of CCR7 contacted with an agent comprising a domain that specifically binds CD3ε (e.g., an anti-CD3 antibody).

In some embodiments, the expression of CD45R, CD95, and CCR7 is measured by determining the level of the protein on the surface of the cell (e.g., as measured by flow cytometry).

In some embodiments, the number of TEMRA T cells in the first population is higher than the number of TEMRA T cells in a comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε (e.g., an anti-CD3ε antibody).

In some embodiments, the number of TEMRA T cells in the first population is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 500, or 1000 fold higher than the number of TEMRA T cells in a comparable population of T cells contacted with an agent comprising a domain that specifically binds CD3ε (e.g., an anti-CD3ε antibody).

In some embodiments, the contacting comprises incubating the plurality of T cells with the first agent.

In some embodiments, contacting comprises incubating or culturing the plurality of T cells with the first agent for at most about 10 minutes, 20 minutes, 30 minutes, 1 hour, 6 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 5 days, 7 days, 10 days, 12 days, 14 days, 15 days, 21 days, 30 days, 45 days, or 60 days.

In some embodiments, contacting comprises incubating or culturing the plurality of T cells with the first agent for from about 10-90 minutes, 10-60 minutes, 10-30 minutes, 1-30 days, 1-21 days, 1-14 days, 1-7 days, 1-5 days, 1-3 days, 21-30 days, 14-30 days, 7-30 days, 5-30 days, or 3-30 days.

In some embodiments, the first agent is coupled to a solid surface (e.g., a bead, a cell culture plate). In some embodiments, said coupling enables cross linking of the TCRs on the surface of the plurality of T cells specifically bound by the first agent.

In some embodiments, the first agent comprises an antibody domain. In some embodiments, the first agent comprises an anti-idiotypic antibody domain. In some embodiments, the first agent comprises a human or humanized antibody domain. In some embodiments, the first agent comprises an antigen binding domain comprising a single chain Fv (scFv) or a Fab. In some embodiments, the first agent comprises an antibody comprising two antibody heavy chains, each of the two heavy chains comprising a variable region and a constant region; and two antibody light chains, each of the two light chains comprising a variable region and a constant region.

In some embodiments, the plurality of T cells comprises a population of T cells that comprise an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid encodes a cell surface receptor. In some embodiments, the cell surface receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the method further comprises introducing an exogenous nucleic acid into at least a portion of T cells of the plurality prior to contacting the plurality of T cells with the first agent. In some embodiments, the method further comprises introducing an exogenous nucleic acid into at least a portion of T cells of the plurality after contacting the plurality of T cells with the first agent. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

In some embodiments, the plurality of T cells are human. In some embodiments, the plurality of T cells comprises T cells from a human subject that was healthy when the cells were removed (e.g., a subject that does not have or has not been diagnosed with a predetermined disease or condition, e.g., a cancer). In some embodiments, the plurality of T cells comprises T cells from a human subject having or diagnosed with a disease or condition when the cells were removed (e.g., diagnosed with a predetermined disease or condition, e.g., cancer). In some embodiments, the disease is a cancer.

In one aspect, provided herein are methods of expanding T cells ex vivo comprising contacting a plurality of T cells to a plurality of agents, wherein the plurality of agents comprises at least a first and a second agent, wherein each agent of the plurality comprises a domain that specifically binds to a different T cell receptor variable beta chain (TCRβV) region, thereby generating a first population of T cells.

In some embodiments, said first agent or said second agent or both specifically binds to at least two TCRβVs belonging to different subfamilies.

In some embodiments, said first agent or said second agent or both specifically binds to at least three, four, five, or six TCRβVs belonging to different subfamilies.

In some embodiments, said first agent or said second agent or both specifically binds to at least two different members of the same TCRβV subfamily.

In some embodiments, said first agent or said second agent or both specifically binds to at least three, four, five, six, or seven different members of the same TCRβV subfamily.

In some embodiments, the plurality comprises at least three, four, five, six, seven, eight, nine, or ten agents, wherein each agent of the plurality comprises a domain that specifically binds to a different T cell receptor variable beta chain (TCRβV) region.

In some embodiments, each agent of the plurality specifically binds to a different TCRβV, wherein each TCRβV belongs to a different TCRβV subfamily or are different members of the same TCRβV subfamily.

In some embodiments, each agent of the plurality comprises a domain that specifically binds to a TCRβV region of a TCRβV belonging to a TCRβV6 subfamily, a TCRβV10 subfamily, a TCRβV12 subfamily, a TCRβV5 subfamily, a TCRβV7 subfamily, a TCRβV11 subfamily, a TCRβV14 subfamily, a TCRβV16 subfamily, a TCRβV18 subfamily, a TCRβV9 subfamily, a TCRβV13 subfamily, a TCRβV4 subfamily, a TCRβV3 subfamily, a TCRβV2 subfamily, a TCRβV15 subfamily, a TCRβV30 subfamily, a TCRβV19 subfamily, a TCRβV27 subfamily, a TCRβV28 subfamily, a TCRβV24 subfamily, a TCRβV20 subfamily, TCRβV25 subfamily, or a TCRβV29 subfamily.

In some embodiments, at least one agent of said plurality comprises a domain that specifically binds to a TCRβV region of a TCRβV belonging to a TCRβV12 subfamily.

In some embodiments, each agent of the plurality specifically binds to a different TCRβV, wherein each TCRβV belongs to a different TCRβV subfamily.

In some embodiments, each agent of the plurality specifically binds to a different TCRβV, wherein each TCRβV or are different members of the same TCRβV subfamily.

In some embodiments, the first population of T cells exhibit at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) of: a lower level of IL-1β expression, a lower level of IL-6 expression, a lower level of TNFα expression, a lower level of IFNγ expression, a lower level of IL-10 expression, a lower level of IL-17 expression, a higher level of IL-2 expression, or a higher level of IL-15 expression, relative to a comparable population of T cells that contacted with an agent comprising a domain that specifically binds CD3ε (e.g., an anti-CD3ε antibody).

In some embodiments, expression is measured by determining the level of the protein secreted from the population of T cells, as measured by an assay described herein.

In some embodiments, the expression of CD45R, CD95, and CCR7 is measured by determining the level of the protein on the surface of the cell (e.g., as measured by flow cytometry).

In some embodiments, the contacting comprises incubating the plurality of T cells with the first agent.

In one aspect, provided herein are methods of treating cancer in a subject, the method comprising administering at least a portion of the first population of cells described herein or a pharmaceutical composition comprising at least a portion of the first population of cells described herein.

In some embodiments, the plurality of T cells express an exogenous cell surface receptor. In some embodiments, the exogenous cell surface receptor is a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

In some embodiments, the cell is autologous or allogenic to the subject administered said cell.

In some embodiments, the cancer is a solid cancer or hematological cancer.

The method of any one of claim 81, wherein the cancer is a solid cancer.

In some embodiments, the solid cancer is a prostate cancer, lung cancer, renal cancer, stomach cancer, colon cancer, ovarian cancer, bladder cancer, breast cancer, cervical cancer, esophageal cancer, testicular cancer, liver cancer, pancreatic cancer, rectal cancer, thyroid cancer, uterine cancer, skin cancer, muscle cancer, cartilage cancer, bone cancer, endothelial cancer, epithelial cancer, dermal cancer, basal cancer, retinal cancer, skin cancer, or brain cancer.

In some embodiments, the cancer is a hematologic cancer.

In some embodiments, the hematologic cancer is a leukemia, lymphoma, or myeloma.

In some embodiments, the hematologic cancer is B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), acute lymphoblastic leukemia (ALL); chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell-follicular lymphoma, large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

In one aspect, provided herein are methods of treating cancer in a subject, the method comprising: removing a plurality of T cells from a human subject, expanding at least a portion of the plurality of T cells from the human subject by the method described herein, to thereby generate the first population of T cells, administering at least a portion of the first population of T cells into the human subject, to thereby treat the cancer in the subject.

In some embodiments, the cell is autologous or allogenic to the subject administered said cell.

In some embodiments, the cancer is a solid cancer or hematological cancer.

The method of any one of claim 81, wherein the cancer is a solid cancer.

In some embodiments, the cancer is a hematologic cancer.

In some embodiments, the hematologic cancer is a leukemia, lymphoma, or myeloma.

In one aspect, provided herein are methods of preventing or lessening cytokine release syndrome (CRS) in a human subject, the method comprising: removing a plurality of T cells from a human subject, expanding at least a portion of the plurality of T cells from the human subject by a method described herein, to thereby generate the first population of T cells, administering at least a portion of the first population of T cells into the human subject, wherein after the administration (e.g., within 24 hours, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 14 days, 21 days, or 30 days) the subject shows no symptoms of cytokine release syndrome or at least one symptom of CRS is less severe relative to a human subject administered with at least a portion of a comparable population of T cells generated by expanding the T cells by contacting the plurality of T cells with an agent that binds CD3ε (e.g., an anti-CD3ε antibody).

In some embodiments, the at least one symptom is selected from those listed in Table 8, Table 9, or Table 10.

In some embodiments, the at least one symptom is selected from hemophagocytic lymphohistiocytosis (HLH), fever, nausea, vomiting, chills, hypotension, tachycardia, arrhythmia, cardiomyopathy, acute heart failure, asthenia, headache, rash, dyspnea, encephalopathy, aphasia, tremor, ataxia, hemiparesis, palsy, dysmetria, seizure, motor weakness, loss of consciousness, hallucinations, cerebral edema, hepatomegaly, hypofibrinogeniemia, liver failure, diarrhea, edema, rigor, arthralgia, myalgia, acute kidney failure, splenomegaly, respiratory failure, pulmonary edema, hypoxia, capillary leak syndrome, macrophage activation syndrome, or tachypnea.

The method of any one of claims 87-89, wherein the subject does not exhibit at least one symptom of CRS (e.g., as described herein) within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 30 days of administration of the at least a portion of the first population of T cells.

In some embodiments, the subject does not exhibit at least one symptom grade 4 or grade 5 CRS (e.g., as described herein).

In some embodiments, the subject does not exhibit any symptom grade 4 or grade 5 CRS (e.g., as described herein).

In some embodiments, the level of one or more protein selected from the group consisting of IL-6, IL-10, IL-8, IL-10, IFNγ, TNFα, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF, in the serum of the subject post administration (e.g., 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5, days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days) of the at least a portion of the first population of T cells is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of the level of the one or more protein in the serum of the subject prior to administration (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours) of the at least a portion of the first population of T cells.

In some embodiments, the method further comprises selecting the subject for administration of the first population of T cells described herein based on a determination of at least one of the following: the subject's risk of developing CRS, the subject's risk of developing CRS if administered a cell expressing a CAR comprising a CD3ζ signaling domain, the subject's diagnosis of CRS, or the subject's diagnosis of CRS associated with or induced by administration of a cell expressing a CAR comprising a CD3ζ signaling domain.

In some embodiments, the subject is selected for administration if the subject is at risk of developing CRS, if the subject is at risk of developing CRS if administered a CAR comprising a cell expressing a CAR CD3ζ signaling domain, if the subject has been diagnosed with CRS, or if the subject has been diagnosed with CRS associated with or induced by administration of a cell expressing a CAR comprising a CD3ζ signaling domain.

In some embodiments, the cell is autologous or allogenic to the subject administered said cell.

In some embodiments, the cancer is a solid cancer or hematological cancer.

In some embodiments, the cancer is a solid cancer.

In some embodiments, the cancer is a hematologic cancer.

In some embodiments, the hematologic cancer is a leukemia, lymphoma, or myeloma.

In one aspect, provided herein are recombinant nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) an antigen binding domain, wherein the antigen binding domain does not contain a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising a TCRβ constant region intracellular domain; wherein the intracellular signaling domain does not contain a functional CD3ζ signaling domain.

In some embodiments, the chimeric antigen receptor (CAR) does not contain a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region. In some embodiments, the antigen binding domain, transmembrane domain, and intracellular signaling domain are operatively linked. In some embodiments, the CAR further comprises a TCRβ 1 constant domain or a TCRβ 2 constant domain. In some embodiments, the transmembrane domain comprises a TCRβ constant 1 domain or a TCRβ constant 2 domain. In some embodiments, the antigen binding domain is connected to the transmembrane domain by a linker. In some embodiments, the TCRβ constant intracellular domain comprises a TCRβ constant 1 intracellular domain or a TCRβ constant 2 intracellular domain. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the antigen binding domain is a human or humanized single chain variable fragment (scFv) or single domain antibody (sdAb). In some embodiments, the antigen binding domain specifically binds to a tumor associated antigen. In some embodiments, the encoded chimeric antigen receptor (CAR) is expressed in frame and as a single polypeptide chain.

In one aspect, provided herein are recombinant nucleic acids encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) an antigen binding domain, wherein the antigen binding domain is a single chain variable fragment (scFv) or a single domain antibody; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising a TCRβ intracellular domain; wherein the intracellular signaling domain does not contain a functional CD3 signaling domain.

In one aspect, provided herein are polypeptides encoded by a recombinant nucleic acid described herein.

In one aspect, provided herein are vectors comprising a recombinant nucleic acid molecule described herein.

In one aspect, provided herein are methods of making a population of immune effector cells, comprising transducing a plurality of immune effector cells with a vector described herein.

In one aspect, provided herein are populations of immune effector cells, wherein the immune effector cells comprise a recombinant nucleic acid described herein.

In some embodiments, the population of immune effector cells are made by the method described herein.

In some embodiments, the population of immune effector cells upon binding of the antigen binding domain of the CAR to a cognate antigen expressed by a cell, the level of expression of at least one proinflammatory cytokine by the population immune effector cells is lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of immune effector cells that comprise a nucleic acid encoding a CAR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, the population of immune effector cells upon binding of the antigen binding domain of the CAR to a cognate antigen expressed by a cell, the level of expression of at least one proinflammatory cytokine by the population of immune effector cells is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of immune effector cells that comprise a nucleic acid encoding a CAR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, the population of immune effector cells upon binding of the antigen binding domain of the CAR to a cognate antigen expressed by a cell in the presence of a population of antigen presenting cells, the level of expression of at least one proinflammatory cytokine by the population of antigen presenting cells is lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of antigen presenting cells in the presence of a comparable population of immune effector cells that comprise a nucleic acid encoding a CAR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, the population of immune effector cells upon binding of the antigen binding domain of the CAR to a cognate antigen expressed by a cell in the presence of a population of antigen presenting cells, the level of expression of at least one proinflammatory cytokine by the antigen presenting cell is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of antigen presenting cells in the presence of a population of comparable immune effector cells that comprise a nucleic acid encoding a CAR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, the at least one proinflammatory cytokine is selected from the group consisting of IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, IL-17, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF.

In some embodiments, expression of the at least one proinflammatory cytokine is measured by determining the level of the cytokine secreted from the population of immune effector cells, as measured by an assay described herein.

In some embodiments, expression of the at least one proinflammatory cytokine is measured by determining the level of the cytokine secreted from the population of antigen presenting cells, as measured by an assay described herein.

In some embodiments, said population of antigen presenting cells comprises dendritic cells, macrophages, or monocytes.

In one aspect, provided herein are pharmaceutical compositions comprising at least a portion of the population of immune effector cells described herein.

In one aspect, provided herein are methods of treating a cancer in a subject, the method comprising: administering to the subject at least a portion of the population of immune effector cells described herein.

In one aspect, provided herein are methods of preventing or lessening the severity of cytokine release syndrome (CRS) in a human subject, the method comprising: administering to the subject at least a portion of the population of immune effector cells described herein.

In some embodiments, the subject has cancer.

In some embodiments, the subject does not exhibit at least one symptom of CRS (e.g., as described herein) within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 30 days of administration of the immune cell.

In some embodiments, the subject does not exhibit at least one symptom grade 4 or grade 5 CRS (e.g., as described herein).

In some embodiments, the subject does not exhibit any symptom grade 4 or grade 5 CRS (e.g., as described herein).

In some embodiments, the level of one or more protein selected from the group consisting of IL-6, IL-1β, IL-8, IL-10, IFNγ, TNFα, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF, in the serum of the subject post administration (e.g., 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5, days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days) of the cell (e.g., population of cells, e.g., population of immune effector cells) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of the level of the one or more protein in the serum of the subject prior to administration (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours) of the immune cell.

In some embodiments, the method further comprises selecting the subject for administration of the immune cell of any one of claims 86-100 based on a determination of at least one of the following: the subject's risk of developing CRS, the subject's risk of developing CRS if administered a cell expressing a CAR comprising a CD3ζ signaling domain, the subject's diagnosis of CRS, or the subject's diagnosis of CRS associated with or induced by administration of a cell expressing a CAR comprising a CD3ζ signaling domain.

In some embodiments, the cell is autologous or allogenic to the subject administered said cell.

In some embodiments, the cancer is a solid cancer or hematological cancer.

In some embodiments, the cancer is a solid cancer.

In some embodiments, the cancer is a hematologic cancer.

In some embodiments, the hematologic cancer is a leukemia, lymphoma, or myeloma.

In one aspect, provided herein are recombinant nucleic acids encoding an exogenous T cell receptor (TCR), wherein the TCR comprises: a TCRα chain comprising i) an immunoglobulin variable heavy domain, ii) a TCRα transmembrane domain, and iii) an intracellular signaling domain comprising optionally a TCRα intracellular domain; a TCRβ chain comprising i) an immunoglobulin variable light domain, ii) a TCRβ transmembrane domain, and iii) an intracellular signaling domain comprising a TCRβ intracellular domain; wherein the immunoglobulin variable heavy domain and the immunoglobulin variable light domain form an antigen binding domain; wherein the TCR does not contain a functional CD3ζ intracellular signaling domain; and wherein the TCR does not comprise a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

In some embodiments, the TCRα chain further comprises a TCRα constant domain.

In one aspect, provided herein are recombinant nucleic acids encoding an exogenous T cell receptor (TCR), wherein the TCR comprises: a TCRα chain comprising i) an immunoglobulin variable light domain, ii) a TCRα transmembrane domain, and iii) an intracellular signaling domain comprising optionally a TCRα intracellular domain; a TCRβ chain comprising i) an immunoglobulin variable heavy domain, ii) a TCRβ transmembrane domain, and iii) an intracellular signaling domain comprising a TCRβ intracellular domain; wherein the immunoglobulin variable heavy domain and the immunoglobulin variable light domain form an antigen binding domain; wherein the TCR does not contain a functional CD3ζ intracellular signaling domain; and wherein the TCR does not comprise a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

In some embodiments, the TCRα chain further comprises a TCRα constant domain.

In one aspect, provided herein are recombinant nucleic acids encoding an exogenous T cell receptor (TCR), wherein the TCR comprises: a TCRα chain comprising i) an antigen binding domain (e.g., a scFv), ii) a TCRα variable domain, iii) a TCRα constant domain, iv) a TCRα transmembrane domain, and iii) an intracellular signaling domain comprising optionally a TCRα intracellular domain; a TCRβ chain comprising i) an TCRβ variable domain, ii) a TCRβ constant domain, iii) a TCRβ transmembrane domain, and iv) an intracellular signaling domain comprising a TCRβ intracellular domain; and wherein the TCR does not contain a functional CD3ζ intracellular signaling domain.

In one aspect, provided herein are recombinant nucleic acids encoding an exogenous T cell receptor (TCR), wherein the TCR comprises: a TCRα chain comprising i) a TCRα variable domain, ii) a TCRα constant domain, iii) a TCRα transmembrane domain, and iv) an intracellular signaling domain comprising optionally a TCRα intracellular domain; a TCRβ chain comprising i) an antigen binding domain (e.g., a scFv), ii) an TCRβ variable domain, iii) a TCRβ constant domain, iii) a TCRβ transmembrane domain, and iv) an intracellular signaling domain comprising a TCRβ intracellular domain; and wherein the TCR does not contain a functional CD3ζ intracellular signaling domain.

In one aspect, provided herein are polypeptides encoded by the recombinant nucleic acid described herein.

In one aspect, provided herein are vectors comprising the recombinant nucleic acid described herein.

In one aspect, provided herein are methods of making a population of immune effector cells, comprising transducing the population of immune effector cells with a vector described herein.

In one aspect, provided herein are populations of immune effector cells, wherein the immune effector cells comprise a recombinant nucleic acid described herein.

In some embodiments, the immune effector cells are made by the method described herein.

In some embodiments, the immune effector cells upon binding of the antigen binding domain of the TCR to a cognate antigen expressed by a cell, the level of expression of at least one proinflammatory cytokine by the population immune effector cells is lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of immune effector cells that comprise a nucleic acid encoding a TCR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, the immune effector cells upon binding of the antigen binding domain of the TCR to a cognate antigen expressed by a cell, the level of expression of at least one proinflammatory cytokine by the population of immune effector cells is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of immune effector cells that comprise a nucleic acid encoding a TCR that comprises a CD3 intracellular signaling domain.

In some embodiments, the immune effector cells upon binding of the antigen binding domain of the TCR to a cognate antigen expressed by a cell in the presence of a population of antigen presenting cells, the level of expression of at least one proinflammatory cytokine by the population of antigen presenting cells is lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of antigen presenting cells in the presence of a comparable population of immune effector cells that comprise a nucleic acid encoding a TCR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, the immune effector cells upon binding of the antigen binding domain of the TCR to a cognate antigen expressed by a cell in the presence of a population of antigen presenting cells, the level of expression of at least one proinflammatory cytokine by the antigen presenting cell is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower relative to the level of expression of the at least one proinflammatory cytokine by a comparable population of antigen presenting cells in the presence of a population of comparable immune effector cells that comprise a nucleic acid encoding a TCR that comprises a CD3ζ intracellular signaling domain.

In some embodiments, said population of antigen presenting cells comprises dendritic cells, macrophages, or monocytes.

In one aspect, provided herein are pharmaceutical compositions comprising at least a portion of the population of immune effector cells described herein.

In some embodiments, the subject has cancer.

In some embodiments, the subject does not exhibit at least one symptom of CRS (e.g., as described herein) within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 30 days of administration of the immune cell.

In some embodiments, the subject does not exhibit at least one symptom grade 4 or grade 5 CRS (e.g., as described herein).

In some embodiments, the subject does not exhibit any symptom grade 4 or grade 5 CRS (e.g., as described herein).

In some embodiments, the level of one or more protein selected from the group consisting of IL-6, IL-10, IL-8, IL-10, IFNγ, TNFα, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF, in the serum of the subject post administration (e.g., 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5, days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days) of the cell (e.g., population of cells, e.g., population of immune effector cells) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of the level of the one or more protein in the serum of the subject prior to administration (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours) of the immune cell.

In some embodiments, the method further comprises selecting the subject for administration of the immune cell described herein based on a determination of at least one of the following: the subject's risk of developing CRS, the subject's risk of developing CRS if administered a cell expressing a CAR comprising a CD3ζ signaling domain, the subject's diagnosis of CRS, or the subject's diagnosis of CRS associated with or induced by administration of a cell expressing a CAR comprising a CD3ζ signaling domain.

In some embodiments, the cell is autologous or allogenic to the subject administered said cell.

In some embodiments, the cancer is a solid cancer or hematological cancer.

In some embodiments, the cancer is a solid cancer.

In some embodiments, the cancer is a hematologic cancer.

In some embodiments, the hematologic cancer is a leukemia, lymphoma, or myeloma.

In one aspect, provided herein are methods of expanding a T cell population ex vivo comprising contacting the T cell population with one or more anti-TCRβV antibody, and methods of treating a disease or disorder, e.g., cancer, using the aforesaid expanded cell populations.

Methods described herein include, methods of activating or expanding (or both activating and expanding) T cells ex vivo comprising contacting a plurality of T cells to a first agent, wherein the first agent comprises a first domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, thereby generating a first population of T cells.

In some embodiments, the method further comprises contacting the plurality of T cells with a second agent, wherein the second agent comprises a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, wherein the first and the second agents specifically bind to different TCRβV regions.

In some embodiments, the first agent comprises a domain that specifically binds to a TCRβV region of a first TCRβV belonging to a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily; the second agent comprises a domain that specifically binds to a second TCRβV region of a TCRβV belonging to a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily, and wherein the first and the second agent each specifically bind a TCRβV belonging to a different subfamily or different members of the same TCRβV subfamily.

In some embodiments, the first and the second agent each specifically bind a TCRβV belonging to a different subfamily. In some embodiments, the first and the second agent each specifically bind different members of the same TCRβV subfamily.

In some embodiments, the methods further comprise contacting the plurality of T cells with one more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) agents, wherein each of the one or more agents comprises a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, wherein each of the one or more agents specifically binds to a different T cell receptor beta variable chain (TCRβV) region, and wherein each one of the TCRβV regions the one or more agents specifically bind is different from the TCRβV regions the first and the second agents specifically bind.

In some embodiments, each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) agents specifically bind to a TCRβV belonging to different TCRβV subfamily or that are different members of the same TCRβV subfamily; and wherein each of the one or more agents specifically bind to a TCRβV belonging to different TCRβV subfamily than the TCRβVs bound by the first agent and the second agent or each of the one or more agents specifically bind different members of the same TCRβV subfamily as the TCRβVs bound by the first agent, the second agent, or both.

In some embodiments, the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) agents each comprise a domain that specifically binds to a TCRβV region of a TCRβV belonging to a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily.

In some embodiments, each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) agents specifically bind a TCRβV belonging to a different subfamily, and wherein each of the one or more agents specifically bind a TCRβV that belongs to a different subfamily than the TCRβVs bound by the first agent and the second agent.

In some embodiments, the first agent further comprises a second domain that binds to a protein expressed on the surface of one more T-cells in the plurality. In some embodiments, the first agent is a bispecific antibody molecule.

In some embodiments, the second domain specifically binds to a T cell receptor beta variable chain (TCRβV) region. In some embodiments, the second domain and the first domain specifically bind different T cell receptor beta variable chain (TCRβV) regions. In some embodiments, the second domain and the first domain specifically bind TCRβVs belonging to different subfamilies or different members of the same TCRβV subfamily. In some embodiments, the first domain specifically binds specifically binds to a TCRβV region of a TCRβV belonging to a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily, and the second domain specifically binds a TCRβV region of a TCRβV belonging to a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily. In some embodiments, the second domain and the first domain specifically bind TCRβVs belonging to different subfamilies. In some embodiments, the second domain and the first domain specifically bind different members of the same TCRβV subfamily.

In some embodiments, the second domain specifically binds to CD19 or 4-1BB.

In some embodiments, the second domain specifically binds to an antibody molecule. In some embodiments, the antibody molecule is expressed by one or more of the T cells in the plurality. In some embodiments, the antibody molecule comprises a variable heavy chain and a variable light chain. In some embodiments, the antibody molecule is a scFv or a Fab. In some embodiments, the second domain specifically binds to a light chain of the antibody molecule. In some embodiments, the second domain specifically binds to a κ light chain region of an antibody molecule. In some embodiments, the second domain comprises a protein L.

In some embodiments, the first population of T cells exhibit one or more of: (i) reduced expression of IL-1β, (ii) reduced expression level of IL-6, (iii) reduced expression of TNFα, (iv) increased expression of IL-2, (v) increased expression of IFNγ, (vi) maintained expression of IFNγ, and (vii) increased expression of 4-1BB, relative to a plurality of T cells contacted with an agent comprising a domain that specifically binds CD3 (e.g., CDRε).

In some embodiments, the contacting comprises incubating the plurality of T cells with the first agent.

In some embodiments, contacting comprises incubating or culturing the plurality of T cells with the first agent for at least about 10 minutes, 20 minutes, 30 minutes, 1 hour, 6 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 5 days, 7 days, 10 days, 14 days, 15 days, or 30 days. In some embodiments, contacting comprises incubating or culturing the plurality of T cells with the first agent for at most about 10 minutes, 20 minutes, 30 minutes, 1 hour, 6 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 5 days, 7 days, 10 days, 12 days, 14 days, 15 days, 21 days, 30 days, 45 days, or 60 days. In some embodiments, contacting comprises incubating or culturing the plurality of T cells with the first agent for about from 10-90 minutes, 10-60 minutes, 10-30 minutes, 1-30 days, 1-21 days, 1-14 days, 1-7 days, 1-5 days, 1-3 days, 21-30 days, 14-30 days, 7-30 days, 5-30 days, or 3-30 days.

In some embodiments, the first agent is coupled to a solid surface (e.g., a bead). In some embodiments, the first agent comprises an antibody domain.

In some embodiments, the first agent comprises an anti-idiotypic antibody domain. In some embodiments, the first agent comprises a mouse antibody domain. In some embodiments, the first agent comprises a human antibody domain. In some embodiments, the first agent comprises a humanized antibody domain. In some embodiments, the first agent comprises an antigen binding domain comprising a single chain Fv (scFv) or a Fab. In some embodiments, the first agent comprises an antibody comprising two antibody heavy chains, each heavy chain comprising a variable region and a constant region; and two antibody light chains, each light chain comprising a variable region and a constant region.

In some embodiments, the plurality of T cells comprises one or more T cell comprising an exogenous nucleic acid.

In some embodiments, the plurality of T cells comprises one or more T cell comprising an exogenous nucleic acid encoding a chimeric polypeptide. In some embodiments, the method comprises introducing an exogenous nucleic acid into one or more T cells of the plurality prior to contacting the plurality of T cells with the first agent. In some embodiments, the method comprises introducing an exogenous nucleic acid into one or more T cells of the plurality after to contacting the plurality of T cells with the first agent. In some embodiments, the method further comprises introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the T cells of the plurality prior to contacting the plurality of T cells with the first agent. In some embodiments, the method further comprises introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the T cells of the plurality after to contacting the plurality of T cells with the first agent. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

In some embodiments, the chimeric polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) comprises and antigen binding region, a transmembrane region, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises one or more signaling domain. In some embodiments, the intracellular signaling domain comprises a signaling domain from CD27, CD28, 4-1BB, ICOS, OX40, DAP10, DAP12, CD134, CD3-zeta or fragment or combination thereof. In some embodiments, the transmembrane region comprises a transmembrane region from CD8, CD28, or CTLA4.

In some embodiments, the antigen binding region comprises an antibody domain. In some embodiments, the antibody domain comprises a scFv or a Fab. In some embodiments, the antigen binding region specifically binds a tumor associated antigen (e.g., as described herein).

In some embodiments, the chimeric polypeptide is a chimeric T cell receptor (TCR). In some embodiments, the chimeric TCR comprises an antigen binding region. In some embodiments, the chimeric TCR further comprises a transmembrane region. In some embodiments, the chimeric TCR further comprises an intracellular signaling region. In some embodiments, the chimeric TCR comprises a TCR α polypeptide and a TCR β polypeptide. In some embodiments, chimeric TCR comprises a TCR γ polypeptide and a TCR δ polypeptide. In some embodiments, the antigen binding region specifically binds a tumor associated antigen.

In some embodiments, the plurality of T cells comprises one or more T cells from a human subject.

In some embodiments, the one or more T cell are removed from the human subject via apheresis.

In some embodiments, the plurality of T cells comprises one or more T cell from a human subject that is healthy (e.g., a subject that does not have or has not been diagnosed with a specified disease or condition, e.g., a cancer). In some embodiments, the plurality of T cells comprises one or more T cells from a mammalian (e.g., human) subject having or diagnosed with a disease or condition (e.g., diagnosed with a specified disease or condition, e.g., cancer). In some embodiments, the disease is a cancer. In some embodiments, the cancer is a solid tumor or hematological cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, prostate, lung, renal, stomach, colon, ovarian, bladder, breast, cervical, esophageal, testicular, liver, pancreatic, rectal, thyroid, uterine, skin, muscle, cartilage, bone, endothelial, epithelial, dermal, basal, retinal, skin, or brain.

In some embodiments, the plurality of T cells comprises one or more autologous T cell. In some embodiments, the plurality of T cells comprises one or more allogeneic T cell.

In some embodiments, the number of cells in the first T cell population is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, or 1000 fold greater than the number of cells in the plurality of T cells prior to be contacted with the first agent.

In some embodiments, the agent that specifically binds CD3 (e.g., CD3ε) comprises an antibody domain (e.g., an anti-CD3 antibody (e.g., an anti-CD3ε antibody)).

In some embodiments, the agent that specifically binds CD3 specifically binds CD3ε.

In some embodiments, the first agent, upon binding to the TCRβV region, results in one, two, three, four, five, six, seven, eight, nine, ten or more (e.g., all) of the following: (i) reduced level, e.g., expression level, and/or activity of IL-1β; (ii) reduced level, e.g., expression level, and/or activity of IL-6; (iii) reduced level, e.g., expression level, and/or activity of TNFα; (iv) increased level, e.g., expression level, and/or activity of IL-2; (v) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours delay, in increased level, e.g., expression level, and/or activity of IL-2; (vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay, in increased level, e.g., expression level, and/or activity of IFNγ; (vii) reduced T cell proliferation kinetics; or (viii) reduced cytokine storm, e.g., cytokine release syndrome (CRS), e.g., as measured by an assay described herein; (ix) cell killing, e.g., target cell killing, e.g. cancer cell killing, e.g., as measured by an assay described herein; (x) increased level, e.g., expression level, and/or activity of IL-15; or (xi) increased Natural Killer (NK) cell proliferation, e.g., expansion, compared to an antibody that binds to: a CD3 molecule, e.g., CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments, the first agent, upon binding to the TCRβV region, results in expansion, e.g., at least about 1.1-10 fold expansion (e.g., at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold expansion), of a population of memory T cells, e.g., T effector memory (TEM) cells, e.g., TEM cells expressing CD45RA (TEMRA) cells.

In some embodiments, expansion of a population of memory effector T cells, e.g., TEM cells, e.g., TEMRA cells, in the first population of T cells a is increased compared to expansion of a similar population of cells with an antibody that binds to a CD3 molecule.

In some embodiments, the population of expanded T effector memory cells comprises cells which: (i) have a detectable level of CD45RA, e.g., express or re-express CD45RA; (ii) have low or no expression of CCR7; and/or (iii) have a detectable level of CD95, e.g., express CD95, e.g., a population of CD45RA+, CCR7−, CD95+ T cells, optionally wherein the T cells comprise CD3+, CD4+ or CD8+ T cells.

In some embodiments, binding of the first agent to the TCRβV region results in one, two, three, four, five, six, seven, eight, nine, ten or more (e.g., all) of the following: (i) reduced level, e.g., expression level, and/or activity of IL-10; (ii) reduced level, e.g., expression level, and/or activity of IL-6; (iii) reduced level, e.g., expression level, and/or activity of TNFα; (iv) increased level, e.g., expression level, and/or activity of IL-2; (v) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours delay, in increased level, e.g., expression level, and/or activity of IL-2; (vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay, in increased level, e.g., expression level, and/or activity of IFNγ; (vii) reduced T cell proliferation kinetics; or (viii) reduced cytokine storm, e.g., cytokine release syndrome (CRS), e.g., as measured by an assay described herein; (ix) cell killing, e.g., target cell killing, e.g. cancer cell killing, e.g., as measured by an assay described herein; (x) increased level, e.g., expression level, and/or activity of IL-15; or (xi) increased Natural Killer (NK) cell proliferation, e.g., expansion, compared to an antibody that binds to: a CD3 molecule, e.g., CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

In some embodiments, binding of the first agent to the TCRβV region results in a reduction of at least 2, 5, 10, 20, 50, 100, or 200 fold, or at least 2-200 fold (e.g., 5-150, 10-100, 20-50 fold) in the expression level and or activity of IL-1β as measured by an assay described herein.

In some embodiments, binding of the first agent to the TCRβV region results in a reduction of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 fold, or at least 2-1000 fold (e.g., 5-900, 10-800, 20-700, 50-600, 100-500, or 200-400 fold) in the expression level and or activity of IL-6 as measured by an assay described herein.

In some embodiments, binding of the first agent to the TCRβV region results in a reduction of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000 fold, or at least 2-2000 fold (e.g., 5-1000, 10-900, 20-800, 50-700, 100-600, 200-500, or 300-400 fold) in the expression level and or activity of TNFα as measured by an assay described herein.

In some embodiments, binding of the first agent to the TCRβV region results in an increase of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000 fold, or at least 2-2000 fold (e.g., 5-1000, 10-900, 20-800, 50-700, 100-600, 200-500, or 300-400 fold) in the expression level and or activity of IL-2 as measured by an assay described herein.

Methods described herein, include, methods of expanding T cells ex vivo comprising contacting a plurality of T cells to a plurality of agents, wherein the plurality of agents comprises two, three, four, five, or more agents, wherein each agent of the plurality comprises a domain that specifically binds to a different T cell receptor beta variable chain (TCRβV) region, thereby generating a first population of T cells.

In some embodiments, each agent of the plurality comprises a domain that specifically binds to a TCRβV region of a TCRβV belonging to a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily.

In some embodiments, each agent of the plurality specifically binds to a different TCRβV, wherein each TCRβV belongs to a different TCRβV subfamily.

Methods described herein, include, methods of expanding T cells ex vivo comprising contacting a plurality of T cells to a plurality of agents, wherein the plurality of agents comprises at least a first and a second agent, wherein each agent of the plurality comprises a domain that specifically binds to a different T cell receptor beta variable chain (TCRβV) region, thereby generating a first population of T cells.

In some embodiments, the plurality comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more agents.

In some embodiments, the method further comprises contacting the plurality of T cells with a second agent, wherein the second agent comprises a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, wherein the first and the second agents specifically bind to different TCRβV regions.

In some embodiments, the first agent and/or the second agent further comprises a second domain that binds to a protein expressed on the surface of one more T-cells in the plurality. In some embodiments, the first agent is a bispecific antibody molecule.

In some embodiments, the second domain specifically binds to CD19 or 4-1BB.

In some embodiments, the contacting comprises incubating the plurality of T cells with the first agent.

In some embodiments, the first agent is coupled to a solid surface (e.g., a bead). In some embodiments, the first agent comprises an antibody domain. In some embodiments, each agent of the plurality is coupled to one or more solid surface (e.g., one or more beads). In some embodiments, each agent of the plurality comprises an antibody domain.

In some embodiments, each agent of the plurality comprises an anti-idiotypic antibody domain. In some embodiments, each agent of the plurality comprises a mouse antibody domain. In some embodiments, each agent of the plurality comprises a human antibody domain. In some embodiments, each agent of the plurality comprises a humanized antibody domain. In some embodiments, each agent of the plurality comprises an antigen binding domain comprising a single chain Fv (scFv) or a Fab. In some embodiments, each agent of the plurality comprises an antibody comprising two antibody heavy chains, each heavy chain comprising a variable region and a constant region; and two antibody light chains, each light chain comprising a variable region and a constant region.

In some embodiments, the plurality of T cells comprises one or more T cell comprising an exogenous nucleic acid.

In some embodiments, the plurality of T cells comprises one or more T cell comprising an exogenous nucleic acid encoding a chimeric polypeptide. In some embodiments, the method comprises introducing an exogenous nucleic acid into one or more T cells of the plurality prior to contacting the plurality of T cells with the first agent. In some embodiments, the method comprises introducing an exogenous nucleic acid into one or more T cells of the plurality after contacting the plurality of T cells with the first agent. In some embodiments, the method further comprises introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the T cells of the plurality prior to contacting the plurality of T cells with the first agent. In some embodiments, the method further comprises introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the T cells of the plurality after to contacting the plurality of T cells with the first agent. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

In some embodiments, the plurality of T cells comprises one or more T cells from a human subject. In some embodiments, the one or more T cell are removed from the human subject via apheresis. In some embodiments, the plurality of T cells comprises one or more T cell from a human subject that is healthy (e.g., a subject that does not have or has not been diagnosed with a specified disease or condition, e.g., a cancer). In some embodiments, the plurality of T cells comprises one or more T cells from a mammalian (e.g., human) subject having or diagnosed with a disease or condition (e.g., diagnosed with a specified disease or condition, e.g., cancer). In some embodiments, the disease is a cancer. In some embodiments, the cancer is a solid tumor or hematological cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, prostate, lung, renal, stomach, colon, ovarian, bladder, breast, cervical, esophageal, testicular, liver, pancreatic, rectal, thyroid, uterine, skin, muscle, cartilage, bone, endothelial, epithelial, dermal, basal, retinal, skin, or brain.

In some embodiments, the plurality of T cells comprises one or more autologous T cell. In some embodiments, the plurality of T cells comprises one or more allogeneic T cell.

In some embodiments, the agent that specifically binds CD3 (e.g., CD3ε) comprises an antibody domain (e.g., an anti-CD3 antibody (e.g., an anti-CD3ε antibody)).

In some embodiments, the agent that specifically binds CD3 specifically binds CD3ε.

In some embodiments, binding of the first agent to the TCRβV region results in one, two, three, four, five, six, seven, eight, nine, ten or more (e.g., all) of the following: (i) reduced level, e.g., expression level, and/or activity of IL-1β; (ii) reduced level, e.g., expression level, and/or activity of IL-6; (iii) reduced level, e.g., expression level, and/or activity of TNFα; (iv) increased level, e.g., expression level, and/or activity of IL-2; (v) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours delay, in increased level, e.g., expression level, and/or activity of IL-2; (vi) a delay, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours delay, in increased level, e.g., expression level, and/or activity of IFNγ; (vii) reduced T cell proliferation kinetics; or (viii) reduced cytokine storm, e.g., cytokine release syndrome (CRS), e.g., as measured by an assay described herein; (ix) cell killing, e.g., target cell killing, e.g. cancer cell killing, e.g., as measured by an assay described herein; (x) increased level, e.g., expression level, and/or activity of IL-15; or (xi) increased Natural Killer (NK) cell proliferation, e.g., expansion, compared to an antibody that binds to: a CD3 molecule, e.g., CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

Methods disclosed herein, include, methods of treating cancer in a subject, the method comprising: removing a plurality of T cells from a human subject, expanding the plurality of T cells from the human subject comprising contacting the plurality of T cells to a first agent, wherein the first agent comprises a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, thereby generating a first population of T cells, infusing at least a portion of the first population of T cells into the human subject, to thereby treat the cancer in the subject.