Anti-TCR antibody molecule and its use

Abstract

The present disclosure provides a method of expanding T cells ex vivo, comprising contacting the T cells with an antibody molecule that binds to the TCR Vβ region. In some embodiments, the T cells comprise one or more nucleic acid molecules encoding an exogenous cell receptor, e.g., a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR).

Description

Technical Field

[0001] Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 788,497, filed Jan. 4, 2019, and U.S. Provisional Patent Application 62 / 803,893, filed Feb. 11, 2019, the disclosures of each of which are hereby incorporated by reference in their entirety.

Background Art

[0002]

[0002] Current molecules designed to activate and expand T cells that encode exogenous receptors (e.g., CAR T cells, T cells expressing exogenous TCRs) 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, this approach has limitations. Previous studies have shown that the use of these anti-CD3ε targeting molecules can produce or stimulate other cells to produce inflammatory cytokines (e.g., IL-1, IL-6, and TNFα) associated with inflammatory conditions such as cytokine release syndrome (CRS), macrophage activation syndrome, neurotoxicity, and tumor lysis syndrome when injected into a subject. Thus, there is a need to develop additional methods for activating and expanding T cells ex vivo that do not pose these significant risks to patients.

Summary of the Invention

Problems to be Solved by the Invention

[0003]

[0003] This disclosure is based, at least in part, on the unexpected discovery that T cells can be activated and expanded ex vivo using anti-TCRVβ antibodies; that these T cells secrete substantially low levels of pro-inflammatory cytokines associated with the induction of cytokine release syndrome (CRS), macrophage activation syndrome, neurotoxicity, and tumor lysis syndrome in vivo, such as IFNγ, IL-10, IL-17A, IL-1α, IL-1β, IL-2, IL-6 and TNFα; while secreting higher or similar levels of IL-2.

[0004]

[0004] In particular, methods for expanding (expanding: proliferating) T cells ex vivo using antibodies directed to the variable chain of the β subunit of the TCR (TCRβV) are disclosed herein. In some embodiments, the methods described herein do not result in significant or any production of cytokines associated with cytokine release syndrome (CRS), such as IL-6, IL-1 beta and TNF alpha; and enhance and / or delay the production of IL-2 and IFNγ. In some embodiments, the methods described herein limit the undesirable side effects of CRS, such as CRS associated with anti-CD3e targeting.

Means for Solving the Problems

[0005]

[0005] Accordingly, in one aspect, provided herein is a method for expanding T cells ex vivo, comprising contacting a plurality of T cells with a first substance, the first substance comprising a first domain that specifically binds to the T cell receptor beta variable β chain (TCRβV) region, thereby generating a first population of T cells.

[0006]

[0006] In some embodiments, the first substance further comprises a second domain that binds to a protein expressed on the surface of a plurality of T cell populations.

[0007] In some embodiments, the first substance is a bispecific antibody molecule.

[0007]

[0008] In some embodiments, the second domain specifically binds to the T cell receptor variable beta chain (TCRβV) region.

[0009] In some embodiments, the second domain and the first domain specifically bind to different T cell receptor variable beta chain (TCRβV) regions.

[0008]

[0010] 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.

[0009]

[0011] In some embodiments, the first domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily or TCRβV29 subfamily, and the second domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily or TCRβV29 subfamily.

[0010]

[0012] In some embodiments, the first domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβV12 subfamily.

[0013] In some embodiments, the second domain and the first domain specifically bind to TCRβV belonging to different subfamilies.

[0011]

[0014] In some embodiments, the second domain and the first domain specifically bind to different members of the same TCRβV subfamily.

[0015] In some embodiments, the second domain specifically binds to an antibody molecule. In some embodiments, the antibody molecule is expressed by a plurality of T cell populations. 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.

[0012]

[0016] In some embodiments, the second domain specifically binds to the light chain region of the antibody molecule. In some embodiments, the second domain specifically binds to the kappa light chain region of the antibody molecule. In some embodiments, the second domain comprises protein L.

[0013]

[0017] In some embodiments, the first domain comprises the LC CDR1, LC CDR2, LC CDR, HC CDR1, HC CDR2, and HC CDR3 of the antibodies described in Table 2, Table 3, Table 4, or Table 5. In some embodiments, the first domain comprises the VH and VL chain sequences of the antibodies disclosed in Table 2, Table 3, Table 4, or Table 5.

[0014]

[0018] In some embodiments, the first substance comprises the LC CDR1, LC CDR2, LC CDR, HC CDR1, HC CDR2, and HC CDR3 of the antibodies described in Table 2, Table 3, Table 4, or Table 5. In some embodiments, the first substance comprises the VH and VL chain sequences of the antibodies disclosed in Table 2, Table 3, Table 4, or Table 5.

[0015]

[0019] In some embodiments, the first substance specifically binds to at least two TCRβVs belonging to different subfamilies.

[0020] In some embodiments, the first substance specifically binds to at least 3, 4, 5, or 6 TCRβVs belonging to different subfamilies.

[0016]

[0021] In some embodiments, the first substance specifically binds to at least two different members of the same TCRβV subfamily.

[0022] In some embodiments, the first substance specifically binds to at least 3, 4, 5, 6, or 7 different members of the same TCRβV subfamily.

[0017]

[0023] In some embodiments, the method further comprises contacting a plurality of T cells with a second substance, the second substance comprising a domain that specifically binds to a T cell receptor variable beta chain (TCRβV) region, and the first and second substances specifically bind to different TCRβV regions.

[0018]

[0024] In some embodiments, the first substance comprises a domain that specifically binds to a TCRβV region of a first TCRβV, the second substance comprises a domain that specifically binds to a TCRβV region of a second TCRβV, and the first substance and the second TCRβV belong to different TCRβV subfamilies or are different members of the same TCRβV subfamily.

[0019]

[0025] In some embodiments, the first domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily or TCRβV29 subfamily, and the second domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily or TCRβV29 subfamily.

[0020]

[0026] In some embodiments, the first substance comprises a domain that specifically binds to the TCRβV region of a first TCRβV belonging to the TCRβV12 subfamily.

[0027] In some embodiments, the first and second substances each specifically bind to TCRβV belonging to different subfamilies.

[0021]

[0028] In some embodiments, the first and second substances each specifically bind to different members of the same TCRβV subfamily.

[0029] In some embodiments, the first T cell population exhibits at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) of low levels of IL-1β expression, low levels of IL-6 expression, low levels of TNFα expression, low levels of IFNγ expression, low levels of IL-10 expression, low levels of IL-17 expression, high levels of IL-2 expression, or high levels of IL-15 expression as compared to an equivalent T cell population contacted with a substance (e.g., an anti-CD3ε antibody) that specifically binds to CD3ε.

[0022]

[0030] In some embodiments, expression is measured by determining the protein levels secreted from the T cell population when measured by the assays described herein.

[0031] In some embodiments, the IL-1β expression level 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 an equivalent T cell population contacted with a substance that specifically binds to CD3ε when measured by the assays described herein.

[0023]

[0032] In some embodiments, the IL-6 expression level 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 an equivalent T cell population contacted with a substance that specifically binds to CD3ε when measured by the assays described herein.

[0024]

[0033] In some embodiments, the IL-10 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0025]

[0034] In some embodiments, the IL-17 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0026]

[0035] In some embodiments, the IFN-γ expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0027]

[0036] In some embodiments, the TNF-α expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0028]

[0037] In some embodiments, the IL-15 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε as measured by the assays described herein.

[0029]

[0038] In some embodiments, the IL-2 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε as measured by the assays described herein.

[0030]

[0039] In some embodiments, the number of T cells in the first T cell population is 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 a plurality of T cells.

[0031]

[0040] In some embodiments, the number of T cells in the first T cell population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 is higher compared to the number of T cells in an equivalent population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 and is contacted with a substance (e.g., an anti-CD3ε antibody) comprising a domain that specifically binds to CD3ε.

[0032]

[0041] In some embodiments, the number of T cells in the first population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 500 or 1000-fold higher compared to the number of T cells in an equivalent population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 and is contacted with a substance (e.g., an anti-CD3 antibody) comprising a domain that specifically binds to CD3ε.

[0033]

[0042] In some embodiments, the expression of CD45R, CD95, and CCR7 is measured by determining the protein level on the cell surface (e.g., measured by flow cytometry).

[0034]

[0043] In some embodiments, the number of TEMRA T cells in the first population is higher than the number of TEMRA T cells in an equivalent T cell population contacted with a substance (e.g., an anti-CD3ε antibody) that contains a domain that specifically binds to CD3ε.

[0035]

[0044] 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 times higher than the number of TEMRA T cells in an equivalent T cell population contacted with a substance (e.g., an anti-CD3ε antibody) that contains a domain that specifically binds to CD3ε.

[0036]

[0045] In some embodiments, the contacting step includes incubating a plurality of T cells with the first substance.

[0046] In some embodiments, the contacting step includes incubating or culturing a plurality of T cells with the first substance 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.

[0037]

[0047] In some embodiments, the contacting step includes incubating or culturing a plurality of T cells with the first substance for up to 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.

[0038]

[0048] In some embodiments, the step of contacting comprises incubating or culturing a plurality of T cells with a first substance for about 10 to 90 minutes, 10 to 60 minutes, 10 to 30 minutes, 1 to 30 days, 1 to 21 days, 1 to 14 days, 1 to 7 days, 1 to 5 days, 1 to 3 days, 21 to 30 days, 14 to 30 days, 7 to 30 days, 5 to 30 days, or 3 to 30 days.

[0039]

[0049] In some embodiments, the first substance is coupled to a solid surface (e.g., beads, cell culture plates). In some embodiments, the coupling enables cross-linking of TCRs on the surface of a plurality of T cells specifically bound by the first substance.

[0040]

[0050] In some embodiments, the first substance comprises an antibody domain. In some embodiments, the first substance comprises an anti-idiotype antibody domain. In some embodiments, the first substance comprises a human or humanized antibody domain. In some embodiments, the first substance comprises an antigen-binding domain comprising a single-chain Fv (scFv) or Fab. In some embodiments, the first substance comprises an antibody comprising two antibody heavy chains each comprising a variable region and a constant region, and two antibody light chains each comprising a variable region and a constant region.

[0041]

[0051] In some embodiments, the plurality of T cells comprises a T cell population comprising 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 exogenous nucleic acid into at least a portion of the plurality of T cells prior to contacting the plurality of T cells with the first substance. In some embodiments, the method further comprises introducing exogenous nucleic acid into at least a portion of the plurality of T cells after contacting the plurality of T cells with the first substance. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

[0042]

[0052] In some embodiments, the plurality of T cells are human. In some embodiments, the plurality of T cells include T cells derived from a human subject who was healthy when the cells were removed (e.g., a subject who does not have or is not diagnosed with a given disease or condition, such as cancer). In some embodiments, the plurality of T cells include T cells derived from a human subject who had or was diagnosed with a disease or condition (e.g., diagnosed with a given disease or condition, such as cancer) when the cells were removed. In some embodiments, the disease is cancer.

[0043]

[0053] In one aspect, the methods provided herein are methods of expanding T cells ex vivo, comprising contacting a plurality of T cells with a plurality of substances, wherein the plurality of substances comprises at least a first and a second substance, each of the plurality of substances comprising a domain that specifically binds to a different T cell receptor variable beta chain (TCRβV) region, thereby generating a first population of T cells.

[0044]

[0054] In some embodiments, the first substance or the second substance or both specifically bind to at least two TCRβVs belonging to different subfamilies.

[0055] In some embodiments, the first substance or the second substance or both specifically bind to at least 3, 4, 5, or 6 TCRβVs belonging to different subfamilies.

[0045]

[0056] In some embodiments, the first substance or the second substance or both specifically bind to at least two different members of the same TCRβV subfamily.

[0057] In some embodiments, the first substance or the second substance or both specifically bind to at least 3, 4, 5, 6, or 7 different members of the same TCRβV subfamily.

[0046]

[0058] In some embodiments, the plurality comprises at least 3, 4, 5, 6, 7, 8, 9, or 10 substances, and each of the plurality of substances comprises a domain that specifically binds to a different T cell receptor variable beta chain (TCRβV) region.

[0047]

[0059] In some embodiments, each of the plurality of substances specifically binds to a different TCRβV, and each TCRβV belongs to a different TCRβV subfamily or is a different member of the same TCRβV subfamily.

[0048]

[0060] In some embodiments, each of the plurality of substances comprises a domain that specifically binds to a TCRβV region of a TCRβV belonging to the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily, or TCRβV29 subfamily.

[0049]

[0061] In some embodiments, at least one of the plurality of agents comprises a domain that specifically binds to a TCRβV region of a TCRβV belonging to the TCRβV12 subfamily.

[0062] In some embodiments, each of the plurality of substances specifically binds to a different TCRβV, and each TCRβV belongs to a different TCRβV subfamily.

[0050]

[0063] In some embodiments, each of the plurality of substances specifically binds to a different TCRβV, and each TCRβV is a different member of the same TCRβV subfamily.

[0064] In some embodiments, the first T cell population exhibits at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) of low-level IL-1β expression, low-level IL-6 expression, low-level TNFα expression, low-level IFNγ expression, low-level IL-10 expression, low-level IL-17 expression, high-level IL-2 expression, or high-level IL-15 expression as compared to an equivalent T cell population contacted with a substance (e.g., an anti-CD3ε antibody) comprising a domain that specifically binds to CD3ε.

[0051]

[0065] In some embodiments, expression is measured by determining the protein level secreted from the T cell population when measured by the assays described herein.

[0066] In some embodiments, the IL-1β expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0052]

[0067] In some embodiments, the IL-6 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0053]

[0068] In some embodiments, the IL-10 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0054]

[0069] In some embodiments, the IL-17 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0055]

[0070] In some embodiments, the IFN-γ expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0056]

[0071] In some embodiments, the TNF-α expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0057]

[0072] In some embodiments, the IL-15 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε when measured by the assays described herein.

[0058]

[0073] In some embodiments, the IL-2 expression level 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 an equivalent T cell population contacted with a substance comprising a domain that specifically binds to CD3ε as measured by the assays described herein.

[0059]

[0074] In some embodiments, the number of T cells in the first T cell population is 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 a plurality of T cells.

[0060]

[0075] In some embodiments, the number of T cells in the first T cell population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 is higher compared to the number of T cells in an equivalent population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 and is contacted with a substance (e.g., an anti-CD3ε antibody) comprising a domain that specifically binds to CD3ε.

[0061]

[0076] In some embodiments, the number of T cells in the first population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 500 or 1000-fold higher compared to the number of T cells in an equivalent population that expresses CD45R, expresses CD95, and exhibits low or undetectable expression of CCR7 and is contacted with a substance (e.g., an anti-CD3 antibody) comprising a domain that specifically binds to CD3ε.

[0062]

[0077] In some embodiments, the expression of CD45R, CD95 and CCR7 is measured by determining the protein level on the cell surface (e.g., measured by flow cytometry).

[0063]

[0078] In some embodiments, the number of TEMRA T cells in the first population is higher than the number of TEMRA T cells in an equivalent T cell population that has been contacted with a substance (e.g., an anti-CD3ε antibody) comprising a domain that specifically binds to CD3ε.

[0064]

[0079] 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 times higher than the number of TEMRA T cells in an equivalent T cell population that has been contacted with a substance (e.g., an anti-CD3ε antibody) comprising a domain that specifically binds to CD3ε.

[0065]

[0080] In some embodiments, the contacting step comprises incubating a plurality of T cells with the first substance.

[0081] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with the first substance 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.

[0066]

[0082] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with the first substance for up to 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.

[0067]

[0083] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with the first substance for 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.

[0068]

[0084] In some embodiments, the first substance is coupled to a solid surface (e.g., beads, cell culture plates). In some embodiments, the coupling enables cross-linking of TCRs on the surface of a plurality of T cells specifically bound by the first substance.

[0069]

[0085] In some embodiments, the first substance comprises an antibody domain. In some embodiments, the first substance comprises an anti-idiotype antibody domain. In some embodiments, the first substance comprises a human or humanized antibody domain. In some embodiments, the first substance comprises an antigen-binding domain comprising a single-chain Fv (scFv) or Fab. In some embodiments, the first substance comprises an antibody comprising two antibody heavy chains each comprising a variable region and a constant region, and two antibody light chains each comprising a variable region and a constant region.

[0070]

[0086] In some embodiments, the plurality of T cells comprises a population of T cells comprising 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 exogenous nucleic acid into at least a portion of the plurality of T cells prior to contacting the plurality of T cells with the first substance. In some embodiments, the method further comprises introducing exogenous nucleic acid into at least a portion of the plurality of T cells after contacting the plurality of T cells with the first substance. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

[0071]

[0087] In some embodiments, the plurality of T cells are human. In some embodiments, the plurality of T cells include T cells derived from a human subject who was healthy when the cells were removed (e.g., a subject without or not diagnosed with a given disease or condition, such as cancer). In some embodiments, the plurality of T cells include T cells derived from a human subject who had or was diagnosed with a disease or condition (e.g., diagnosed with a given disease or condition, such as cancer) when the cells were removed. In some embodiments, the disease is cancer.

[0072]

[0088] In one aspect, provided herein is a method of treating cancer in a subject, comprising administering at least a portion of a first cell population described herein, or a pharmaceutical composition comprising at least a portion of a first cell population described herein.

[0073]

[0089] 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).

[0074]

[0090] In some embodiments, the cells are autologous or allogeneic to the subject to whom the cells are administered.

[0091] In some embodiments, the cancer is a solid cancer or a hematologic cancer.

[0075]

[0092] The method according to any one of claims 81, wherein the cancer is a solid cancer.

[0093] In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney 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.

[0076]

[0094] In some embodiments, the cancer is a hematologic cancer.

[0095] In some embodiments, the blood cancer is leukemia, lymphoma or myeloma.

[0096] In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0077]

[0097] In one aspect, the methods provided herein are methods of treating cancer in a subject, comprising the steps of 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 methods described herein, thereby generating a first population of T cells, and administering at least a portion of the first population of T cells to the human subject, thereby treating cancer in the subject.

[0078]

[0098] 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).

[0079]

[0099] In some embodiments, the cells are autologous or allogeneic to the subject to whom the cells are administered.

[0100] In some embodiments, the cancer is a solid cancer or a blood cancer.

[0080]

[0101] The method according to any one of claims 81, wherein the cancer is a solid cancer.

[0102] In some embodiments, solid cancer is prostate cancer, lung cancer, kidney cancer, gastric 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.

[0081]

[0103] In some embodiments, the cancer is a blood cancer.

[0104] In some embodiments, the blood cancer is leukemia, lymphoma or myeloma.

[0105] In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0082]

[0106] In one aspect, the methods provided herein are methods for preventing or reducing cytokine release syndrome (CRS) in a human subject, the method comprising removing a plurality of T cells from the human subject, expanding at least a portion of the plurality of T cells from the human subject by the methods described herein to thereby generate a first T cell population, and administering at least a portion of the first T cell population to the human subject, wherein after 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 does not exhibit symptoms of cytokine release syndrome or at least one symptom of CRS is less severe compared to a human subject to whom at least a portion of an equivalent T cell population generated by expanding T cells by contacting the plurality of T cells with a substance that binds to CD3ε (e.g., an anti-CD3ε antibody) is administered.

[0083]

[0107] In some embodiments, the at least one symptom is selected from those listed in Table 8, Table 9, or Table 10.

[0108] 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, hemiplegia, paralysis, dysmetria, seizure, motor weakness, loss of consciousness, hallucination, cerebral edema, hepatomegaly, hypofibrinogenemia, liver failure, diarrhea, edema, rigidity, arthralgia, myalgia, acute renal failure, splenomegaly, respiratory failure, pulmonary edema, hypoxia, capillary leak syndrome, macrophage activation syndrome, or tachypnea.

[0084]

[0109] The method according to 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 from administration of at least a portion of the first T cell population.

[0085]

[0110] In some embodiments, the subject does not exhibit CRS with at least one symptom grade 4 or grade 5 (e.g., as described herein).

[0111] In some embodiments, the subject does not exhibit any CRS of grade 4 or grade 5 (e.g., as described herein).

[0086]

[0112] In some embodiments, the level of one or more proteins 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 a subject after administration of at least a portion of the first T cell population (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) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the level of one or more protein levels in the serum of the subject before administration of at least a portion of the first T cell population (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours).

[0087]

[0113] In some embodiments, the method further comprises the step of selecting a subject for administration of the first T cell population described herein based on at least one determination of: the risk of a subject developing CRS, the risk of a subject developing CRS upon administration of cells expressing a CAR comprising a CD3ζ signaling domain, the diagnosis of CRS in a subject, or the diagnosis of CRS in a subject associated with or induced by the administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0088]

[0114] In some embodiments, a subject is selected for administration if the subject is at risk of developing CRS, if the subject is at risk of developing CRS upon administration of a CAR expressing a CAR CD3ζ signaling domain, if the subject is diagnosed with CRS, or if the subject is diagnosed with CRS associated with or induced by the administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0089]

[0115] In some embodiments, the cells are autologous or allogeneic to the subject to which the cells are administered.

[0116] In some embodiments, the cancer is a solid cancer or a hematologic cancer.

[0090]

[0117] In some embodiments, the cancer is a solid cancer.

[0118] In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney cancer, gastric 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.

[0091]

[0119] In some embodiments, the cancer is a hematologic cancer.

[0120] In some embodiments, the hematologic cancer is leukemia, lymphoma or myeloma.

[0121] In some embodiments, the hematologic cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0092]

[0122] In one aspect, the present specification provides a recombinant nucleic acid molecule encoding a chimeric antigen receptor (CAR), the CAR comprising: (a) an antigen-binding domain that does not include 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 that does not include a functional CD3ζ signaling domain.

[0093]

[0123] In some embodiments, the chimeric antigen receptor (CAR) does not include 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 operably linked. In some embodiments, the CAR further includes a TCRβ1 constant domain or a TCRβ2 constant domain. In some embodiments, the transmembrane domain includes a TCRβ constant 1 domain or a TCRβ constant 2 domain. In some embodiments, the antigen-binding domain is linked to the transmembrane domain by a linker. In some embodiments, the TCRβ constant intracellular domain includes a TCRβ constant 1 intracellular domain or a TCRβ constant 2 intracellular domain. In some embodiments, the intracellular signaling domain further includes a co-stimulatory signaling domain. In some embodiments, the antigen-binding domain is a human or humanized single-chain variable fragment (scFv) or a 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 in-frame and expressed as a single polypeptide chain.

[0094]

[0124] In one aspect, provided herein is a recombinant nucleic acid 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 CD3ζ signaling domain.

[0095]

[0125] 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, the transmembrane domain, and the intracellular signaling domain are operably linked. In some embodiments, the CAR further comprises a TCRβ1 constant domain or a TCRβ2 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 linked 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 co-stimulatory signaling domain. In some embodiments, the antigen-binding domain is a human or humanized single-chain variable fragment (scFv) or a 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 in-frame and expressed as a single polypeptide chain.

[0096]

[0126] In one aspect, provided herein is a polypeptide encoded by the recombinant nucleic acid described herein.

[0127] In one aspect, provided herein is a vector comprising the recombinant nucleic acid molecule described herein.

[0097]

[0128] In one aspect, provided herein is a method for generating a population of immune effector cells, comprising the step of transducing a plurality of immune effector cells with a vector described herein.

[0098]

[0129] In one aspect, provided herein is a population of immune effector cells, wherein the immune effector cells comprise a recombinant nucleic acid described herein.

[0130] In some embodiments, the population of immune effector cells is generated by the method described herein.

[0099]

[0131] In some embodiments, upon binding of the antigen-binding domain of a CAR to a cognate antigen expressed by a cell, the level of expression of at least one pro-inflammatory cytokine by the population of immune effector cells is lower compared to the level of expression of at least one pro-inflammatory cytokine by an equivalent population of immune effector cells comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0100]

[0132] In some embodiments, upon binding of the antigen-binding domain of a CAR to a cognate antigen expressed by a cell, the level of expression of at least one pro-inflammatory 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 compared to the level of expression of at least one pro-inflammatory cytokine by an equivalent population of immune effector cells comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0101]

[0133] In some embodiments, upon binding of the antigen-binding domain of a CAR to a cognate antigen expressed by a cell in the presence of an antigen-presenting cell population, the expression level of at least one pro-inflammatory cytokine by the antigen-presenting cell population is lower than the expression level of at least one pro-inflammatory cytokine by an equivalent antigen-presenting cell population in the presence of an equivalent immune effector cell population comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain, and is an immune effector cell population.

[0102]

[0134] In some embodiments, upon binding of the antigen-binding domain of a CAR to a cognate antigen expressed by a cell in the presence of an antigen-presenting cell population, the expression level of at least one pro-inflammatory cytokine by the antigen-presenting cell population 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 than the expression level of at least one pro-inflammatory cytokine by an equivalent antigen-presenting cell population in the presence of an equivalent immune effector cell population comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain, and is an immune effector cell population.

[0103]

[0135] In some embodiments, the at least one pro-inflammatory cytokine is selected from the group consisting of IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, IL-17, sIL-2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β and GM-CSF.

[0104]

[0136] In some embodiments, the expression of the at least one pro-inflammatory cytokine is measured by determining the level of cytokine secreted from a population of immune effector cells when measured by the assays described herein.

[0105]

[0137] In some embodiments, the expression of at least one pro-inflammatory cytokine is measured by determining cytokine levels secreted from a population of antigen-presenting cells as measured by the assays described herein.

[0106]

[0138] In some embodiments, the population of antigen-presenting cells includes dendritic cells, macrophages, or monocytes.

[0139] In one aspect, provided herein is a pharmaceutical composition comprising at least a portion of the population of immune effector cells described herein.

[0107]

[0140] In one aspect, provided herein is a method of treating cancer in a subject, comprising administering to the subject at least a portion of the population of immune effector cells described herein.

[0108]

[0141] In one aspect, provided herein is a method of preventing or reducing the severity of cytokine release syndrome (CRS) in a human subject, comprising administering to the subject at least a portion of the population of immune effector cells described herein.

[0109]

[0142] In some embodiments, the subject has cancer.

[0143] 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 from administration of the immune cells.

[0110]

[0144] In some embodiments, the subject does not exhibit at least one symptom grade 4 or grade 5 CRS (e.g., as described herein).

[0145] In some embodiments, the subject does not exhibit any symptom grade 4 or grade 5 CRS (e.g., as described herein).

[0111]

[0146] In some embodiments, the level of one or more proteins 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 a subject after administration of cells (e.g., a cell population, e.g., an immune effector cell population) (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) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the level of one or more proteins in the serum of the subject before administration of the immune cells (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours).

[0112]

[0147] In some embodiments, the method further comprises selecting a subject for administration of the immune cells according to any one of claims 86 to 100, based on at least one determination of: the risk of a subject developing CRS, the risk of a subject developing CRS when administering cells expressing a CAR comprising a CD3ζ signaling domain, the diagnosis of CRS in a subject, or the diagnosis of CRS in a subject associated with or induced by the administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0113]

[0148] In some embodiments, a subject is selected for administration if the subject is at risk of developing CRS, if the subject is at risk of developing CRS when administered a CAR expressing a CAR CD3ζ signaling domain, if the subject is diagnosed with CRS, or if the subject is diagnosed with CRS associated with or induced by the administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0114]

[0149] In some embodiments, the cells are autologous or allogeneic to the subject to whom the cells are administered.

[0150] In some embodiments, the cancer is a solid cancer or a blood cancer.

[0115]

[0151] In some embodiments, the cancer is a solid cancer.

[0152] In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney cancer, gastric 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.

[0116]

[0153] In some embodiments, the cancer is a blood cancer.

[0154] In some embodiments, the blood cancer is leukemia, lymphoma or myeloma.

[0155] In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0117]

[0156] In one aspect, provided herein is a recombinant nucleic acid 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 optionally comprising a TCRα intracellular domain; and 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, the TCR does not comprise a functional CD3ζ intracellular signaling domain, and the TCR does not comprise a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

[0118]

[0157] In some embodiments, the TCRα chain further comprises a TCRα constant domain.

[0158] In one aspect, provided herein is a recombinant nucleic acid 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 optionally comprising a TCRα intracellular domain; and 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, the TCR does not comprise a functional CD3ζ intracellular signaling domain, and the TCR does not comprise a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

[0119]

[0159] In some embodiments, the TCRα chain further comprises a TCRα constant domain.

[0160] In one aspect, provided herein is a recombinant nucleic acid encoding an exogenous T cell receptor (TCR), wherein the TCR comprises a TCRα chain comprising: i) an antigen-binding domain (e.g., scFv), ii) a TCRα variable domain, iii) a TCRα constant domain, iv) a TCRα transmembrane domain, and iii) an intracellular signaling domain optionally comprising a TCRα intracellular domain; and a TCRβ chain comprising: i) a 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 the TCR does not comprise a functional CD3ζ intracellular signaling domain.

[0120]

[0161] In one aspect, provided herein is a recombinant nucleic acid 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 optionally comprising a TCRα intracellular domain; and a TCRβ chain comprising: i) an antigen-binding domain (e.g., scFv), ii) a 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 the TCR does not comprise a functional CD3ζ intracellular signaling domain.

[0121]

[0162] In one aspect, provided herein is a polypeptide encoded by the recombinant nucleic acid described herein.

[0163] In one aspect, provided herein is a vector comprising the recombinant nucleic acid described herein.

[0122]

[0164] In one aspect, provided herein is a method of generating an immune effector cell population comprising the step of transducing an immune effector cell population with the vector described herein.

[0123]

[0165] In one aspect, provided herein is a population of immune effector cells, wherein the immune effector cells comprise the recombinant nucleic acids described herein.

[0166] In some embodiments, the immune effector cells are produced by the methods described herein.

[0124]

[0167] In some embodiments, upon binding of the antigen-binding domain of the TCR to the cognate antigen expressed by the cell, the level of expression of at least one pro-inflammatory cytokine by the population of immune effector cells is lower compared to the level of expression of at least one pro-inflammatory cytokine by an equivalent population of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0125]

[0168] In some embodiments, upon binding of the antigen-binding domain of the TCR to the cognate antigen expressed by the cell, the level of expression of at least one pro-inflammatory 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 compared to the level of expression of at least one pro-inflammatory cytokine by an equivalent population of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0126]

[0169] In some embodiments, upon binding of the antigen-binding domain of the TCR to the cognate antigen expressed by the cell in the presence of a population of antigen-presenting cells, the level of expression of at least one pro-inflammatory cytokine by the population of antigen-presenting cells is lower compared to the level of expression of at least one pro-inflammatory cytokine by an equivalent population of antigen-presenting cells in the presence of an equivalent population of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0127]

[0170] In some embodiments, upon binding of the antigen-binding domain of a TCR to a cognate antigen expressed by a cell in the presence of an antigen-presenting cell population, the expression level of at least one pro-inflammatory 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 than the expression level of at least one pro-inflammatory cytokine by an equivalent antigen-presenting cell population in the presence of an equivalent immune effector cell population comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0128]

[0171] In some embodiments, the at least one pro-inflammatory 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.

[0129]

[0172] In some embodiments, the expression of the at least one pro-inflammatory cytokine is measured by determining the level of cytokine secreted from a population of immune effector cells when measured by an assay described herein.

[0130]

[0173] In some embodiments, the expression of the at least one pro-inflammatory cytokine is measured by determining the cytokine level secreted from a population of antigen-presenting cells when measured by an assay described herein.

[0131]

[0174] In some embodiments, the antigen-presenting cell population comprises dendritic cells, macrophages or monocytes.

[0175] In one aspect, provided herein is a pharmaceutical composition comprising at least a portion of the immune effector cell population described herein.

[0132]

[0176] In one aspect, provided herein is a method of treating cancer in a subject, comprising administering to at least a portion of the immune effector cell population described herein to the subject.

[0133]

[0177] In one aspect, provided herein is a method of preventing or reducing the severity of cytokine release syndrome (CRS) in a human subject, comprising administering to at least a portion of the immune effector cell population described herein to the subject.

[0134]

[0178] In some embodiments, the subject has cancer.

[0179] 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 from administration of the immune cells.

[0135]

[0180] In some embodiments, the subject does not exhibit at least one symptom grade 4 or grade 5 CRS (e.g., as described herein).

[0181] In some embodiments, the subject does not exhibit any symptom grade 4 or grade 5 CRS (e.g., as described herein).

[0136]

[0182] In some embodiments, the level of one or more proteins 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 after administration of the cells (e.g., cell population, e.g., immune effector cell population) (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) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the level of one or more proteins in the serum of the subject before administration of the immune cells (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours).

[0137]

[0183] In some embodiments, the method further comprises selecting a subject for administration of the immune cells described herein based on at least one determination of: the risk of developing CRS in a subject, the risk of developing CRS in a subject upon administration of cells expressing a CAR comprising a CD3ζ signaling domain, the diagnosis of CRS in a subject, or the diagnosis of CRS in a subject associated with or induced by the administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0138]

[0184] In some embodiments, a subject is selected for administration if the subject is at risk of developing CRS, if the subject is at risk of developing CRS upon administration of a CAR expressing a CAR CD3ζ signaling domain, if the subject is diagnosed with CRS, or if the subject is diagnosed with CRS associated with or induced by the administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0139]

[0185] In some embodiments, the cells are autologous or allogeneic to the subject to whom the cells are administered.

[0186] In some embodiments, the cancer is a solid cancer or a hematological cancer.

[0140]

[0187] In some embodiments, the cancer is a solid cancer.

[0188] In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney cancer, gastric 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.

[0141]

[0189] In some embodiments, the cancer is a hematological cancer.

[0190] In some embodiments, the blood cancer is leukemia, lymphoma or myeloma.

[0191] In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0142]

[0192] In one aspect, provided herein is a method of expanding a T cell population ex vivo, comprising contacting the T cell population with one or more anti-TCRβV antibodies, and a method of treating a disease or disorder, such as cancer, using the expanded cell population described above.

[0143]

[0193] The methods described herein include methods of activating or expanding (or activating and expanding) T cells ex vivo, comprising contacting a plurality of T cells with a first substance, the first substance comprising a first domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, thereby generating a first T cell population.

[0144]

[0194] In some embodiments, the method further comprises contacting the plurality of T cells with a second substance, the second substance comprising a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, and the first and second substances specifically bind to different TCRβV regions.

[0145]

[0195] In some embodiments, the first substance comprises a domain that specifically binds to the TCRβV region of the first TCRβV, the second substance comprises a domain that specifically binds to the TCRβV region of the second TCRβV, and the first and second TCRβVs belong to different TCRβV subfamilies or are different members of the same TCRβV subfamily.

[0146]

[0196] In some embodiments, the first substance comprises a domain that specifically binds to a TCRβV region of a first TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily; the second substance comprises a domain that specifically binds to a second TCRβV region of a TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the first and second substances each specifically bind to TCRβVs belonging to different subfamilies, or different members of the same TCRβV subfamily.

[0147]

[0197] In some embodiments, the first and second substances each specifically bind to TCRβV belonging to different subfamilies. In some embodiments, the first and second substances each specifically bind to different members of the same TCRβV subfamily.

[0148]

[0198] In some embodiments, the method further comprises contacting a plurality of T cells with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances, each of the one or more substances comprising a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, each of the one or more substances specifically binds to a different T cell receptor beta variable chain (TCRβV) region, and each one of the TCRβV regions to which the one or more substances specifically bind is different from the TCRβV regions to which the first and second substances specifically bind.

[0149]

[0199] In some embodiments, each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to a TCRβV belonging to a different TCRβV subfamily or is a different member of the same TCRβV subfamily; each of the one or more substances specifically binds to a TCRβV belonging to a TCRβV subfamily different from the TCRβV bound by the first and second substances, or each of the one or more substances specifically binds to a different member of the same TCRβV subfamily as the TCRβV bound by the first substance, the second substance, or both.

[0150]

[0200] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances each comprise a domain that specifically binds to a TCRβV region of TCRβ belonging to each of the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily.

[0151]

[0201] In some embodiments, each of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to a TCRβV belonging to a different subfamily, and each of the one or more substances specifically binds to a TCRβV belonging to a subfamily different from the TCRβV bound by the first and second substances.

[0152]

[0202] In some embodiments, the first substance further comprises a second domain that binds to a protein expressed on the surface of a plurality of one or more T cells. In some embodiments, the first substance is a bispecific antibody molecule.

[0153]

[0203] In some embodiments, the second domain specifically binds to the T cell receptor beta variable (TCRβV) region. In some embodiments, the second domain and the first domain specifically bind to different T cell receptor beta variable (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 the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the second domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily. In some embodiments, the second domain and the first domain specifically bind to TCRβV belonging to different subfamilies. In some embodiments, the second domain and the first domain specifically bind to different members of the same TCRβV subfamily.

[0154]

[0204] In some embodiments, the second domain specifically binds to CD19 or 4-1BB.

[0205] 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 a plurality of T cells. 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 the light chain of the antibody molecule. In some embodiments, the second domain specifically binds to the κ light chain region of the antibody molecule. In some embodiments, the second domain comprises protein L.

[0155]

[0206] In some embodiments, the first T cell population exhibits 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) maintenance of IFNγ expression, and (vii) increased expression of 4-1BB as compared to a plurality of T cells contacted with a substance comprising a domain that specifically binds to CD3 (e.g., CD3ε).

[0156]

[0207] In some embodiments, the contacting step comprises incubating the plurality of T cells with the first substance.

[0208] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with a first substance 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for up to 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for 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.

[0157]

[0209] In some embodiments, the first substance is coupled to a solid surface (e.g., beads). In some embodiments, the first substance comprises an antibody domain.

[0210] In some embodiments, the first substance comprises an anti - idiotype antibody domain. In some embodiments, the first substance comprises a mouse antibody domain. In some embodiments, the first substance comprises a human antibody domain. In some embodiments, the first substance comprises a humanized antibody domain. In some embodiments, the first substance comprises an antigen - binding domain comprising a single - chain Fv (scFv) or Fab. In some embodiments, the first substance comprises an antibody comprising two antibody heavy chains each comprising a variable region and a constant region, and two antibody light chains each comprising a variable region and a constant region.

[0158]

[0211] In some embodiments, the plurality of T cells comprises one or more T cells comprising exogenous nucleic acid.

[0212] In some embodiments, the plurality of T cells includes one or more T cells comprising an exogenous nucleic acid encoding a chimeric polypeptide. In some embodiments, the method includes introducing an exogenous nucleic acid into one or more of the plurality of T cells before contacting the plurality of T cells with a first substance. In some embodiments, the method includes introducing an exogenous nucleic acid into one or more of the plurality of T cells after contacting the plurality of T cells with a first substance. In some embodiments, the method further includes introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells before contacting the plurality of T cells with a first substance. In some embodiments, the method further includes introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells after contacting the plurality of T cells with a first substance. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

[0159]

[0213] In some embodiments, the chimeric polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) includes an antigen-binding region, a transmembrane region, and an intracellular signaling region. In some embodiments, the intracellular signaling region includes one or more signaling domains. In some embodiments, the intracellular signaling domain includes a signaling domain derived from CD27, CD28, 4-1BB, ICOS, OX40, DAP10, DAP12, CD134, CD3-zeta, or a fragment or combination thereof. In some embodiments, the transmembrane region includes a transmembrane region derived from CD8, CD28, or CTLA4.

[0160]

[0214] In some embodiments, the antigen-binding region includes an antibody domain. In some embodiments, the antibody domain includes an scFv or a Fab. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen (e.g., as described herein).

[0161]

[0215] 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 TCRα polypeptide and a TCRβ polypeptide. In some embodiments, the chimeric TCR comprises a TCRγ polypeptide and a TCRδ polypeptide. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen.

[0162]

[0216] In some embodiments, the plurality of T cells comprises one or more T cells from a human subject.

[0217] In some embodiments, one or more T cells are removed from a human subject via apheresis.

[0163]

[0218] In some embodiments, the plurality of T cells comprises one or more T cells from a healthy human subject (e.g., a subject without or not diagnosed with a particular disease or condition, e.g., 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., a particular disease or condition, e.g., diagnosed with cancer). In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor or a hematologic cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, prostate, lung, kidney, stomach, colon, ovary, bladder, breast, cervical, esophageal, testicular, liver, pancreas, rectal, thyroid, uterine, skin, muscle, cartilage, bone, endothelial, epithelial, dermal, basal, retinal, cutaneous or brain.

[0164]

[0219] In some embodiments, the plurality of T cells comprises one or more autologous T cells. In some embodiments, the plurality of T cells comprises one or more allogeneic T cells.

[0220] 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 times greater than the number of cells in the plurality of T cells before contacting with the first substance.

[0165]

[0221] In some embodiments, the substance that specifically binds to CD3 (e.g., CD3ε) comprises an antibody domain (e.g., an anti-CD3 antibody (e.g., an anti-CD3ε antibody)).

[0222] In some embodiments, the substance that specifically binds to CD3 specifically binds to CD3ε.

[0166]

[0223] In some embodiments, upon binding to the TCRβV region, the first substance causes the following: (i) a reduction in the level of IL-1β, e.g., expression level and / or activity; (ii) a reduction in the level of IL-6, e.g., expression level and / or activity; (iii) a reduction in the level of TNFα, e.g., expression level and / or activity; (iv) an increase in the level of IL-2, e.g., expression level and / or activity; (v) a delay in the increase in the level of IL-2, e.g., expression level and / or activity, e.g., a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, e.g., expression level and / or activity, e.g., a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics; or (viii) a reduction in cytokine storm, e.g., cytokine release syndrome (CRS), as measured by an assay described herein, e.g., an assay described herein; (ix) cell killing, e.g., target cell killing, e.g., cancer cell killing, as measured by an assay described herein, e.g., an assay described herein; (x) an increase in the level of IL-15, e.g., expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, e.g., expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, e.g., a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0167]

[0224] In some embodiments, the first substance, upon binding to the TCRβV region, results in an expansion of a memory T cell population, such as a T effector memory (TEM) cell population, such as a TEM cell expressing CD45RA (TEMRA), such as an expansion of at least about 1.1- to 10-fold (e.g., an expansion of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold).

[0168]

[0225] In some embodiments, the expansion of the population of memory effector T cells, such as TEM cells, such as TEMRA cells, in the first T cell population is increased compared to the expansion of a similar cell population having an antibody that binds to the CD3 molecule.

[0169]

[0226] In some embodiments, the expanded T effector memory cell population has (i) a detectable level of CD45RA, such as expressing or re-expressing CD45RA; (ii) low or no expression of CCR7; and / or (iii) a detectable level of CD95, such as CD95, such as a population of CD45RA+, CCR7-, CD95+ T cells, and the T cells optionally include CD3+, CD4+, or CD8+ T cells.

[0170]

[0227] In some embodiments, binding of the first substance to the TCRβV region results in the following: (i) a reduction in the level of IL-1β, such as a reduction in the expression level and / or activity; (ii) a reduction in the level of IL-6, such as a reduction in the expression level and / or activity; (iii) a reduction in the level of TNFα, such as a reduction in the expression level and / or activity; (iv) an increase in the level of IL-2, such as an increase in the expression level and / or activity; (v) a delay in the increase in the level of IL-2, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics, or (viii) a reduction in cytokine storm, such as cytokine release syndrome (CRS), when measured by an assay as described herein; (ix) cell killing, such as target cell killing, such as cancer cell killing, when measured by an assay as described herein; (x) an increase in the level of IL-15, such as an increase in the expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, such as expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, such as a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0171]

[0228] In some embodiments, binding of the first substance to the TCRβV region results in a reduction in the expression level and / or activity of IL-1β by 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) when measured by an assay as described herein.

[0172]

[0229] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or 1000-fold, or at least a 2- to 1000-fold (e.g., 5- to 900-, 10- to 800-, 20- to 700-, 50- to 600-, 100- to 500-, or 200- to 400-fold) reduction in the expression level and / or activity of IL-6 when measured by the assays described herein.

[0173]

[0230] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-, 10- to 900-, 20- to 800-, 50- to 700-, 100- to 600-, 200- to 500-, or 300- to 400-fold) reduction in the expression level and / or activity of TNFα when measured by the assays described herein.

[0174]

[0231] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-, 10- to 900-, 20- to 800-, 50- to 700-, 100- to 600-, 200- to 500-, or 300- to 400-fold) increase in the expression level and / or activity of IL-2 when measured by the assays described herein.

[0175]

[0232] The methods described herein include methods of expanding T cells ex vivo that include contacting a plurality of T cells with a plurality of substances, the plurality of substances including 2, 3, 4, 5, or more substances, each of the plurality of substances including a domain that specifically binds to a different T cell receptor beta variable chain (TCRβV) region, thereby generating a first population of T cells.

[0176]

[0233] In some embodiments, each of the plurality of substances specifically binds to a different TCRβV, and each TCRβV belongs to a different TCRβV subfamily or is a different member of the same TCRβV subfamily.

[0177]

[0234] In some embodiments, each of the plurality of substances includes a domain that specifically binds to a TCRβV belonging to the TCRβV6 subfamily, TCRβV10 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, TCRβV7 subfamily, TCRβV11 subfamily, TCRβV14 subfamily, TCRβV16 subfamily, TCRβV18 subfamily, TCRβV9 subfamily, TCRβV13 subfamily, TCRβV4 subfamily, TCRβV3 subfamily, TCRβV2 subfamily, TCRβV15 subfamily, TCRβV30 subfamily, TCRβV19 subfamily, TCRβV27 subfamily, TCRβV28 subfamily, TCRβV24 subfamily, TCRβV20 subfamily, TCRβV25 subfamily, or TCRβV29 subfamily.

[0178]

[0235] In some embodiments, each of the plurality of substances specifically binds to a different TCRβV, and each TCRβV belongs to a different TCRβV subfamily.

[0236] The methods described herein include methods for expanding T cells ex vivo that include contacting a plurality of T cells with a plurality of substances, the plurality of substances including at least a first and a second substance, each of the plurality of substances including a domain that specifically binds to a different T cell receptor beta variable chain (TCRβV) region, thereby generating a first population of T cells.

[0179]

[0237] In some embodiments, the plurality includes at least 3, 4, 5, 6, 7, 8, 9, 10 or more substances.

[0238] In some embodiments, the method further comprises contacting a plurality of T cells with a second substance, the second substance comprising a domain that specifically binds to a T cell receptor beta variable (TCRβV) region, and the first and second substances specifically bind to different TCRβV regions.

[0180]

[0239] In some embodiments, the first substance comprises a domain that specifically binds to the TCRβV region of a first TCRβV, the second substance comprises a domain that specifically binds to the TCRβV region of a second TCRβV, and the first and second TCRβVs belong to different TCRβV subfamilies or are different members of the same TCRβV subfamily.

[0181]

[0240] In some embodiments, the first substance comprises a domain that specifically binds to a TCRβV region of a first TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily; the second substance comprises a domain that specifically binds to a second TCRβV region of a TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the first and second substances specifically bind to TCRβVs belonging to different subfamilies, or different members of the same TCRβV subfamily, respectively.

[0182]

[0241] In some embodiments, the first and second substances each specifically bind to TCRβVs belonging to different subfamilies. In some embodiments, the first and second substances each specifically bind to different members of the same TCRβV subfamily.

[0183]

[0242] In some embodiments, the method further comprises contacting a plurality of T cells with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances, each of the one or more substances comprising a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, each of the one or more substances specifically binds to a different T cell receptor beta variable chain (TCRβV) region, and each one of the TCRβV regions to which the one or more substances specifically bind is different from the TCRβV regions to which the first and second substances specifically bind.

[0184]

[0243] In some embodiments, each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to a TCRβV belonging to a different TCRβV subfamily or is a different member of the same TCRβV subfamily; each of the one or more substances specifically binds to a TCRβV belonging to a TCRβV subfamily different from the TCRβV bound by the first and second substances, or each of the one or more substances specifically binds to a different member of the same TCRβV subfamily as the TCRβV bound by the first substance, the second substance, or both.

[0185]

[0244] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances each comprise a domain that specifically binds to a TCRβV region of TCRβV belonging to each of the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily.

[0186]

[0245] In some embodiments, each of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to a TCRβV belonging to a different subfamily, and each of the one or more substances specifically binds to a TCRβV belonging to a subfamily different from the TCRβV bound by the first substance and the second substance.

[0187]

[0246] In some embodiments, the first substance and / or the second substance comprise a second domain that binds to a protein expressed on the surface of a plurality of one or more T cells. In some embodiments, the first substance is a bispecific antibody molecule.

[0188]

[0247] In some embodiments, the second domain specifically binds to the T cell receptor beta variable (TCRβV) region. In some embodiments, the second domain and the first domain specifically bind to different T cell receptor beta variable (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 the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the second domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily. In some embodiments, the second domain and the first domain specifically bind to TCRβV belonging to different subfamilies. In some embodiments, the second domain and the first domain specifically bind to different members of the same TCRβV subfamily.

[0189]

[0248] In some embodiments, the second domain specifically binds to CD19 or 4-1BB.

[0249] 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 a plurality of T cells. 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 the light chain of the antibody molecule. In some embodiments, the second domain specifically binds to the κ light chain region of the antibody molecule. In some embodiments, the second domain comprises protein L.

[0190]

[0250] In some embodiments, the first T cell population exhibits 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) maintenance of IFNγ expression, and (vii) increased expression of 4-1BB as compared to a plurality of T cells contacted with a substance comprising a domain that specifically binds to CD3 (e.g., CD3ε).

[0191]

[0251] In some embodiments, the contacting step comprises incubating the plurality of T cells with the first substance.

[0252] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with a first substance 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for up to 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for 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.

[0192]

[0253] In some embodiments, the first substance is coupled to a solid surface (e.g., beads). In some embodiments, the first substance comprises an antibody domain. In some embodiments, each of the plurality of substances is bound to one or more solid surfaces (e.g., one or more beads). In some embodiments, each of the plurality of substances comprises an antibody domain.

[0193]

[0254] In some embodiments, the first substance comprises an anti - idiotype antibody domain. In some embodiments, the first substance comprises a murine antibody domain. In some embodiments, the first substance comprises a human antibody domain. In some embodiments, the first substance comprises a humanized antibody domain. In some embodiments, the first substance comprises an antigen - binding domain comprising a single - chain Fv (scFv) or Fab. In some embodiments, the first substance comprises an antibody comprising two antibody heavy chains each having a variable region and a constant region, and two antibody light chains each having a variable region and a constant region.

[0194]

[0255] In some embodiments, each of the plurality of substances comprises an anti-idiotype antibody domain. In some embodiments, each of the plurality of substances comprises a murine antibody domain. In some embodiments, each of the plurality of substances comprises a human antibody domain. In some embodiments, each of the plurality of substances comprises a humanized antibody domain. In some embodiments, each of the plurality of substances comprises an antigen-binding domain comprising a single-chain Fv (scFv) or a Fab. In some embodiments, each of the plurality of substances comprises an antibody comprising two antibody heavy chains each comprising a variable region and a constant region, and two antibody light chains each comprising a variable region and a constant region.

[0195]

[0256] In some embodiments, the plurality of T cells comprises one or more T cells comprising exogenous nucleic acid.

[0257] In some embodiments, the plurality of T cells comprises one or more T cells comprising exogenous nucleic acid encoding a chimeric polypeptide. In some embodiments, the method comprises introducing exogenous nucleic acid into one or more of the plurality of T cells prior to contacting the plurality of T cells with the first substance. In some embodiments, the method comprises introducing exogenous nucleic acid into one or more of the plurality of T cells after contacting the plurality of T cells with the first substance. In some embodiments, the method further comprises introducing exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells prior to contacting the plurality of T cells with the first substance. In some embodiments, the method further comprises introducing exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells after contacting the plurality of T cells with the first substance. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

[0196]

[0258] In some embodiments, the chimeric polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) comprises an antigen-binding region, a transmembrane region, and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises one or more signaling domains. In some embodiments, the intracellular signaling domain comprises a signaling domain derived from CD27, CD28, 4-1BB, ICOS, OX40, DAP10, DAP12, CD134, CD3-zeta, or a fragment or combination thereof. In some embodiments, the transmembrane region comprises a transmembrane region derived from CD8, CD28, or CTLA4.

[0197]

[0259] In some embodiments, the antigen-binding region comprises an antibody domain. In some embodiments, the antibody domain comprises an scFv or a Fab. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen.

[0198]

[0260] 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 TCRα polypeptide and a TCRβ polypeptide. In some embodiments, the chimeric TCR comprises a TCRγ polypeptide and a TCRδ polypeptide. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen.

[0199]

[0261] 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 cells are removed from the human subject via apheresis. In some embodiments, the plurality of T cells comprises one or more T cells from a healthy human subject (e.g., a subject without or not diagnosed with a particular disease or condition, e.g., 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., a particular disease or condition, e.g., diagnosed with cancer). In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor or a hematologic cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, prostate, lung, kidney, stomach, colon, ovary, bladder, breast, cervical, esophagus, testis, liver, pancreas, rectum, thyroid, uterus, skin, muscle, cartilage, bone, endothelium, epithelium, dermis, basal, retina, skin, or brain.

[0200]

[0262] In some embodiments, the plurality of T cells comprises one or more autologous T cells. In some embodiments, the plurality of T cells comprises one or more allogeneic T cells.

[0263] 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 times greater than the number of cells in the plurality of T cells prior to contacting with the first substance.

[0201]

[0264] In some embodiments, the substance that specifically binds to CD3 (e.g., CD3ε) comprises an antibody domain (e.g., an anti-CD3 antibody (e.g., an anti-CD3ε antibody)).

[0265] In some embodiments, the substance that specifically binds to CD3 specifically binds to CD3ε.

[0202]

[0266] In some embodiments, upon binding to the TCRβV region, the first substance causes the following: (i) a reduction in the level of IL-1β, such as a reduction in the expression level and / or activity; (ii) a reduction in the level of IL-6, such as a reduction in the expression level and / or activity; (iii) a reduction in the level of TNFα, such as a reduction in the expression level and / or activity; (iv) an increase in the level of IL-2, such as an increase in the expression level and / or activity; (v) a delay in the increase in the level of IL-2, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics; or (viii) a reduction in cytokine storm, such as cytokine release syndrome (CRS), as measured, for example, by an assay described herein; (ix) cell killing, such as target cell killing, such as cancer cell killing, as measured, for example, by an assay described herein; (x) an increase in the level of IL-15, such as an increase in the expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, such as expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, such as a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0203]

[0267] In some embodiments, upon binding to the TCRβV region, the first substance causes an expansion of a memory T cell population, such as T effector memory (TEM) cells, such as TEM cells expressing CD45RA (TEMRA), for example, an expansion of at least about 1.1 to 10-fold (e.g., an expansion of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold).

[0204]

[0268] In some embodiments, the expansion of the population of memory effector T cells, such as TEM cells, such as TEMRA cells, in the first T cell population is increased compared to the expansion of a similar cell population having an antibody that binds to the CD3 molecule.

[0205]

[0269] In some embodiments, the expanded T effector memory cell population has (i) a detectable level of CD45RA, such as expressing or re-expressing CD45RA; (ii) low or no expression of CCR7; and / or (iii) a detectable level of CD95, such as CD95, such as a population of CD45RA+, CCR7−, CD95+ T cells, and the T cells optionally include CD3+, CD4+ or CD8+ T cells.

[0206]

[0270] In some embodiments, binding of the first substance to the TCRβV region results in the following: (i) a reduction in the level of IL-1β, such as a reduction in the expression level and / or activity; (ii) a reduction in the level of IL-6, such as a reduction in the expression level and / or activity; (iii) a reduction in the level of TNFα, such as a reduction in the expression level and / or activity; (iv) an increase in the level of IL-2, such as an increase in the expression level and / or activity; (v) a delay in the increase in the level of IL-2, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics, or (viii) a reduction in cytokine storm, such as cytokine release syndrome (CRS), as measured by an assay described herein, for example; (ix) cell killing, such as target cell killing, such as cancer cell killing, as measured by an assay described herein, for example; (x) an increase in the level of IL-15, such as an increase in the expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, such as expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, such as a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0207]

[0271] In some embodiments, binding of the first substance to the TCRβV region results in a reduction in the expression level and / or activity of IL-1β by 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), as measured by an assay described herein.

[0208]

[0272] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900- or 1000-fold, or at least a 2- to 1000-fold (e.g., 5- to 900-fold, 10- to 800-fold, 20- to 700-fold, 50- to 600-fold, 100- to 500-fold or 200- to 400-fold) reduction in the expression level and / or activity of IL-6 when measured by the assays described herein.

[0209]

[0273] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000- or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-fold, 10- to 900-fold, 20- to 800-fold, 50- to 700-fold, 100- to 600-fold, 200- to 500-fold or 300- to 400-fold) reduction in the expression level and / or activity of TNFα when measured by the assays described herein.

[0210]

[0274] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000- or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-fold, 10- to 900-fold, 20- to 800-fold, 50- to 700-fold, 100- to 600-fold, 200- to 500-fold or 300- to 400-fold) increase in the expression level and / or activity of IL-2 when measured by the assays described herein.

[0211]

[0275] The method disclosed in this specification includes a method for treating cancer in a subject, the method comprising the steps of removing a plurality of T cells from a human subject, expanding a plurality of T cells from the human subject, the step of expanding the plurality of T cells including contacting the plurality of T cells with a first substance, the first substance including a domain that specifically binds to the T cell receptor beta variable chain (TCRβV) region, thereby generating a first T cell population, and injecting at least a portion of the first T cell population into the human subject, thereby treating cancer in the subject.

[0212]

[0276] In some embodiments, the method further comprises the step of contacting the plurality of T cells with a second substance, the second substance including a domain that specifically binds to the T cell receptor beta variable chain (TCRβV) region, and the first and second substances specifically bind to different TCRβV regions.

[0213]

[0277] In some embodiments, the first substance includes a domain that specifically binds to the TCRβV region of a first TCRβV, the second substance includes a domain that specifically binds to the TCRβV region of a second TCRβV, and the first and second TCRβVs belong to different TCRβV subfamilies or are different members of the same TCRβV subfamily.

[0214]

[0278] In some embodiments, the first substance comprises a domain that specifically binds to a TCRβV region of a first TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily; the second substance comprises a domain that specifically binds to a second TCRβV region of a TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the first and second substances each specifically bind to TCRβVs belonging to different subfamilies, or different members of the same TCRβV subfamily.

[0215]

[0279] In some embodiments, the first and second substances each specifically bind to a TCRβV belonging to a different subfamily. In some embodiments, the first and second substances each specifically bind to different members of the same TCRβV subfamily.

[0216]

[0280] In some embodiments, the method further comprises contacting a plurality of T cells with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances, each of the one or more substances comprising a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, each of the one or more substances specifically binds to a different T cell receptor beta variable chain (TCRβV) region, and each one of the TCRβV regions to which the one or more substances specifically bind is different from the TCRβV regions to which the first and second substances specifically bind.

[0217]

[0281] In some embodiments, each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to a TCRβV belonging to a different TCRβV subfamily or is a different member of the same TCRβV subfamily; each of the one or more substances specifically binds to a TCRβV belonging to a TCRβV subfamily different from the TCRβV bound by the first and second substances, or each of the one or more substances specifically binds to a different member of the same TCRβV subfamily as the TCRβV bound by the first substance, the second substance or both.

[0218]

[0282] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances each comprise a domain that specifically binds to the TCRβV region of TCRβV belonging to each of the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily.

[0219]

[0283] In some embodiments, each of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to TCRβV belonging to different subfamilies, and each of the one or more substances specifically binds to TCRβV belonging to a subfamily different from the TCRβV bound by the first substance and the second substance.

[0220]

[0284] In some embodiments, the first substance further comprises a second domain that binds to a protein expressed on the surface of a plurality of one or more T cells. In some embodiments, the first substance is a bispecific antibody molecule.

[0221]

[0285] In some embodiments, the second domain specifically binds to the T cell receptor beta variable chain (TCRβV) region. In some embodiments, the second domain and the first domain specifically bind to different T cell receptor beta variable 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 the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the second domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily. In some embodiments, the second domain and the first domain specifically bind to TCRβV belonging to different subfamilies. In some embodiments, the second domain and the first domain specifically bind to different members of the same TCRβV subfamily.

[0222]

[0286] In some embodiments, the second domain specifically binds to CD19 or 4-1BB.

[0287] 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 a plurality of T cells. In some embodiments, the antibody molecule comprises a variable heavy chain and a variable light chain. In some embodiments, the antibody molecule is an scFv or a Fab. In some embodiments, the second domain specifically binds to the light chain of the antibody molecule. In some embodiments, the second domain specifically binds to the κ light chain region of the antibody molecule. In some embodiments, the second domain comprises protein L.

[0223]

[0288] In some embodiments, the first T cell population exhibits 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) maintenance of IFNγ expression, and (vii) increased expression of 4-1BB as compared to a plurality of T cells contacted with a substance comprising a domain that specifically binds to CD3 (e.g., CD3ε).

[0224]

[0289] In some embodiments, the contacting step comprises incubating the plurality of T cells with the first substance.

[0290] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with a first substance 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for up to 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for 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.

[0225]

[0291] In some embodiments, the first substance is coupled to a solid surface (e.g., beads). In some embodiments, the first substance comprises an antibody domain.

[0292] In some embodiments, the first substance comprises an anti - idiotype antibody domain. In some embodiments, the first substance comprises a mouse antibody domain. In some embodiments, the first substance comprises a human antibody domain. In some embodiments, the first substance comprises a humanized antibody domain. In some embodiments, the first substance comprises an antigen - binding domain comprising a single - chain Fv (scFv) or Fab. In some embodiments, the first substance comprises an antibody comprising two antibody heavy chains each comprising a variable region and a constant region, and two antibody light chains each comprising a variable region and a constant region.

[0226]

[0293] In some embodiments, the plurality of T cells comprises one or more T cells comprising exogenous nucleic acid.

[0294] In some embodiments, the plurality of T cells includes one or more T cells comprising an exogenous nucleic acid encoding a chimeric polypeptide. In some embodiments, the method includes introducing an exogenous nucleic acid into one or more of the plurality of T cells prior to contacting the plurality of T cells with a first substance. In some embodiments, the method includes introducing an exogenous nucleic acid into one or more of the plurality of T cells after contacting the plurality of T cells with a first substance. In some embodiments, the method further includes introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells prior to contacting the plurality of T cells with a first substance. In some embodiments, the method further includes introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells after contacting the plurality of T cells with a first substance. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

[0227]

[0295] In some embodiments, the chimeric polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) includes an antigen-binding region, a transmembrane region, and an intracellular signaling region. In some embodiments, the intracellular signaling region includes one or more signaling domains. In some embodiments, the intracellular signaling domain includes a signaling domain derived from CD27, CD28, 4-1BB, ICOS, OX40, DAP10, DAP12, CD134, CD3-zeta, or a fragment or combination thereof. In some embodiments, the transmembrane region includes a transmembrane region derived from CD8, CD28, or CTLA4.

[0228]

[0296] In some embodiments, the antigen-binding region includes an antibody domain. In some embodiments, the antibody domain includes an scFv or a Fab. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen.

[0229]

[0297] 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 TCRα polypeptide and a TCRβ polypeptide. In some embodiments, the chimeric TCR comprises a TCRγ polypeptide and a TCRδ polypeptide. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen.

[0230]

[0298] 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 cells are removed from a human subject via apheresis. In some embodiments, the plurality of T cells comprises one or more T cells from a healthy human subject (e.g., a subject without or not diagnosed with a particular disease or condition, e.g., 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., a particular disease or condition, e.g., diagnosed with cancer). In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor or a hematologic cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, prostate, lung, kidney, stomach, colon, ovary, bladder, breast, cervix, esophagus, testis, liver, pancreas, rectum, thyroid, uterus, skin, muscle, cartilage, bone, endothelium, epithelium, dermis, basal, retina, skin, or brain.

[0231]

[0299] In some embodiments, the plurality of T cells comprises one or more autologous T cells. In some embodiments, the plurality of T cells comprises one or more allogeneic T cells.

[0300] 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 times greater than the number of cells in the plurality of T cells prior to contact with the first substance.

[0232]

[0301] In some embodiments, the substance that specifically binds to CD3 (e.g., CD3ε) includes an antibody domain (e.g., an anti-CD3 antibody (e.g., an anti-CD3ε antibody)).

[0302] In some embodiments, the substance that specifically binds to CD3 specifically binds to CD3ε.

[0233]

[0303] In some embodiments, upon binding to the TCRβV region, the first substance is as follows: (i) reduction in the level of IL-1β, e.g., expression level and / or activity; (ii) reduction in the level of IL-6, e.g., expression level and / or activity; (iii) reduction in the level of TNFα, e.g., expression level and / or activity; (iv) increase in the level of IL-2, e.g., expression level and / or activity; (v) delay in the increase in the level of IL-2, e.g., expression level and / or activity, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more of delay; (vi) delay in the increase in the level of IFNγ, e.g., expression level and / or activity, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours of delay; (vii) reduction in T cell proliferation kinetics; or (viii) reduction in cytokine storm, e.g., cytokine release syndrome (CRS), as measured by an assay described herein, for example; (ix) cell killing, e.g., target cell killing, e.g., cancer cell killing, as measured by an assay described herein, for example; (x) increase in the level of IL-15, e.g., expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, e.g., expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, e.g., a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0234]

[0304] In some embodiments, the first substance, upon binding to the TCRβV region, results in the expansion of a memory T cell population, such as a T effector memory (TEM) cell population, such as a TEM cell expressing CD45RA (TEMRA), such as an expansion of at least about 1.1- to 10-fold (e.g., an expansion of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold).

[0235]

[0305] In some embodiments, the expansion of the population of memory effector T cells, such as TEM cells, such as TEMRA cells, in the first T cell population is increased compared to the expansion of a similar cell population having an antibody that binds to the CD3 molecule.

[0236]

[0306] In some embodiments, the expanded T effector memory cell population has (i) a detectable level of CD45RA, such as expressing or re-expressing CD45RA; (ii) low or no expression of CCR7; and / or (iii) a detectable level of CD95, such as CD95, such as a population of CD45RA+, CCR7−, CD95+ T cells, and the T cells optionally include CD3+, CD4+, or CD8+ T cells.

[0237]

[0307] In some embodiments, binding of the first substance to the TCRβV region results in the following: (i) a reduction in the level of IL-1β, such as a reduction in the expression level and / or activity; (ii) a reduction in the level of IL-6, such as a reduction in the expression level and / or activity; (iii) a reduction in the level of TNFα, such as a reduction in the expression level and / or activity; (iv) an increase in the level of IL-2, such as an increase in the expression level and / or activity; (v) a delay in the increase in the level of IL-2, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics, or (viii) a reduction in cytokine storm, such as cytokine release syndrome (CRS), as measured by an assay described herein, for example; (ix) cell killing, such as target cell killing, such as cancer cell killing, as measured by an assay described herein, for example; (x) an increase in the level of IL-15, such as an increase in the expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, such as expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, such as a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0238]

[0308] In some embodiments, binding of the first substance 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.

[0239]

[0309] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or 1000-fold, or at least a 2- to 1000-fold (e.g., 5- to 900-fold, 10- to 800-fold, 20- to 700-fold, 50- to 600-fold, 100- to 500-fold, or 200- to 400-fold) reduction in the expression level and / or activity of IL-6 when measured by the assays described herein.

[0240]

[0310] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-fold, 10- to 900-fold, 20- to 800-fold, 50- to 700-fold, 100- to 600-fold, 200- to 500-fold, or 300- to 400-fold) reduction in the expression level and / or activity of TNFα when measured by the assays described herein.

[0241]

[0311] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2-, 5-, 10-, 20-, 50-, 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-fold, 10- to 900-fold, 20- to 800-fold, 50- to 700-fold, 100- to 600-fold, 200- to 500-fold, or 300- to 400-fold) increase in the expression level and / or activity of IL-2 when measured by the assays described herein.

[0242]

[0312] The methods described herein include methods for preventing or reducing cytokine release syndrome (CRS) in a human subject, the method comprising removing a plurality of T cells from the human subject, expanding the plurality of T cells from the human subject, the expanding step comprising contacting the plurality of T cells with a first substance, the first substance comprising a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, thereby generating a first T cell population, injecting at least a portion of the first T cell population into the human subject, wherein the subject exhibits no symptoms of CRS or fewer severe symptoms (one or more of the symptoms described herein) compared to the human subject injected with at least the first T cell population generated by removing a plurality of T cells from the subject, and expanding the plurality of T cells by contacting the plurality of T cells with a substance that binds to CD3 (e.g., CD3e).

[0243]

[0313] In some embodiments, the human subject has cancer.

[0314] In some embodiments, the first substance comprises a domain that specifically binds to the TCRβV region of a first TCRβV, the second substance comprises a domain that specifically binds to the TCRβV region of a second TCRβV, and the first and second TCRβVs belong to different TCRβV subfamilies or are different members of the same TCRβV subfamily.

[0244]

[0315] In some embodiments, the first substance comprises a domain that specifically binds to a TCRβV region of a first TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily; the second substance comprises a domain that specifically binds to a second TCRβV region of a TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the first and second substances each specifically bind to TCRβVs belonging to different subfamilies, or different members of the same TCRβV subfamily.

[0245]

[0316] In some embodiments, the first and second substances each specifically bind to TCRβV belonging to different subfamilies. In some embodiments, the first and second substances each specifically bind to different members of the same TCRβV subfamily.

[0246]

[0317] In some embodiments, the method further comprises contacting a plurality of T cells with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances, each of the one or more substances comprising a domain that specifically binds to a T cell receptor beta variable chain (TCRβV) region, each of the one or more substances specifically binds to a different T cell receptor beta variable chain (TCRβV) region, and each one of the TCRβV regions to which the one or more substances specifically bind is different from the TCRβV regions to which the first and second substances specifically bind.

[0247]

[0318] In some embodiments, each of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to a TCRβV belonging to a different TCRβV subfamily or is a different member of the same TCRβV subfamily; each of the one or more substances specifically binds to a TCRβV belonging to a TCRβV subfamily different from the TCRβV bound by the first and second substances, or each of the one or more substances specifically binds to a different member of the same TCRβV subfamily as the TCRβV bound by the first substance, the second substance, or both.

[0248]

[0319] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances each comprise a domain that specifically binds to the TCRβV region of TCRβV belonging to each of the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily.

[0249]

[0320] In some embodiments, each of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) substances specifically binds to TCRβV belonging to different subfamilies, and each of the one or more substances specifically binds to TCRβV belonging to a subfamily different from the TCRβV bound by the first substance and the second substance.

[0250]

[0321] In some embodiments, the first substance further comprises a second domain that binds to a protein expressed on the surface of a plurality of one or more T cells. In some embodiments, the first substance is a bispecific antibody molecule.

[0251]

[0322] In some embodiments, the second domain specifically binds to the T cell receptor beta variable chain (TCRβV) region. In some embodiments, the second domain and the first domain specifically bind to different T cell receptor beta variable 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 the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily, and the second domain specifically binds to the TCRβV region of TCRβV belonging to the TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily or TCRβ V29 subfamily. In some embodiments, the second domain and the first domain specifically bind to TCRβV belonging to different subfamilies. In some embodiments, the second domain and the first domain specifically bind to different members of the same TCRβV subfamily.

[0252]

[0323] In some embodiments, the second domain specifically binds to CD19 or 4-1BB.

[0324] 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 a plurality of T cells. 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 the light chain of the antibody molecule. In some embodiments, the second domain specifically binds to the κ light chain region of the antibody molecule. In some embodiments, the second domain comprises protein L.

[0253]

[0325] In some embodiments, the first T cell population exhibits 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) maintenance of IFNγ expression, and (vii) increased expression of 4-1BB as compared to a plurality of T cells contacted with a substance comprising a domain that specifically binds to CD3 (e.g., CD3ε).

[0254]

[0326] In some embodiments, the contacting step comprises incubating the plurality of T cells with the first substance.

[0327] In some embodiments, the contacting step comprises incubating or culturing a plurality of T cells with a first substance 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for up to 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, the contacting step comprises incubating or culturing a plurality of T cells with a first substance for 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.

[0255]

[0328] In some embodiments, the first substance is coupled to a solid surface (e.g., beads). In some embodiments, the first substance comprises an antibody domain.

[0329] In some embodiments, the first substance comprises an anti - idiotype antibody domain. In some embodiments, the first substance comprises a mouse antibody domain. In some embodiments, the first substance comprises a human antibody domain. In some embodiments, the first substance comprises a humanized antibody domain. In some embodiments, the first substance comprises an antigen - binding domain comprising a single - chain Fv (scFv) or Fab. In some embodiments, the first substance comprises an antibody comprising two antibody heavy chains each comprising a variable region and a constant region, and two antibody light chains each comprising a variable region and a constant region.

[0256]

[0330] In some embodiments, the plurality of T cells comprises one or more T cells comprising exogenous nucleic acid.

[0331] In some embodiments, the plurality of T cells includes one or more T cells comprising an exogenous nucleic acid encoding a chimeric polypeptide. In some embodiments, the method includes introducing an exogenous nucleic acid into one or more of the plurality of T cells before contacting the plurality of T cells with a first substance. In some embodiments, the method includes introducing an exogenous nucleic acid into one or more of the plurality of T cells after contacting the plurality of T cells with a first substance. In some embodiments, the method further includes introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells before contacting the plurality of T cells with a first substance. In some embodiments, the method further includes introducing an exogenous nucleic acid encoding a chimeric polypeptide into one or more of the plurality of T cells after contacting the plurality of T cells with a first substance. In some embodiments, the exogenous nucleic acid is introduced by transduction or transfection.

[0257]

[0332] In some embodiments, the chimeric polypeptide is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor (CAR) includes an antigen-binding region, a transmembrane region, and an intracellular signaling region. In some embodiments, the intracellular signaling region includes one or more signaling domains. In some embodiments, the intracellular signaling domain includes a signaling domain derived from CD27, CD28, 4-1BB, ICOS, OX40, DAP10, DAP12, CD134, CD3-zeta, or a fragment or combination thereof. In some embodiments, the transmembrane region includes a transmembrane region derived from CD8, CD28, or CTLA4.

[0258]

[0333] In some embodiments, the antigen-binding region includes an antibody domain. In some embodiments, the antibody domain includes an scFv or a Fab. In some embodiments, the antigen-binding region specifically binds to a tumor-associated antigen.

[0259]

[0334] 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 TCRα polypeptide and a TCRβ polypeptide. In some embodiments, the chimeric TCR comprises a TCRγ polypeptide and a TCRδ polypeptide.

[0260]

[0335] 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 cells are removed from a human subject via apheresis. In some embodiments, the plurality of T cells comprises one or more T cells from a healthy human subject (e.g., a subject without or not diagnosed with a particular disease or condition, e.g., 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., a particular disease or condition, e.g., diagnosed with cancer). In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor or a hematologic cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, prostate, lung, kidney, stomach, colon, ovary, bladder, breast, cervix, esophagus, testis, liver, pancreas, rectum, thyroid, uterus, skin, muscle, cartilage, bone, endothelium, epithelium, dermis, basal, retina, skin, or brain.

[0261]

[0336] In some embodiments, the plurality of T cells comprises one or more autologous T cells. In some embodiments, the plurality of T cells comprises one or more allogeneic T cells.

[0337] 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 times greater than the number of cells in the plurality of T cells before contacting with the first substance.

[0262]

[0338] In some embodiments, the substance that specifically binds to CD3 (e.g., CD3ε) includes an antibody domain (e.g., an anti-CD3 antibody (e.g., an anti-CD3ε antibody)).

[0339] In some embodiments, the substance that specifically binds to CD3 specifically binds to CD3ε.

[0263]

[0340] In some embodiments, upon binding to the TCRβV region, the first substance is as follows: (i) a reduction in the level of IL-1β, such as the expression level and / or activity; (ii) a reduction in the level of IL-6, such as the expression level and / or activity; (iii) a reduction in the level of TNFα, such as the expression level and / or activity; (iv) an increase in the level of IL-2, such as the expression level and / or activity; (v) a delay in the increase in the level of IL-2, such as the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, such as the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics; or (viii) a reduction in cytokine storm, such as cytokine release syndrome (CRS), as measured by an assay described herein, for example; (ix) cell killing, such as target cell killing, such as cancer cell killing, as measured by an assay described herein, for example; (x) an increase in the level of IL-15, such as the expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, such as expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, such as a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0264]

[0341] In some embodiments, the first substance, upon binding to the TCRβV region, results in the expansion of a memory T cell population, such as a T effector memory (TEM) cell population, such as a TEM cell expressing CD45RA (TEMRA), such as an expansion of at least about 1.1 - 10 fold (e.g., an expansion of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold).

[0265]

[0342] In some embodiments, the expansion of the population of memory effector T cells, such as TEM cells, such as TEMRA cells, in the first T cell population is increased as compared to the expansion of a similar cell population having an antibody that binds to the CD3 molecule.

[0266]

[0343] In some embodiments, the expanded T effector memory cell population has (i) a detectable level of CD45RA, such as expressing or re-expressing CD45RA; (ii) low or no expression of CCR7; and / or (iii) a detectable level of CD95, such as CD95, such as a population of CD45RA+, CCR7-, CD95+ T cells, and the T cells optionally include CD3+, CD4+, or CD8+ T cells.

[0267]

[0344] In some embodiments, binding of the first substance to the TCRβV region results in the following: (i) a reduction in the level of IL-1β, such as a reduction in the expression level and / or activity; (ii) a reduction in the level of IL-6, such as a reduction in the expression level and / or activity; (iii) a reduction in the level of TNFα, such as a reduction in the expression level and / or activity; (iv) an increase in the level of IL-2, such as an increase in the expression level and / or activity; (v) a delay in the increase in the level of IL-2, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more; (vi) a delay in the increase in the level of IFNγ, such as an increase in the expression level and / or activity, for example, a delay of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours; (vii) a reduction in T cell proliferation kinetics, or (viii) a reduction in cytokine storm, such as cytokine release syndrome (CRS), as measured by an assay described herein, for example; (ix) cell killing, such as target cell killing, such as cancer cell killing, as measured by an assay described herein, for example; (x) an increase in the level of IL-15, such as an increase in the expression level and / or activity; or (xi) an increase in natural killer (NK) cell proliferation, such as expansion, of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., all) compared to an antibody that binds to a CD3 molecule, such as a CD3 epsilon (CD3e) molecule; or a TCR alpha (TCRα) molecule.

[0268]

[0345] In some embodiments, binding of the first substance to the TCRβV region results in a reduction in the expression level and / or activity of IL-1β by 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), as measured by an assay described herein.

[0269]

[0346] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000-fold, or at least a 2- to 1000-fold (e.g., 5- to 900-fold, 10- to 800-fold, 20- to 700-fold, 50- to 600-fold, 100- to 500-fold, or 200- to 400-fold) reduction in the expression level and / or activity of IL-6 when measured by the assays described herein.

[0270]

[0347] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-fold, 10- to 900-fold, 20- to 800-fold, 50- to 700-fold, 100- to 600-fold, 200- to 500-fold, or 300- to 400-fold) reduction in the expression level and / or activity of TNFα when measured by the assays described herein.

[0271]

[0348] In some embodiments, binding of the first substance to the TCRβV region results in at least a 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 2000-fold, or at least a 2- to 2000-fold (e.g., 5- to 1000-fold, 10- to 900-fold, 20- to 800-fold, 50- to 700-fold, 100- to 600-fold, 200- to 500-fold, or 300- to 400-fold) increase in the expression level and / or activity of IL-2 when measured by the assays described herein.

[0272]

[0349] In this specification, in particular, recombinant nucleic acids encoding chimeric antigen receptors (CARs) are provided, wherein the CARs comprise: (a) an antigen-binding domain that 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 that contains a TCRβ constant region intracellular domain and does not contain a functional CD3ζ signaling domain. Also, in this specification, in particular, recombinant nucleic acids encoding chimeric antigen receptors (CARs) are provided, wherein the CARs comprise: (a) an antigen-binding domain that is a single-chain variable fragment (scFv) or a single-domain antibody; (b) a transmembrane domain; and (c) an intracellular signaling domain that contains a TCRβ intracellular domain and does not contain a functional CD3ζ signaling domain.

[0273]

[0350] 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.

[0351] In some embodiments, the antigen-binding domain, transmembrane domain, and intracellular signaling domain are operably linked.

[0274]

[0352] In some embodiments, the CAR further comprises a TCRβ constant domain. In some embodiments, the TCRβ constant domain is the TCRβ1 constant domain. In some embodiments, the TCRβ constant domain is the TCRβ2 constant domain. In some embodiments, the CAR comprises a TCRβ constant domain 1 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 296.

[0275]

[0353] In some embodiments, the CAR comprises a TCRβ constant domain 1 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 297.

[0276]

[0354] In some embodiments, the CAR comprises a TCRβ constant domain 2 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300.

[0277]

[0355] In some embodiments, the CAR comprises a TCRβ constant domain 2 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 301.

[0278]

[0356] In some embodiments, the transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of the T cell receptor β chain, the T cell receptor α chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

[0279]

[0357] In some embodiments, the transmembrane domain comprises the TCRβ constant 1 domain. In some embodiments, the transmembrane domain comprises the TCRβ constant 2 domain.

[0358] In some embodiments, the transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 302.

[0280]

[0359] In some embodiments, the transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 298.

[0281]

[0360] In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a linker. In some embodiments, the linker comprises or consists of glycine and serine.

[0282]

[0361] In some embodiments, the TCRβ constant intracellular domain comprises the TCRβ constant 1 intracellular domain. In some embodiments, the TCRβ constant intracellular domain comprises the TCRβ constant 2 intracellular domain.

[0283]

[0362] In some embodiments, the TCRβ intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299.

[0284]

[0363] In some embodiments, the TCRβ intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 303.

[0285]

[0364] In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises one or more costimulatory signaling domains and the TCRβ constant region intracellular domain from the N-terminus to the C-terminus.

[0286]

[0365] In some embodiments, the co-stimulatory signaling domain is one or more functional signaling domains of one or more proteins selected from the group consisting of 4-1BB (CD137), OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, a ligand that specifically binds to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, and Toll ligand receptors.

[0287]

[0366] In some embodiments, the antigen-binding domain is a human or humanized single-chain variable fragment (scFv) or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises a single-domain antibody (sdAb).

[0288]

[0367] In some embodiments, the antigen-binding domain binds to a tumor-associated antigen.

[0368] In some embodiments, the encoded chimeric antigen receptor (CAR) is in-frame and expressed as a single polypeptide chain.

[0289]

[0369] Provided herein are, in particular, vectors comprising the nucleic acid molecules described herein. In some embodiments, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

[0290]

[0370] Provided herein are, in particular, methods of generating immune effector cells comprising the step of transducing immune effector cells with the vectors described herein. In some embodiments, the immune effector cells are T cells or NK cells. In some embodiments, the immune effector cells are autologous or allogeneic immune effector cells.

[0291]

[0371] Provided herein are, in particular, immune effector cells comprising the nucleic acid molecules described herein.

[0372] In some embodiments, the immune effector cells are generated by the methods described herein. In some embodiments, the immune effector cells are T cells or NK cells. In some embodiments, the immune effector cells are autologous or allogeneic immune effector cells.

[0292]

[0373] In some embodiments, upon binding of the antigen-binding domain to the cognate antigen expressed by the cell, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by the immune effector cell is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by the immune effector cell comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0293]

[0374] In some embodiments, upon binding of the antigen-binding domain to the cognate antigen expressed by the cell, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by the immune effector 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by the immune effector cell comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0294]

[0375] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0295]

[0376] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0296]

[0377] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in vitro in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0297]

[0378] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in vitro in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a CAR comprising a CD3ζ intracellular signaling domain.

[0298]

[0379] In particular, provided herein are pharmaceutical compositions comprising the immune effector cells described herein.

[0380] In particular, provided herein are polypeptides encoded by the recombinant nucleic acids described herein.

[0299]

[0381] In particular, provided herein is a method of generating an RNA-engineered cell population comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, wherein the RNA comprises a nucleic acid molecule described herein.

[0300]

[0382] In particular, provided herein is a chimeric antigen receptor (CAR) comprising: (a) an antigen-binding domain that 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 that contains a TCRβ constant region intracellular domain, wherein the intracellular signaling domain does not contain a functional CD3ζ intracellular signaling domain. Also provided herein is, in particular, a chimeric antigen receptor (CAR) comprising: (a) an antigen-binding domain that is a single-chain variable fragment (scFv) or a single-domain antibody; (b) a transmembrane domain; and (c) an intracellular signaling domain that contains a TCRβ intracellular domain, wherein the intracellular signaling domain does not contain a functional CD3ζ intracellular signaling domain.

[0301]

[0383] In some embodiments, the CAR does not contain a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

[0384] In some embodiments, the antigen-binding domain, the transmembrane domain, and the intracellular signaling domain are operably linked. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the T cell receptor β chain, the T cell receptor α chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0302]

[0385] In some embodiments, the transmembrane domain comprises the TCRβ transmembrane domain. In some embodiments, the transmembrane domain comprises the TCRβ1 transmembrane domain. In some embodiments, the transmembrane domain comprises the TCRβ2 transmembrane domain.

[0303]

[0386] In some embodiments, the transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 298.

[0304]

[0387] In some embodiments, the transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 302.

[0305]

[0388] In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a linker. In some embodiments, the linker comprises glycine and serine.

[0389] In some embodiments, the TCRβ constant intracellular domain comprises the TCRβ constant 1 intracellular domain. In some embodiments, the TCRβ constant intracellular domain comprises the TCRβ constant 2 intracellular domain.

[0306]

[0390] In some embodiments, the intracellular signaling domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299.

[0307]

[0391] In some embodiments, the intracellular signaling domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 303.

[0308]

[0392] In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises one or more costimulatory signaling domains and the intracellular domain of the TCRβ constant region from the N-terminus to the C-terminus. In some embodiments, the costimulatory signaling domain specifically binds to one or more functional signaling domains of one or more proteins selected from the group consisting of 4-1BB (CD137), OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha a, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, a ligand that specifically binds to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, and Toll ligand receptors.

[0309]

[0393] In some embodiments, the antigen-binding domain is a human or humanized single-chain variable fragment (scFv) and a single-domain antibody.

[0394] In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises a single-domain antibody (sdAb).

[0310]

[0395] In some embodiments, the antigen-binding domain binds to a tumor-associated antigen.

[0396] In some embodiments, the CAR is produced by the methods described herein.

[0311]

[0397] Provided herein is, in particular, a method of treating cancer in a subject, comprising administering to the subject a cell (e.g., a cell population, e.g., an immune effector cell population) expressing a chimeric antigen receptor (CAR) described herein.

[0312]

[0398] In some embodiments, the chimeric antigen receptor (CAR) is encoded by a nucleic acid molecule described herein.

[0399] Provided herein is, in particular, a method of preventing cytokine release syndrome (CRS) (e.g., CRS associated with or induced by the administration of chimeric antigen receptor (CAR) cell therapy) in a subject having cancer, the method comprising administering to the subject a cell (e.g., a cell population, e.g., an immune effector cell population) expressing a chimeric antigen receptor (CAR) described herein.

[0313]

[0400] In some embodiments, the chimeric antigen receptor (CAR) is encoded by a nucleic acid molecule described herein.

[0401] In some embodiments, the subject does not exhibit one or more symptoms 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 cells (e.g., cell population, e.g., immune effector cell population). In some embodiments, the subject does not exhibit one or more grade 4 or grade 5 symptoms of CRS (e.g., as described herein). In some embodiments, the subject does not exhibit any grade 4 or grade 5 symptoms of CRS (e.g., as described herein). In some embodiments, the level of one or more proteins 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 after administration of the cells (e.g., cell population, e.g., immune effector cell population) (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) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the level of one or more proteins in the serum of the subject before administration of the cells (e.g., cell population, e.g., immune effector cell population) (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours).

[0314]

[0402] In some embodiments, the method further comprises selecting a subject for administration of the cells (e.g., cell population, e.g., immune effector cell population) based on at least one determination of: the risk of a subject developing CRS, the risk of a subject developing CRS upon administration of cells expressing a CAR comprising a CD3ζ signaling domain, the diagnosis of CRS in a subject, or the diagnosis of CRS in a subject associated with or induced by administration of cells expressing a CAR comprising a CD3ζ signaling domain.

[0315]

[0403] In some embodiments, the subject is selected for administration if the subject is at risk of developing CRS, if the subject is diagnosed with CRS, or if the subject is diagnosed with CRS associated with or induced by administration of cells expressing a CAR comprising a CD3ζ signaling domain when the subject is at risk of developing CRS upon administration of the CAR expressing the CAR CD3ζ signaling domain.

[0316]

[0404] In some embodiments, the cell (e.g., cell population, e.g., immune effector cell population) is a cell as described herein. In some embodiments, the cell (e.g., cell population, e.g., immune effector cell population) is a T cell or an NK cell. In some embodiments, the cell (e.g., cell population, e.g., immune effector cell population) is an autologous or allogeneic immune effector cell.

[0317]

[0405] In some embodiments, the subject is a mammal, such as a human.

[0406] In some embodiments, the cell (e.g., cell population, e.g., immune effector cell population) is administered in combination with a further therapeutic agent.

[0318]

[0407] In some embodiments, the cancer is a solid cancer or a blood cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney 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 blood cancer is leukemia, lymphoma or myeloma. In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0319]

[0408] Provided herein is a recombinant T cell receptor (TCR) comprising (a) a TCRα chain comprising an intracellular signaling domain comprising i) an immunoglobulin variable heavy domain, ii) a TCRα transmembrane domain, and iii) optionally a TCRα intracellular domain, and (b) a TCRβ chain comprising an intracellular signaling domain comprising i) an immunoglobulin variable light domain, ii) a TCRβ transmembrane domain, and iii) a TCRβ intracellular domain, wherein the immunoglobulin variable heavy domain and the immunoglobulin variable light domain form an antigen-binding domain, the recombinant TCR does not comprise a functional CD3ζ intracellular signaling domain, and the recombinant TCR does not comprise a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

[0320]

[0409] Also, in the present specification, in particular, a recombinant T cell receptor (TCR) is provided that includes (a) a TCRα chain containing an intracellular signaling domain that includes i) an immunoglobulin variable light domain, ii) a TCRα transmembrane domain, and iii) optionally a TCRα intracellular domain, and (b) a TCRβ chain containing an intracellular signaling domain that includes i) an immunoglobulin variable heavy domain, ii) a TCRβ transmembrane domain, and iii) a TCRβ intracellular domain, wherein the immunoglobulin variable heavy domain and the immunoglobulin variable light domain form an antigen-binding domain, the recombinant TCR does not include a functional CD3ζ intracellular signaling domain, and the recombinant TCR does not include a T cell receptor α (TCRα) variable region or a T cell receptor β (TCRβ) variable region.

[0321]

[0410] In some embodiments, the TCRα chain further includes a TCRα constant domain.

[0411] In some embodiments, the TCRα chain further includes a TCRα constant domain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 293.

[0322]

[0412] In some embodiments, the TCRβ chain further includes a TCRβ constant domain.

[0413] In some embodiments, the TCRβ constant domain includes TCRβ constant domain 1.

[0414] In some embodiments, the TCRβ constant domain includes TCRβ constant domain 2.

[0323]

[0415] In some embodiments, the TCRβ chain includes a TCRβ constant domain 1 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 296.

[0324]

[0416] In some embodiments, the TCRβ chain comprises a TCRβ constant domain 1 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 297.

[0325]

[0417] In some embodiments, the TCRβ chain further comprises a TCRβ constant domain 2 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300.

[0326]

[0418] In some embodiments, the TCRβ chain comprises a TCRβ constant domain 2 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 301.

[0327]

[0419] In some embodiments, the TCRβ transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 302.

[0328]

[0420] In some embodiments, the TCRβ transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 298.

[0329]

[0421] In some embodiments, the TCRα transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 294.

[0330]

[0422] In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a linker. In some embodiments, being connected comprises or consists of glycine and serine.

[0331]

[0423] In some embodiments, the TCRβ intracellular domain comprises a TCRβ1 intracellular domain. In some embodiments, the TCRβ intracellular domain comprises a TCRβ2 intracellular domain.

[0424] In some embodiments, the TCRβ intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299.

[0332]

[0425] In some embodiments, the TCRβ intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 303.

[0333]

[0426] In some embodiments, the TCRα intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 295.

[0334]

[0427] In some embodiments, the TCRα intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the TCRβ intracellular signaling domain further comprises a co-stimulatory signaling domain.

[0335]

[0428] In some embodiments, the co-stimulatory signaling domain is one or more functional signaling domains of one or more proteins selected from the group consisting of 4-1BB (CD137), OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, a ligand that specifically binds thereto, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activated NK cell receptor, BTLA and a Toll ligand receptor.

[0336]

[0429] In some embodiments, i) the immunoglobulin variable heavy domain and the immunoglobulin variable light domain are humanized; or ii) the immunoglobulin variable heavy domain and the immunoglobulin variable light domain are human.

[0337]

[0430] In some embodiments, the antigen-binding domain binds to a tumor-associated antigen.

[0431] In some embodiments, the recombinant T cell receptor (TCR) is produced by the methods described herein.

[0338]

[0432] Specifically provided herein is a recombinant nucleic acid encoding the recombinant TCR described herein.

[0433] Specifically provided herein is a polypeptide encoding the recombinant TCR described herein, encoded by the nucleic acid described herein.

[0339]

[0434] Specifically provided herein is a vector comprising a nucleic acid molecule encoding the recombinant TCR described herein. In some embodiments, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0340]

[0435] Specifically provided herein is a method for producing an immune effector cell, comprising the step of transducing an immune effector cell with the vector described herein. In some embodiments, the immune effector cell is a T cell or an NK cell. In some embodiments, the immune effector cell is an autologous or allogeneic immune effector cell.

[0341]

[0436] Specifically provided herein is an immune effector cell comprising the nucleic acid molecule described herein encoding the recombinant TCR described herein.

[0437] In some embodiments, the immune effector cells are produced by the methods described herein. In some embodiments, the immune effector cells are T cells or NK cells. In some embodiments, the immune effector cells are autologous or allogeneic immune effector cells. In some embodiments, upon binding of the antigen-binding domain to the cognate antigen expressed by the cell, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by the immune effector cells is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0342]

[0438] In some embodiments, upon binding of the antigen-binding domain to the cognate antigen expressed by the cell, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by the 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0343]

[0439] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells (e.g., dendritic cells or macrophages) is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0344]

[0440] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells (e.g., dendritic cells or macrophages) 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0345]

[0441] In some embodiments, in the presence of one or more antigen-presenting cells, upon binding of an antigen-binding domain to a cognate antigen expressed by cells in vitro, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0346]

[0442] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by cells in vitro in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0347]

[0443] In particular, the present specification provides a pharmaceutical composition comprising the immune effector cells described herein.

[0444] In particular, the present specification provides a method for treating cancer in a subject, the method comprising administering to the subject a cell (e.g., a cell population, e.g., a population of immune effector cells) expressing a TCR described herein.

[0348]

[0445] In some embodiments, the recombinant T cell receptor (TCR) is encoded by a nucleic acid molecule described herein.

[0446] Disclosed herein is, in particular, a method for preventing cytokine release syndrome (CRS) (e.g., CRS associated with or induced by administration of recombinant T cell receptor (TCR) cell therapy) in a subject having cancer, the method comprising administering to the subject cells (e.g., a cell population, e.g., an immune effector cell population) that express a recombinant T cell receptor (TCR) described herein.

[0349]

[0447] In some embodiments, the recombinant T cell receptor (TCR) is encoded by a nucleic acid molecule described herein.

[0448] In some embodiments, the subject does not exhibit one or more symptoms of CRS (e.g., as described herein) within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 30 days from administration of the cells (e.g., a cell population, e.g., an immune effector cell population). In some embodiments, the subject does not exhibit one or more grade 4 or grade 5 CRS symptoms (e.g., as described herein). In some embodiments, the subject does not exhibit any grade 4 or grade 5 CRS symptoms (e.g., as described herein). In some embodiments, the level of one or more proteins 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 after administration of the cells (e.g., a cell population, e.g., an immune effector cell population) (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) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the level of one or more proteins in the serum of the subject before administration of the cells (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours).

[0350]

[0449] In some embodiments, the method further comprises selecting a subject for administration of a cell (e.g., a cell population, e.g., an immune effector cell population) based on one or more determinations of: the risk of developing CRS in a subject, the risk of developing CRS in a subject when administered a cell expressing a recombinant TCR comprising a CD3ζ signaling domain, the diagnosis of CRS in the subject, the diagnosis of CRS in the subject associated with or induced by the administration of a cell expressing a recombinant TCR comprising a CD3ζ signaling domain.

[0351]

[0450] In some embodiments, a subject is selected for administration if the subject is at risk of developing CRS, if the subject is at risk of developing CRS when administered a cell expressing a recombinant TCR comprising a CD3ζ signaling domain, if the subject is diagnosed with CRS, or if the subject is diagnosed with CRS associated with or induced by the administration of a cell expressing a recombinant TCR comprising a CD3ζ signaling domain.

[0352]

[0451] In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is a cell described herein. In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is a T cell or an NK cell. In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is an autologous or allogeneic immune effector cell.

[0353]

[0452] In some embodiments, the subject is a mammal, e.g., a human.

[0453] In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is administered in combination with a further therapeutic agent.

[0354]

[0454] In some embodiments, the cancer is a solid cancer or a blood cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney 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 blood cancer. In some embodiments, the blood cancer is leukemia, lymphoma or myeloma. In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

[0355]

[0455] In particular, provided herein is a recombinant T cell receptor (TCR) comprising: (a) a TCRα chain comprising an intracellular signaling domain that includes i) an antigen-binding domain (e.g., scFv), ii) a TCRα variable domain, iii) a TCRα constant domain, iv) a TCRα transmembrane domain, and optionally iii) a TCRα intracellular domain; and (b) a TCRβ chain comprising an intracellular signaling domain that includes i) a TCRβ variable domain, ii) a TCRβ constant domain, iii) a TCRβ transmembrane domain, and iv) a TCRβ intracellular domain, wherein the recombinant TCR does not include a functional CD3ζ intracellular signaling domain. In particular, provided herein is a recombinant T cell receptor (TCR) comprising the following.

[0356]

[0456] (a)i) The TCRα variable domain, ii) the TCRα constant domain, iii) the TCRα transmembrane domain, and iv) optionally the TCRα intracellular domain, the TCRα chain comprising an intracellular signaling domain; (b) i) an antigen-binding domain (e.g., scFv), ii) the TCRβ variable domain, iii) the TCRβ constant domain, iii) the TCRβ transmembrane domain, and iv) the TCRβ chain comprising an intracellular signaling domain comprising the TCRβ intracellular domain; the recombinant TCR does not comprise a functional CD3ζ intracellular signaling domain.

[0357]

[0457] In some embodiments, the TCRα constant domain is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 293.

[0358]

[0458] In some embodiments, the TCRβ constant domain comprises TCRβ constant domain 1.

[0459] In some embodiments, the TCRβ constant domain comprises TCRβ constant domain 2.

[0460] In some embodiments, TCRβ constant domain 1 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 296.

[0359]

[0461] In some embodiments, TCRβ constant domain 1 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 297.

[0360]

[0462] In some embodiments, TCRβ constant domain 2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 300.

[0361]

[0463] In some embodiments, the TCRβ constant domain 2 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 301.

[0362]

[0464] In some embodiments, the TCRβ transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 302.

[0363]

[0465] In some embodiments, the TCRβ transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 298.

[0364]

[0466] In some embodiments, the TCRα transmembrane domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 294.

[0365]

[0467] In some embodiments, the antigen-binding domain is connected to the transmembrane domain by a linker. In some embodiments, being connected comprises or consists of glycine and serine.

[0366]

[0468] In some embodiments, the TCRβ intracellular domain comprises the TCRβ1 intracellular domain. In some embodiments, the TCRβ intracellular domain comprises the TCRβ2 intracellular domain.

[0469] In some embodiments, the TCRβ intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 299.

[0367]

[0470] In some embodiments, the TCRβ intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 303.

[0368]

[0471] In some embodiments, the TCRα intracellular domain comprises a nucleic acid encoding an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 295.

[0369]

[0472] In some embodiments, the TCRα intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the TCRβ intracellular signaling domain further comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain specifically binds to one or more functional signaling domains of one or more proteins selected from the group consisting of 4-1BB (CD137), OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha a, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, a ligand that specifically binds to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, and Toll ligand receptors.

[0370]

[0473] In some embodiments, the antigen-binding domain is a scFv, a single-domain antibody or a nanobody. In some embodiments, the antigen-binding domain binds to a tumor-associated antigen.

[0474] In some embodiments, the TCR is produced by the methods described herein.

[0371]

[0475] In particular, provided herein is a recombinant nucleic acid encoding a recombinant TCR described herein.

[0476] In particular, provided herein is a polypeptide encoded by a nucleic acid described herein.

[0372]

[0477] In particular, provided herein is a vector comprising a nucleic acid molecule described herein.

[0478] In some embodiments, the vector is a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector or a retroviral vector.

[0373]

[0479] In particular, provided herein is a method for producing an immune effector cell comprising the step of transducing an immune effector cell with a vector described herein. In some embodiments, the immune effector cell is a T cell or an NK cell. In some embodiments, the immune effector cell is an autologous or allogeneic immune effector cell.

[0374]

[0480] In particular, provided herein is an immune effector cell comprising a nucleic acid molecule described herein. In some embodiments, the immune effector cell is produced by the methods described herein. In some embodiments, the immune effector cell is a T cell or an NK cell. In some embodiments, the immune effector cell is an autologous or allogeneic immune effector cell.

[0375]

[0481] In some embodiments, upon binding of the antigen-binding domain to the cognate antigen expressed by the cell, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by immune effector cells is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0376]

[0482] In some embodiments, upon binding of the antigen-binding domain to the cognate antigen expressed by the cell, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0377]

[0483] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells (e.g., dendritic cells or macrophages) is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0378]

[0484] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells (e.g., dendritic cells or macrophages) 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0379]

[0485] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in vitro in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) is lower compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0380]

[0486] In some embodiments, upon binding of an antigen-binding domain to a cognate antigen expressed by a cell in vitro in the presence of one or more antigen-presenting cells, the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells (e.g., dendritic cells or macrophages) 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 compared to the expression (e.g., release) level of one or more pro-inflammatory cytokines (e.g., IL-6, IFNγ, TNFα, IL-6, IL-1β, IL-8, IL-10, sIL2Rα, sgp130, sIL6R, MCP1, MIP1α, MIP1β, and GM-CSF) by one or more (e.g., a population of) antigen-presenting cells in the presence of immune effector cells comprising a nucleic acid encoding a TCR comprising a CD3ζ intracellular signaling domain.

[0381]

[0487] In particular, provided herein are pharmaceutical compositions comprising the immune effector cells described herein.

[0488] In particular, provided herein is a method of treating cancer in a subject, the method comprising administering to the subject cells (e.g., a cell population, e.g., a population of immune effector cells) expressing a TCR described herein. In some embodiments, the recombinant T cell receptor (TCR) is encoded by a nucleic acid molecule described herein.

[0382]

[0489] Disclosed herein is a method for preventing cytokine release syndrome (CRS) in a subject having cancer (e.g., CRS associated with or induced by administration of recombinant T cell receptor (TCR) cell therapy), the method comprising administering to the subject a cell (e.g., a cell population, e.g., an immune effector cell population) expressing a recombinant T cell receptor (TCR) described herein.

[0383]

[0490] In some embodiments, the recombinant T cell receptor (TCR) is encoded by a nucleic acid molecule described herein.

[0491] In some embodiments, the subject does not exhibit one or more symptoms of CRS (e.g., as described herein) within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 30 days from administration of the cell (e.g., a cell population, e.g., an immune effector cell population). In some embodiments, the subject does not exhibit one or more grade 4 or grade 5 CRS symptoms (e.g., as described herein). In some embodiments, the subject does not exhibit any grade 4 or grade 5 CRS symptoms (e.g., as described herein). In some embodiments, the level of one or more proteins 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 after administration of the cell (e.g., a cell population, e.g., an immune effector cell population) (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) is within ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the level of one or more proteins in the serum of the subject before administration of the cell (e.g., a cell population, e.g., an immune effector cell population) (e.g., 10 minutes, 30 minutes, 1 hour, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours).

[0384]

[0492] In some embodiments, the method further comprises selecting a subject for administration of a cell (e.g., a cell population, e.g., an immune effector cell population) based on one or more determinations of: the risk of a subject developing CRS, the risk of a subject developing CRS when administered a cell expressing a recombinant TCR comprising a CD3ζ signaling domain, the diagnosis of CRS in the subject, and the diagnosis of CRS in the subject associated with or induced by the administration of a cell expressing a recombinant TCR comprising a CD3ζ signaling domain.

[0385]

[0493] In some embodiments, a subject is selected for administration if the subject is at risk of developing CRS, if the subject is at risk of developing CRS when administered a cell expressing a recombinant TCR comprising a CD3ζ signaling domain, if the subject is diagnosed with CRS, or if the subject is diagnosed with CRS associated with or induced by the administration of a cell expressing a recombinant TCR comprising a CD3ζ signaling domain.

[0386]

[0494] In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is a cell described herein. In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is a T cell or an NK cell. In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is an autologous or allogeneic immune effector cell.

[0387]

[0495] In some embodiments, the subject is a mammal, such as a human.

[0496] In some embodiments, the cell (e.g., a cell population, e.g., an immune effector cell population) is administered in combination with a further therapeutic agent.

[0388]

[0497] In some embodiments, the cancer is a solid cancer or a blood cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is prostate cancer, lung cancer, kidney 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 blood cancer. In some embodiments, the blood cancer is leukemia, lymphoma or myeloma. In some embodiments, the blood cancer is B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia or preleukemia.

Brief Description of the Drawings

[0389]

Figure 1

[0498] Figure 1 shows a phylogenetic tree of the gene families and subfamilies of TCRβV that map the corresponding antibodies. The identities of the subfamilies are as follows. Subfamily A: TCRβV6; Subfamily B: TCRβV10; Subfamily C: TCRβV12; Subfamily D: TCRβV5; Subfamily E: TCRβV7; Subfamily F: TCRβV11; Subfamily G: TCRβV14; Subfamily H: TCRβV16; Subfamily I: TCRβV18; Subfamily J: TCRβV9; Subfamily K: TCRβV13; Subfamily L: TCRβV4; Subfamily M: TCRβV3; Subfamily N: TCRβV2; Subfamily O: TCRβV15; Subfamily P: TCRβV30; Subfamily Q: TCRβV19; Subfamily R: TCRβV27; Subfamily S: TCRβV28; Subfamily T: TCRβV24; Subfamily U: TCRβV20; Subfamily V: TCRβV25; and Subfamily W: TCRβV29. Subfamily members are described in detail in the section entitled "TCR Beta V (TCRβV)" in this specification.

Figure 2A

[0499] Figures 2A - 2C show human CD3+ T cells activated for 6 days with the anti - TCR Vβ13.1 antibody (BHM1709). Human CD3+ T cells were isolated using magnetic bead separation (negative selection) and activated for 6 days with 100 nM of immobilized (plate - coated) anti - TCR Vβ13.1 antibody (BHM1709) or anti - CD3ε (OKT3) antibody. Figure 2A shows two enlarged scatter plots of T cells (left: activated with OKT3, right: activated with BHM1709) evaluating surface expression of TCR Vβ13.1 using anti - TCR Vβ13.1 (BHM1709) and subsequent secondary fluorochrome - conjugated antibodies for flow cytometry analysis.

Figure 2B

Figure 2C

Figure 3A

[0500] Figures 3A - 3B show the cytolytic activity of human CD3+ T cells activated by anti-TCR Vβ13.1 antibody (BHM1709) against the transformed cell line RPMI8226. Figure 3A represents the target cell lysis of human CD3+ T cells activated by BHM1709 or OKT3. Human CD3+ T cells were isolated using magnetic bead separation (negative selection), activated with immobilized (plate-coated) BHM1709 or OKT3 at the indicated concentrations for 4 days, and then co-cultured with RPMI8226 cells at an (E:T) ratio of 5:1 for 2 days. Samples were then analyzed for lysis of RPMI8226 cells by FACS staining for CFSE / CD138 labeling and the membrane-impermeable DNA dye (DRAQ7) using flow cytometry analysis.

Figure 3B

Figure 4A

[0501] Figures 4A-4B show the production of IFNγ by human PBMCs activated by the indicated antibodies. Human PBMCs were isolated from whole blood from the indicated number of donors and subjected to solid-phase (plate-coated) stimulation with the indicated antibody at 100 nM. Supernatants were collected on days 1, 2, 3, 5, or 6. Figure 4A is a graph comparing the production of IFNγ in human PBMCs activated by the indicated antibody, anti-TCR Vβ13.1 antibody (BHM1709 or BHM1710), or anti-CD3e antibody (OKT3 or SP34-2) at 1, 2, 3, 5, or 6 days after activation.

Figure 4B

Figure 5A

[0502] Figure 5A shows the production of IL-2 by human PBMCs activated by the indicated antibody. The same experimental setup as described for Figures 4A-4B was used.

Figure 5B

Figure 6A

[0503] Figure 6A shows the production of IL-6 by human PBMCs activated by the indicated antibody. The same experimental setup as described for Figures 4A-4B was used.

Figure 6B

Figure 7A

[0504] Figure 7A shows the production of TNF-alpha by human PBMCs activated by the indicated antibody. The same experimental setup as described for Figures 4A-4B was used.

Figure 7B

Figure 8A

[0505] Figure 8A is a diagram showing the production of IL-1 beta by human PBMCs activated by the indicated antibodies.

Figure 8B

Figure 9A

[0506] Figure 9A is a graph showing the delay in the kinetics of IFNγ secretion in human PBMCs from 4 donors activated by the anti-TCR Vβ13.1 antibody BHM1709 compared to PBMCs activated by the anti-CD3e antibody OKT3.

Figure 9B

Figure 10

[0507] Figure 10 represents the increase in subsets of CD8+ TSCM and TEMRA T cells in human PBMCs activated by anti-TCR Vβ13.1 antibodies (BHM1709 or BHM1710) compared to PBMCs activated by anti-CD3e antibodies (OKT3 or SP34-2).

Figure 11A

[0508] Figure 11A represents an exemplary T cell stimulation method.

Figure 11B

Figure 11C

Figure 12A

[0509] Figure 12A depicts an exemplary T cell stimulation method.

Figure 12B

Figure 12C

Figure 12D

Figure 13

[0510] Figure 13 depicts an exemplary T cell receptor (TCR) described herein. The TCR includes a TCRα polypeptide chain comprising an immunoglobulin variable heavy chain or immunoglobulin variable light chain, a TCRα constant domain, a TCRα transmembrane domain, a TCRα intracellular domain, and optionally one or more (e.g., 2 or 3) co-stimulatory domains, and a TCRβ polypeptide chain comprising an immunoglobulin variable heavy chain or immunoglobulin variable light chain, a TCRβ constant domain (TCRβ constant 1 domain or TCRβ constant 2 domain), a TCRα transmembrane domain, a TCRβ intracellular domain, and optionally one or more (e.g., 2 or 3) co-stimulatory domains, wherein the immunoglobulin domains form an antigen-binding domain.

Figure 14

[0511] Figure 14 depicts an exemplary chimeric antigen receptor (CAR) described herein. The CAR includes an antigen-binding domain (e.g., scFv), a TCRβ constant domain (TCRβ constant 1 domain or TCRβ constant 2 domain), a TCRα transmembrane domain, a TCRβ intracellular domain, and optionally one or more (e.g., 2 or 3) co-stimulatory domains.

Figure 15

[0512] Figure 15 depicts the anti-CD19 chimeric antigen receptor (CAR) cassette used in Example 3. The CAR includes an EF1A promoter, a CD8α signal peptide, an FMC63 single-chain Fv that binds to CD19, a FLAG tag, a CD28 intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain.

Figure 16

[0513] Figure 16 is a bar graph showing the number of viable cells on day 6 after activation of the culture of T cells or CAR T cells from one of three donors (donor 010, donor 541, donor 871). One of three activation conditions was used. Condition 1: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS). Condition 2: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS) and IL2 (culture medium containing 300 U / mL of rIL-2 (cat #Pr21269, ProMab)). Condition 3: Activation using equal amounts of anti-CD3ε antibody and anti-CD28 antibody (50 nM each in PBS). The number of viable cells was determined by FACS analysis.

Figure 17

[0514] Figure 17 is a bar graph showing the number of viable cells on day 9 after activation of the culture of T cells or CAR T cells from one of three donors (donor 010, donor 541, donor 871). One of three activation conditions was used. Condition 1: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS). Condition 2: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS) and IL2 (culture medium containing 300 U / mL of rIL-2 (cat #Pr21269, ProMab)). Condition 3: Activation using equal amounts of anti-CD3ε antibody and anti-CD28 antibody (50 nM each in PBS). The number of viable cells was determined by FACS analysis.

Figure 18

[0515] Figure 18 is a bar graph showing the number of CD3+ cells on day 9 after activation of the culture of T cells or CAR T cells from one of three donors (Donor 010, Donor 541, Donor 871). One of three activation conditions was used. Condition 1: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS). Condition 2: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS) and IL2 (culture medium containing 300 U / mL of rIL-2 (cat #Pr21269, ProMab)). Condition 3: Activation using equal amounts of anti-CD3ε antibody and anti-CD28 antibody (50 nM each in PBS). The number of CD3+ cells was determined by FACS analysis.

Figure 19

[0516] Figure 19 is a bar graph showing the ratio of CD4+ T cells to CD8+ T cells on day 9 after activation of the culture of T cells or CAR T cells from one of three donors (Donor 010, Donor 541, Donor 871). One of three activation conditions was used. Condition 1: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS). Condition 2: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS) and IL2 (culture medium containing 300 U / mL of rIL-2 (cat #Pr21269, ProMab)). Condition 3: Activation using equal amounts of anti-CD3ε antibody and anti-CD28 antibody (50 nM each in PBS). The ratio of CD4+ T cells to CD8+ T cells was determined by FACS analysis.

Figure 20

[0517] Figure 20 is a bar graph showing the percentage of TCRβV+ cells on day 9 after activation of T cells or CAR T cells from one of three donors (donor 010, donor 541, donor 871). One of three activation conditions was used. Condition 1: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS). Condition 2: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS) and IL2 (culture medium containing 300 U / mL of rIL-2 (cat #Pr21269, ProMab)). Condition 3: Activation using equal amounts of anti-CD3ε antibody and anti-CD28 antibody (50 nM each in PBS). The ratio of CD4+ T cells to CD8+ T cells was determined by FACS analysis using the 16G8-PE-labeled antibody.

Figure 21

[0518] Figure 21 is a bar graph showing the percentage of CAR+ T cells on day 9 after activation of T cells from one of three donors (donor 010, donor 541, donor 871). One of three activation conditions was used. Condition 1: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS). Condition 2: Activation using equal amounts of TCRβV clonotype-specific antibodies H131 and 16G8 (50 nM each in PBS) and IL2 (culture medium containing 300 U / mL of rIL-2 (cat #Pr21269, ProMab)). Condition 3: Activation using equal amounts of anti-CD3ε antibody and anti-CD28 antibody (50 nM each in PBS). The ratio of CD4+ T cells to CD8+ T cells was determined by FACS analysis using FLAG staining as the CAR construct containing the FLAG tag shown in Figure 15.

Figure 22

[0519] Figure 22 is a graphical depiction of the protocol for the expansion of T cells (e.g., CAR T cells) described herein. In contrast to the use of anti-CD3ε antibody that activates all T cells, T cells expanded using a clonotype anti-TCRβV antibody were expanded targeting only a specific subset of T cells. Activation and expansion of T cells using anti-TCRβV antibody prevents systemic release of cytokines that can result in toxicity (e.g., CRS) when administered to a subject.

Figure 23

[0520] Figure 23 is a FACS plot showing the expansion of TCRβV 6-5+ T cells over 8 days using anti-TCRvb 6-5 v1.

Figure 24

[0521] Figure 24 is a bar graph showing the expansion of TCRβV 6-5+ CD4+ T cells and TCRβV 6-5+ CD8+ T cells over 8 days using anti-CD3ε antibody OKT3 (100 nM).

Figure 25

[0522] Figure 25 is a bar graph showing the expansion of TCRβV 6-5+ CD4+ T cells and TCRβV 6-5+ CD8+ T cells over 8 days using anti-TCRvb 6-5 v1 antibody (100 nM).

Figure 26

[0523] Figure 26 is a FACS plot showing the expansion of TCRβV 6-5+ T cells over 8 days using anti-TCRvb 6-5 v1 or anti-CD3ε antibody OKT3.

Figure 27A

[0524] Figure 27A is a bar graph showing the percentage of TCRβV 6-5+ T cells in PBMC cultures after 8 days of culture with the indicated antibodies. Data for 5 replicate experiments are shown.

Figure 27B

Figure 28A

[0525] Figure 28A is a bar graph showing the relative numbers of TCRβV 6-5+ T cells in PBMC cultures after 8 days of culture with the indicated antibodies.

Figure 28B

Figure 29A

[0526] Figure 29A is a bar graph showing the relative numbers of TCRβV 6-5+ T cells in purified T cell cultures after 8 days of culture with the indicated antibodies.

Figure 29B

Figure 30

[0527] Figure 30 is a line graph showing the total CD3+ T cell numbers (fold increase) after 8 days of T cell culture with either anti-CD3ε antibody OKT3 or anti-TCRvb 6-5 v1 antibody.

Figure 31

[0528] Figure 31 is a series of line graphs showing the kinetics of target cells by TCRβV 6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells. T cells from 3 different donors were used (Donor 6769, Donor 9880, Donor 5411).

Figure 32A

[0529] Figure 32A is a scatter plot showing the percent of target cell lysis by TCRβV 6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells without prior activation of T cells. Data are presented on day 6 of co-culture of target cells and effector T cells.

Figure 32B

Figure 33

[0530] Figure 33 is a scatter plot showing the percentage of target cell lysis by TCRβV 6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells with 4-day pre-activation of T cells. The data are presented on day 2 of the co-culture of target cells and effector T cells (4 days after pre-activation of T cells).

Figure 34

[0531] Figure 34 is a bar graph showing target cell lysis by TCRβV 6-5 v1-activated T cells or anti-CD3ε (OKT3)-activated T cells (100 nM of each antibody). The data include 7 replicate experiments under each experimental condition.

Figure 35

[0532] Figure 35 is a series of FACS plots showing cell surface expression of CD3ε at days 0, 1, 2, 4, 6, or 8 after antibody activation in CD4+ TCRβV 6-5- or CD4+ TCRβV 6-5+ T cells activated by either SP34-2 (anti-CD3ε antibody) or anti-TCRβV 6-5 v1 (anti-TCRβV 6-5 antibody).

Figure 36

[0533] Figure 36 is a series of FACS plots showing cell surface expression of CD3ε at 0, 1, 2, 4, 6, or 8 days after antibody activation in CD8+ TCRβV 6-5- or CD8+ TCRβV 6-5+ T cells activated by either SP34-2 (anti-CD3ε antibody) or anti-TCRβV 6-5 v1 (anti-TCRβV 6-5 antibody).

Figure 37

[0534] Figure 37 is a series of FACS plots showing cell surface expression of TCRβV at days 0, 1, 2, 4, 6, or 8 after antibody activation in CD4+ TCRβV 6-5- or CD4+ TCRβV 6-5+ T cells activated by either SP34-2 (anti-CD3ε antibody) or anti-TCRβV 6-5 v1 (anti-TCRβV 6-5 antibody).

Figure 38

[0535] Figure 38 is a series of FACS plots showing cell surface expression of TCRβV on CD8+ TCRβV 6-5- or CD8+ TCRβV 6-5+ T cells activated by either SP34-2 (anti-CD3ε antibody) or anti-TCRβV 6-5 v1 (anti-TCRβV 6-5 antibody) on days 0, 1, 2, 4, 6, or 8 of antibody activation.

Figure 39A

[0536] Figure 39A shows FACS plots of TCRβV 6-5+ cynomolgus T cell expansion 7 days after activation of unstimulated (left) or anti-TCRβV 6-5 v1-stimulated (right) cynomolgus PBMC. PBMC from donor DW8N (fresh PBMC sample, male, 8 years old, body weight 7.9 kg) were used.

Figure 39B

Figure 40

[0537] Figure 40 shows FACS plots of TCRβV 6-5+ cynomolgus T cell expansion after activation of cryopreserved donor DW8N cynomolgus PBMC that were unstimulated (left), stimulated with SP34-2 (anti-CD3ε antibody) (middle), or stimulated with anti-TCRβV 6-5 v1 (right), and the corresponding microscopic images. The microscopic images show the formation of cell clusters (represented by circles).

Figure 41

[0538] Figure 41 shows a schematic diagram of a FACS plot showing FACS gating / staining of PBMC before purification of γδ T cells.

Figure 42

[0539] Figure 42 shows a schematic diagram of a FACS plot showing FACS gating / staining of the purified γδ T cell population.

Figure 43

[0540] Figure 43 shows activation of the purified γδ T cell population by anti-CD3ε antibody (SP34-2) (left) or anti-TCRβV antibody (anti-TCRβV 6-5 V1) (right).

Figure 44A

[0541] Figure 44A shows the release of IFNγ from purified γδ T cell populations activated by anti-CD3ε antibody (SP34-2), activated by anti-TCRβV antibody (anti-TCRβV 6-5 V1), or not stimulated.

Figure 44B

Figure 44C

Figure 44D

Figure 44E

Figure 44F

Figure 44G

Figure 44H

Figure 45

[0542] Figure 45 shows the relative expression of all TCR alpha V fragments (TRAV gene groups of the gene) and their variants (top), all TCR beta V fragment 6-5 variants (TRBV6-5 gene) (bottom left), and all TCR beta V fragments and variants excluding 6-5 (bottom right).

Figure 46A

[0543] Figure 46A is a FACS plot showing the phenotypic markers of CD4+ T cells expanded by anti-TCRβV antibody (anti-TCRβV 6-5 v1). Defined phenotypes include TEMRA (top left), naive / TSCM (top right), TEM (bottom left), and TCM (bottom right).

Figure 46B

Figure 47A

[0544] Figure 47A is a FACS plot showing the phenotypic markers of CD8+ T cells expanded by anti-TCRβV antibody (anti-TCRβV 6-5 v1). Defined phenotypes include TEMRA (top left), naive / TSCM (top right), TEM (bottom left), and TCM (bottom right).

Figure 47B

Figure 48A

[0545] Figure 48A is a bar graph showing the percentage of PD1-expressing CD4+ T cells from T cell cultures activated by an anti-TCRβV antibody (anti-TCRβV 6-5 v1), activated by an anti-CD3ε antibody (OKT3), or not stimulated.

Figure 48B

Figure 49A

[0546] Figure 49A is a bar graph showing the expression of Ki-67 by CD4+ T cells from T cell cultures activated by an anti-TCRβV antibody (anti-TCRβV 6-5 v1), activated by an anti-CD3ε antibody (OKT3), or not stimulated.

Figure 49B

Figure 50A

[0547] Figure 50A is a FACS plot showing the percentage of TEMRA-like CD8+ T cells expressing CD57 (18.7%) activated using an anti-TCRβV antibody (anti-TCRβV 6-5 v1).

Figure 50B

Figure 51

[0548] Figure 51 shows a series of FACS plots depicting the expression of CD27 by CD4+ (top) or CD8+ (bottom) T cells from T cell cultures activated by an anti-TCRβV antibody (anti-TCRβV 6-5 v1), an anti-CD3ε antibody (OKT3), or not stimulated.

Figure 52

[0549] Figure 52 shows a series of FACS plots depicting the expression of OX40, 41BB, and ICOS by CD4+ (top) or CD8+ (bottom) T cells from T cell cultures activated by an anti-TCRβV antibody (anti-TCRβV 6-5 v1), an anti-CD3ε antibody (OKT3), or not stimulated.

Figure 53

[0550] Figure 53 shows a series of FACS plots depicting the expression levels of TCRβV6-5 by Jurkat cells passaged 11 times (P11), 15 times (P15), and 21 times (P21).

Figure 54

[0551] Figure 54 shows a series of FACS plots depicting the percentages of CD3+(CD4 gate) TCRβV 6-5+ T cells at 1, 2, 3, 4, 5, 6, and 8 days after activation by BCMA and an anti-TCR Vβ antibody (anti-TCRVβ 6-5 v1).

Figure 55A

[0552] Figure 55A shows a series of FACS plots depicting the percentages of CD4+ T cells at day 0 after expansion and activation using an isotype control (IgG1 N297A), an anti-TCRβV (anti-TCRβV 6-5 v1), or an anti-CD3ε (OKT3) antibody.

Figure 55B

Figure 55C

Figure 55D

Figure 55E

Figure 55F

Figure 55G

Figure 55H

Figure 56A

[0553] Figure 56A is a map showing the difference in gene expression between cells activated by the anti-TCRvb 6-5 v1 antibody and unstimulated cells.

Figure 56B

Figure 56C

Figure 56D

Figure 56E

Figure 57A

[0554] Figure 57A shows the distribution of genes that were differentially upregulated after stimulation of T cells with the indicated antibodies.

Figure 57B

Figure 57C

Figure 57D

Figure 58

[0555] Figure 58 shows a heat map of pathway scores for genes that are differentially regulated and associated with various cellular pathways. Samples of purified T cells included unstimulated (n = 3), OKT3-stimulated (n = 3), SP34-2-stimulated (n = 3), and anti-TCRβV 6-5 v1-stimulated (n = 3).

Figure 59A

[0556] Figure 59A shows a plot of the pathways of cytokines and chemokines that were upregulated or downregulated or unstimulated by activation with the indicated antibodies.

Figure 59B

Figure 59C

Figure 59D

Figure 60A

[0557] Figure 60A shows plots of T cell function pathways that are upregulated, downregulated, or not stimulated by activation with the indicated antibodies.

Figure 60B

Figure 61A

[0558] Figure 61A shows differences in granzyme B regulation in cells activated or not stimulated by the indicated antibodies. P≤0.01****; p≤0.05***; p≤0.5*; p≤0.5 ns.

Figure 61B

Figure 61C

Figure 61D

Figure 61E

Figure 61F

Figure 61G

Figure 61H

Figure 61I

Figure 61J

Figure 62

[0559] Figure 62 shows a graph from the principal component analysis (PCA) of genes related to the activation and exhaustion of T cells that were differentially expressed after activation of T cells by the indicated antibodies.

Figure 63

[0560] Figure 63 shows a graph from the principal component analysis (PCA) of genes related to co-stimulatory molecules that were expressed after activation of T cells by the indicated antibodies.

Figure 64

[0561] Figure 64 shows a graph from the principal component analysis (PCA) of genes related to regulatory functions that were expressed after activation of T cells by the indicated antibodies.

Figure 65A

[0562] Figure 65A is a bar graph showing the glycolytic ATP production of T cell cultures activated by the indicated antibodies.

Figure 65B

Figure 66

[0563] Figure 66 is a line graph showing the oxygen consumption rate (OCR) of T cells activated by the indicated antibodies for approximately 0 - 75 minutes.

Figure 67A

[0564] Figure 67A shows the oxygen consumption rate (OCR) during the basal respiration of T cells activated by the indicated antibodies.

Figure 67B

Figure 67C

Figure 67D

Figure 68A

[0565] Figure 68A is a bar graph showing the glycolytic ATP production of T cell cultures activated with anti - TCRβV 6 - 5 v1 and restimulated with the indicated antibodies.

Figure 68B

Figure 69A

[0566] Figure 69A is a FACS plot showing the percentage of CMV(pp65) - specific anti - TCRβV 6 - 5 v1 - activated TCRvβ 6 - 5+ CD8+ T cells from the indicated donors (donor 14497 or donor 14693).

Figure 69B

Figure 69C

Figure 69D

Figure 69E

Figure 69F

Figure 70

[0567] Figure 70 is a FACS plot showing the percentage of NK cells expanded from T cell cultures activated by the indicated antibody.

Figure 71

[0568] Figure 71 is a bar graph showing the number of NK cells expanded from T cell cultures activated by the indicated antibody.

Figure 72

[0569] Figure 72 shows a series of FACS plots demonstrating the expansion of NK cells induced by T cell cultures activated by the indicated antibody.

Figure 73

[0570] Figure 73 is a schematic diagram showing the assay described in the examples for determining NK cell-mediated cell lysis of target K562 cells.

Figure 74

[0571] Figure 74 is a bar graph showing the percentage of target cell lysis mediated by NK cells activated by PBMC activated by the indicated antibody.

Figure 75

[0572] Figure 75 is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5 days).

Figure 76

[0573] Figure 76 is a bar graph showing the levels of IL-2 secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5 days).

Figure 77

[0574] Figure 77 is a bar graph showing the levels of IL-15 secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5 days).

Figure 78

[0575] Figure 78 is a bar graph showing the levels of IL-1β secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5 days).

Figure 79

[0576] Figure 79 is a bar graph showing the levels of IL-6 secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5 days).

Figure 80

[0577] Figure 80 is a bar graph showing the levels of IL-10 secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34) and cultured with the antibody for the indicated number of days (1, 3, or 5 days).

Figure 81

[0578] Figure 81 is a bar graph showing the levels of the indicated cytokines secreted by T cells activated / expanded by the indicated antibodies (anti-TCRβV 6-5 v1 or SP34). Data include the use of 17 individual PBMC donors.

Figure 82A

[0579] Figure 82A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibodies (anti-TCRβV 6-5 v1 or OKT3) and cultured with the indicated antibodies for the indicated number of days (1, 2, 3, 5, or 6 days).

Figure 82B

Figure 82C

Figure 82D

Figure 82E

Figure 82F

Figure 82G

Figure 83A

[0580] Figure 83A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, SP34-2, or isotype control) and cultured with the antibody for the indicated number of days (1, 2, 3, 5, or 6 days).

Figure 83B

Figure 83C

Figure 83D

Figure 83E

Figure 83F

Figure 83G

Figure 84A

[0581] Figure 84A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the indicated antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8 days).

Figure 84B

Figure 84C

Figure 84D

Figure 84E

Figure 84F

Figure 84G

Figure 85A

[0582] Figure 85A is a bar graph showing the levels of IL-17A secreted by T cells activated / expanded by the indicated antibody (anti-TCRβV 6-5 v1, OKT3, or SP34-2) and cultured with the antibody for the indicated number of days (2, 5, or 7 days).

Figure 85B

Figure 85C

Figure 85D

Figure 86A

[0583] Figure 86A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibody (isotype control, anti-TCRβV 6-5 v1 and anti-BCMA antibody, anti-TCRβV 6-5 v1, anti-TCRβV 123 / 4 v1, or SP34-2) and cultured with the antibody for the indicated number of days (1, 2, 3, 4, 5, 6, or 8 days).

Figure 86B

Figure 86C

Figure 86D

Figure 86E

Figure 86F

Figure 86G

Figure 86H

Figure 86I

Figure 86J

Figure 86K

Figure 86L

Figure 86M

Figure 86N

Figure 86O

Figure 86P

Figure 86Q

Figure 86R

Figure 86S

Figure 86T

Figure 86U

Figure 86V

Figure 86W

Figure 86X

Figure 86Y

Figure 86Z

Figure 86AA

Figure 86BB

Figure 86CC

Figure 86DD

Figure 87A

[0584] Figure 87A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibodies (anti-TCRβV 6-5 v1 (plate-coated), anti-CD3ε (plate-coated), anti-TCRβV 6-5 v1 (in solution), or anti-CD3ε (in solution)) and cultured with the indicated antibodies for the indicated number of days (1, 3, 5, or 7 days).

Figure 87B

Figure 87C

Figure 87D

Figure 87E

Figure 87F

Figure 87G

Figure 87H

Figure 87I

Figure 87J

Figure 87K

Figure 87L

Figure 88

[0585] Figure 88 shows a schematic representation of the sequence relationships among different TCRVB clonotype subfamilies.

Figure 89A

[0586] Figure 89A is a bar graph showing the percentage of cytokine release from PBMCs activated / expanded for 8 days using the indicated antibodies (anti-TCRβV 12-3 / 4 v1 or SP34-2).

Figure 89B

Figure 89C

Figure 90

[0587] Figure 90 shows a series of FACS plots showing the proliferation of NK cells from PBMC cultures activated / expanded by the indicated antibody (isotype control or OKT3). PBMC from three donors (D1, D2, and D3) were analyzed.

Figure 91

[0588] Figure 91 shows a series of FACS plots showing the proliferation of NK cells from PBMC cultures activated / expanded by the indicated antibody (anti-TCRvβ 12-3 / 4 v1 or anti-TCRvβ 12-3 / 4 v2). PBMC from three donors (D1, D2, and D3) were analyzed.

Figure 92

[0589] Figure 92 shows a series of FACS plots showing the proliferation of NK cells from PBMC cultures activated / expanded by the indicated antibody (anti-TCRvβ 12-3 / 4 v3 or SP34-2). PBMC from three donors (D1, D2, and D3) were analyzed.

Figure 93A

[0590] Figure 93A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibody for the indicated number of days (3 or 6 days).

Figure 93B

Figure 93C

Figure 93D

Figure 93E

Figure 93F

Figure 93G

Figure 93H

Figure 94

[0591] Figure 94 is a bar graph summarizing data from FACS analysis of PBMCs activated / expanded for 6 days using the indicated anti-TCRVβ antibodies.

Figure 95A

[0592] Figure 95A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibodies for the indicated number of days (1, 3, 5, or 7 days).

Figure 95B

Figure 95C

Figure 95D

Figure 95E

Figure 95F

Figure 95G

Figure 95H

Figure 96

[0593] Figure 96 is a bar graph summarizing data from FACS analysis of PBMCs activated / expanded for 7 days using the indicated anti-TCRVβ antibodies.

Figure 97A

[0594] Figure 97A is a bar graph showing the levels of IFNγ secreted by T cells activated / expanded by the indicated antibodies over the indicated number of days (3 or 6).

Figure 97B

Figure 97C

Figure 97D

Figure 97E

Figure 97F

Figure 97G

Figure 97H

Figure 97I

Figure 98

[0595] Figure 98 is a FACS plot showing the ability of MH3-2 to bind to PBMC from one of two donors when the PBMC were pre-incubated with TM23 or not (MH3-2 alone).

Figure 99

[0596] Figure 99 is a FACS plot showing the ability of MH3-2 to bind to PBMC from one of two donors when the PBMC were pre-incubated with TM23 or not (MH3-2 alone).

Figure 100A

[0597] Figure 100A is a bar graph showing the multifunctional intensity index (PSI) of CD4+ T cells in PBMC, CD4+ T cells expanded by anti-CD3 antibody (CD3-expanded T cells), and CD4+ T cells expanded by anti-TCRVβ 6-5 antibody (drug-expanded T cells). Effector mediators are granzyme B, IFNγ, MIP-1α, perforin, TNFα, and TNFβ. The stimulatory mediator is IL-5. The chemoattractant mediator is MIP-1b.

Figure 100B

Figure 101A

[0598] Figure 101A is a line graph showing the cell numbers of CAR T cells produced from the PBMC of donor 177 and cultured with the indicated antibodies and medium (or virus-free control) on days 0, 7, 9, and 11.

Figure 101B

Figure 101C

Figure 102

[0599] Figure 102 is a schematic diagram of the protocol for flow cytometry staining of CAR-T cells on day 11.

Figure 103

[0600] Figure 103 is a bar graph showing the frequency of CAR-T cells on day 11 of CAR T cells cultured with the indicated antibodies and medium (or virus-free control).

Figure 104A

[0601] Figure 104A is a bar graph showing the percentages of CAR-T cells of Teff, Tem, Tcm, and Tn phenotypes based on CD45RO-APC and CD62L-FITC staining of CAR-T cells produced from the PBMC of donor 177 on day 11.

Figure 104B

Figure 104C

Figure 105A

[0602] Figure 105A shows the cytotoxicity of CAR-T cells prepared by activation with the indicated antibodies and medium from PBMCs of donor 177.

Figure 105B

Figure 105C

Figure 105D

Figure 105E

Figure 106

[0603] Figure 106 is a bar graph showing the production of IFNγ by CAR-T cells activated with the indicated antibodies and used in the cytotoxicity assay.

Figure 107

[0604] Figure 107 shows the sequence alignment of eight functional human TCRVβ6 family sequences. The box indicates three unique amino acids in subfamily 6-5.

Figure 108A

[0605] FIG. 108A is a line graph showing the binding of the H131 antibody to the WT TCR receptor.

Figure 108B

Figure 108C

Figure 108D

[0390]

[0606] Current methods for expanding T cells ex vivo involve contacting the T cells with an antibody molecule that specifically binds to the CD3ε subunit of the T cell receptor (TCR) alone or in combination with targeting to the costimulatory receptor CD28. However, this approach has limitations that can impede the full realization of the therapeutic potential of such T cell therapies. Previous studies have shown that even low "activation" doses of anti-CD3ε monoclonal antibodies (mAbs) can cause long-term T cell dysfunction and have an immunosuppressive effect. Furthermore, administration of T cells activated / expanded by anti-CD3ε antibodies has been associated with inflammatory side effects including cytokine release syndrome (CRS), macrophage activation syndrome, neurotoxicity, and tumor lysis syndrome. T cells activated by anti-CD3ε antibodies secrete pro-inflammatory cytokines such as IFNγ, IL-1, IL-6, and TNF-α, or activate antigen-presenting cells such as macrophages that secrete pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α that induce cytokine release syndrome (CRS), macrophage activation syndrome, neurotoxicity, or tumor lysis syndrome (e.g., IFNγ). Thus, there is a need for the development of antibodies that can bind to and activate only a subset of effector T cells, for example, to reduce CRS.

[0391]

[0607] The present disclosure is at least partially based on the unexpected discovery that anti-TCRVβ antibodies can be used to activate and expand T cells ex vivo, and that these T cells secrete substantially low levels of inflammatory cytokines such as IFNγ, IL-10, IL-17A, IL-1α, IL-1β, IL-2, IL-6, and TNFα, etc. associated with cytokine release syndrome (CRS), macrophage activation syndrome, neurotoxicity, and tumor lysis syndrome, while also secreting high or similar levels of IL-2. The present disclosure provides, inter alia, methods of using antibodies directed to the use of anti-TCRVβ antibodies for expanding T cells ex vivo. By using the anti-TCRβV antibody molecules disclosed herein, cytokines associated with CRS, such as IL-6, IL-1 beta, and TNF alpha, are produced less or not at all, and the production of IL-2 and IFNγ is enhanced and / or delayed. In some embodiments, the anti-TCRβV antibodies disclosed herein result in the expansion of a subset of memory effector T cells known as EMRA T. In some embodiments, the expanded cells are injected into a subject for the treatment of a disease (e.g., cancer). In some embodiments, compositions comprising the anti-TCRβV antibody molecules of the present disclosure can be used to expand T cells (e.g., CAR-T cells) ex vivo to promote tumor cell lysis for cancer immunotherapy. In some embodiments, a method of expanding T cells ex vivo comprising contacting the T cells with an anti-TCRβV antibody molecule as disclosed herein limits the adverse side effects of CRS, such as CRS associated with anti-CD3e targets and / or CD28 targets.

[0392] Incorporation by reference

[0608] All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0393] Definitions

[0609] 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.

[0394]

[0610] Range: Throughout this disclosure, various aspects may be presented in a range format. The description in range format is for convenience and brevity only and should not be construed as an immovable limitation on the range. Accordingly, the description of a range should be considered to specifically disclose all the possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1 - 6 should be considered to specifically disclose sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, etc. As another example, a range such as 95 - 99% identity includes those having 95%, 96%, 97%, 98%, or 99% identity and includes sub-ranges such as 96 - 99%, 96 - 98%, 96 - 97%, 97 - 99%, 97 - 98%, and 98 - 99% identity. This applies regardless of the width of the range.

[0395]

[0611] The terms "a" and "an" refer to one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0396]

[0612] As used herein, the terms "T cell receptor beta variable chain", "TCRβV", "TCRβ V", "TCR βV", "TCRβV", "TCR βv", "TCRβ v", "T cell receptor variable beta chain", "TCRβV", "TCR Vβ", "TCRV β", "TCRβV", "TCRv β", or "TCR vβ" are used interchangeably herein and refer to the extracellular region of the T cell receptor beta chain that includes the antigen recognition domain of the T cell receptor. The term TCRβV includes isoforms, mammals such as human TCRβV, human species homologs and analogs that include at least one common epitope with TCRβV. Human TCRβV includes, but is not limited to, gene families that include subfamilies such as TCRβ V6 subfamily, TCRβ V10 subfamily, TCRβ V12 subfamily, TCRβ V5 subfamily, TCRβ V7 subfamily, TCRβ V11 subfamily, TCRβ V14 subfamily, TCRβ V16 subfamily, TCRβ V18 subfamily, TCRβ V9 subfamily, TCRβ V13 subfamily, TCRβ V4 subfamily, TCRβ V3 subfamily, TCRβ V2 subfamily, TCRβ V15 subfamily, TCRβ V30 subfamily, TCRβ V19 subfamily, TCRβ V27 subfamily, TCRβ V28 subfamily, TCRβ V24 subfamily, TCRβ V20 subfamily, TCRβ V25 subfamily, or TCRβ V29 subfamily. In some embodiments, the TCRβ V6 subfamily includes TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01, or TCRβ V6-1*01. In some embodiments, TCRβV includes TCRβ V6-5*01. TCRβ V6-5*01 is also known as TRBV65, TCRβV 6S5, TCRβV 13S1, or TCRβV 13.1.The amino acid sequence of TCRβ V6-5*01, for example human TCRβ V6-5*01, is known in the art as provided, for example, by IMGT ID L36092.

[0397]

[0613] As used herein, the term "molecule" includes full-length, naturally occurring molecules as well as variants, such as functional variants (e.g., truncated, fragmented, mutated (e.g., substantially similar sequences) or their derived forms), as long as at least one function and / or activity of the unmodified (e.g., full-length, naturally occurring) molecule remains.

[0398]

[0614] The terms "antibody" and "antibody molecule" are used interchangeably herein and refer to a protein comprising at least one immunoglobulin variable domain sequence. The term "antibody" encompasses full-length antibodies, antibody fragments (e.g., functional fragments thereof), and variants (e.g., functional variants thereof). Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural sources or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. In one embodiment, an antibody molecule comprises an antigen-binding fragment or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that exists naturally or is formed by a recombinant process of normal immunoglobulin gene fragments. In embodiments, an antibody molecule refers to the antigen-binding portion of an immunologically active immunoglobulin molecule such as an antibody fragment. The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen-binding domain sufficient to allow the antibody fragment to recognize and specifically bind a target such as an antigen, e.g., the antigenic determinant variable region of the intact antibody. Antibody fragments, e.g., functional fragments, are portions of an antibody, e.g., Fab, Fab’, F(ab’)2, F(ab)2, variable fragment (Fv), domain antibody (dAb), or single-chain variable fragment (scFv). Functional antibody fragments bind to the same antigen recognized by the intact (e.g., full-length) antibody. The term "antibody fragment" or "functional fragment" also includes an isolated fragment consisting of the variable regions, e.g., an "Fv" fragment consisting of the variable regions of the heavy and light chains or a recombinant single-chain polypeptide molecule in which the variable regions of the light and heavy chains are linked by a peptide linker ("scFv protein"). In some embodiments, an antibody fragment does not include portions of an antibody that do not have antigen-binding activity, such as an Fc fragment or a single amino acid residue. Exemplary antibody molecules include full-length antibodies and antibody fragments, e.g., dAb (domain antibody), single-chain, Fab, Fab’, and F(ab’)2 fragments, and single-chain variable fragments (scFv). Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments, scFv antibody fragments, linear antibodies, sdAb (V L or VH ) single domain antibodies such as, camelid V H H domains, and bispecific antibodies formed from antibody fragments such as bivalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region, and isolated CDRs or other antibody epitope-binding fragments, but are not limited thereto. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, tribodies, tetrabodies, v-NAR, and bis-scFv. Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as type III fibronectin (Fn3) (see, for example, U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies and is incorporated herein by reference). Antigen-binding domains may include nanobodies. In some embodiments, the antigen-binding domain may be a non-antibody target domain. In some embodiments, the antigen-binding domain may be a nanobody.

[0399]

[0615] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing the variable region of the light chain and at least one antibody fragment containing the variable region of the heavy chain, wherein the variable regions of the light and heavy chains are adjacent and linked via a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the original antibody from which it was derived. Unless otherwise specified, as used herein, scFv may have the variable regions V L and V H in either order with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may comprise V L -linker-V H and may also comprise V H -linker-V L .

[0400]

[0616] The term "complementary determining region" or "CDR" is used interchangeably herein and refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region has three CDRs (e.g., HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (e.g., LCDR1, LCDR2, and LCDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of several known schemes, including those described by Kabat et al., (1991), "Sequences of Proteins of Immunological Interest," 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (the "Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (the "Chothia" numbering scheme), or a combination thereof. In the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (V H ) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in the light chain variable domain (V L ) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). In the Chothia numbering scheme, in some embodiments, the CDR amino acids in V H are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues in V L are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDR corresponds to amino acid residues that are part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDR is V H , e.g., mammalian V H, for example, human V H amino acid residues 26 - 35 (HCDR1), 50 - 65 (HCDR2), and 95 - 102 (HCDR3), and V L , for example, mammalian V L , for example, human V L corresponds to amino acid residues 24 - 34 (LCDR1), 50 - 56 (LCDR2), and 89 - 97 (LCDR3).

[0401]

[0617] The "humanized" form of a non - human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab’, F(ab’)2, or other antigen - binding subsequence of an antibody) that contains a minimal sequence derived from a non - human immunoglobulin. Humanized antibodies and antibody fragments are mostly human immunoglobulins (recipient antibody or antibody fragment) in which the residues of the recipient's complementarity - determining regions (CDRs) are replaced by the residues of the CDRs (donor antibody) of a non - human species such as mouse, rat, or rabbit that have the desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non - human residues. Furthermore, the humanized antibody / antibody fragment may contain residues not found in either the CDR or the framework sequences introduced into the recipient antibody. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, a humanized antibody or its antibody fragment will comprise substantially all of at least one, typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non - human immunoglobulin and all or a substantial portion of the FR regions are those of a human immunoglobulin sequence. A humanized antibody or antibody fragment may include the constant region (Fc) of an immunoglobulin, typically at least a portion of the constant region of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522 - 525, 1986; Reichmann et al., Nature, 332: 323 - 329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593 - 596, 1992.

[0402]

[0618] "Fully human" refers to an immunoglobulin such as an antibody or antibody fragment whose entire molecule is of human origin or consists of the same amino acid sequence as a human - shaped antibody or immunoglobulin.

[0403]

[0619] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a relevant binding partner present in a sample (e.g., a stimulatory and / or co - stimulatory molecule present on a T cell), but does not substantially recognize or bind to other molecules in the sample.

[0404]

[0620] As used herein, "immune cell" refers to any of a variety of cells that function in the immune system to protect against, for example, infectious agents and foreign substances. In embodiments, the term includes white blood cells such as neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Native white blood cells include phagocytic cells (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Native white blood cells attack larger pathogens by contact or identify and eliminate pathogens by engulfing and killing microorganisms. White blood cells are mediators of the activation of the adaptive immune response. The cells of the adaptive immune response are a special type of white blood cell called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response and T cells are involved in the cell - mediated immune response. The term "immune cell" includes immune effector cells.

[0405]

[0621] As used herein, the term "immune effector cell" refers to a cell involved in promoting an immune response, such as an immune effector response. Examples of immune effector cells include, but are not limited to, T cells (e.g., alpha / beta T cells, gamma / delta T cells, CD4+ T cells, CD8+ T cells), B cells, natural killer (NK) cells, natural killer T (NK T) cells, monocytes, macrophages, neutrophils, basophils, dendritic cells, and mast cells.

[0406]

[0622] The term "effector function" or "effector response" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity (e.g., CD8+ T cells) or helper activity (e.g., CD4+ T cells) including cytokine secretion.

[0407]

[0623] The term "antigen-presenting cell" or "APC" refers to an immune system cell, such as an accessory cell (e.g., B cells, dendritic cells, etc.) that presents a foreign antigen complexed with a major histocompatibility complex (MHC) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0408]

[0624] The term "substantially purified cell" or "substantially purified cell population" refers to a cell or cell population that is substantially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types that normally accompany it in its natural state. In some cases, a substantially purified cell population refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that has been separated from the cells that naturally accompany it in its natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0409]

[0625] "Derived from" when used in this specification, indicates the relationship between a first and a second molecule. This generally refers to a structural similarity between the first and second molecules and does not imply or include a limitation on the process or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure for it to have the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation on the specific process for generating the intracellular signaling domain. For example, this does not mean that unwanted sequences have to be removed or mutations imposed starting from the CD3 zeta sequence to arrive at the intracellular signaling domain.

[0410]

[0626] The term "encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in a biological process having a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA encodes a protein if that protein is produced intracellularly or within another biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is usually provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0411]

[0627] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may include introns in some versions.

[0412]

[0628] As used herein, the term "isolated" refers to a material that has been removed from its original or natural environment (e.g., the natural environment in which it naturally occurs). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated by human intervention from some or all of the materials coexisting in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotid...

Claims

**Claim 1** A method for expanding and activating T cells expressing a T cell receptor (TCR) comprising a T cell receptor beta variable (TCRβV) region in an ex vivo T cell population, comprising: contacting the T cells of the T cell population with a first substance comprising a first domain comprising an antibody domain or antigen-binding domain that binds to the TCRβV region of the TCR expressed by the T cells of the T cell population, and culturing the T cells in the absence of an anti-CD28 antibody, wherein the T cells expressing a TCR comprising the TCRβV region of the T cell population expand and activate when contacted with the first substance, the T cell population is an ex vivo T cell population; the T cells of the expanded and activated T cell population are: (i) compared to an expanded and activated T cell population obtained by a method of expanding and activating a T cell population with an anti-CD3 agent, showing a more activated signature based on principal component analysis of CD27, CD28, CD96, CD40LG, ICOS, TNFRSF9 (4-1BB), CD276, CSF2 (GM-CSF), CD80, CD86, CCL3, CCL4, CXCR3, CXCL9, CXCL10, or a combination thereof, or (ii) compared to an expanded T cell population obtained by a method of expanding and activating a T cell population with an anti-CD3 agent, showing a lower signature based on principal component analysis of PD-1 (PDCD1), LAG3, Tim-3 (HAVCR2), CTLA4, BTLA, CD244 (2B4), CD160, CD39 (ENTPD1), TIGIT, or a combination thereof, and the T cells of the expanded and activated T cell population are cytotoxic, said method. **Claim 2** The method of claim 1, further comprising culturing the T cell population in the presence of the first substance for at least 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. **Claim 3** The method of claim 1 or 2, wherein the T cells of the T cell population express a chimeric antigen receptor (CAR) or a recombinant T cell receptor. **Claim 4** The method according to any one of claims 1 to 3, wherein the first domain binds to the TCRβV region of TCRβV belonging to the TCRβV6 subfamily, TCRβV12 subfamily, TCRβV5 subfamily, or TCRβV20 subfamily.

5. The method according to any one of claims 1 to 4, wherein the first domain binds to the TCRβV region of TCRβV6-5, TCRβV20-1, or TCRβV5-1. 1 item as described.

6. The method according to any one of claims 1 to 3, wherein TCRβV is TCRβV2, TCRβV3, TCRβV4, TCRβV5, TCRβV6, TCRβV7, TCRβV8, TCRβV9, TCRβV10, TCRβV11, TCRβV12, TCRβV19, TCRβV20, TCRβV24, TCRβV25, TCRβV27, TCRβV28, TCRβV29, or TCRβV30.

7. The method according to any one of claims 1 to 3 and 6, wherein TCRβV is TCRβV2, TCRβV4-1, TCRβV4-2, TCRβV5-1, TCRβV5-5, TCRβV5-6, TCRβV6, TCRβ6-5, TCRβV6-6, TCRβV6-9, TCRβV7-2, TCRβV7-3, TCRβV7-8, TCRβV7-9, TCRβV9, TCRβV10-1, TCRβV10-2, TCRβV10-3, TCRβV11-2, TCRβV12-3, TCRβV12-4, TCRβV12-5, TCRβV19, TCRβV20-1, TCRβV24-1, TCRβV25-1, or TCRβV28.

8. The method according to any one of claims 1 to 3 and 6, wherein TCRβV is TCRβV2, TCRβV3-1, TCRβV4-1, TCRβV4-2, TCRβV5-1, TCRβV5-4, TCRβV5-5, TCRβV5-6, TCRβV6-1, TCRβV6-5, TCRβV6-6, TCRβV7-3, TCRβV7-6, TCRβV7-8, TCRβV9, TCRβV11-2, TCRβV19, TCRβV20-1, TCRβV24-1, TCRβV27, TCRβV28, TCRβV29-1, or TCRβV30.

9. The method according to any one of claims 1 to 8, wherein the antigen-binding domain comprises a single-chain Fv (scFv) or Fab.

10. The method according to any one of claims 1 to 9, further comprising the step of contacting the T cell population with a second substance, wherein the second substance comprises a domain that binds to the TCRβV region, and the first and second substances bind to TCRβVs belonging to different TCRβV subfamilies, or TCRβV regions that are different members of the same TCRβV subfamily.

11. The method according to any one of claims 1 to 10, wherein the T cell population is derived from blood, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, or a tumor of a human subject.

12. The first substance further comprises a second domain that binds to a protein expressed on the surface of T cells in the T cell population The method according to any one of claims 1 to 11.

13. The method according to claim 12, wherein the second domain binds to the T cell receptor beta chain variable (TCRβV) region.

14. The method according to claim 13, wherein the second domain and the first domain bind to TCRβVs belonging to different subfamilies, or different members of the same TCRβV subfamily.

15. The method according to claim 12, wherein the second domain binds to CD19, 4-1BB, or an antibody molecule.

16. The second domain binds to an antibody molecule, (i) the second domain binds to an antibody molecule expressed by T cells; (ii) the second domain binds to an antibody molecule comprising a variable heavy chain and a variable light chain; (iii) the second domain binds to an antibody molecule that is scFv or Fab; (iv) the second domain binds to the light chain of an antibody molecule; (v) the second domain binds to the κ light chain region of an antibody molecule, or (vi) the second domain comprises protein L, the method according to claim 15.

17. The method according to any one of claims 1 to 16, wherein the first substance is coupled to a solid surface.

18. The method according to claim 17, wherein the solid surface is beads or a cell culture plate.

19. A pharmaceutical composition for use in a method of treating cancer in a patient in need of treatment for cancer, comprising at least a portion of an ex vivo T cell population produced by the method according to any one of claims 1 to 18, The pharmaceutical composition, which is administered to the subject in an effective amount for treating cancer in the subject.

20. (i) the cancer is a solid cancer or a blood cancer; (ii) the cancer is a solid cancer selected from the group consisting of prostate cancer, lung cancer, kidney 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, and brain cancer; (iii) the cancer is a blood cancer selected from the group consisting of leukemia, lymphoma, and myeloma; or (iv) the cancer is a blood cancer selected from the group consisting of B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic 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 lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell follicular lymphoma, large cell follicular lymphoma, malignant lymphoproliferative state, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, and preleukemia The pharmaceutical composition according to claim 19.

21. A pharmaceutical composition comprising at least a part of an ex vivo T cell population produced by the method according to any one of claims 1 to 18 for use in a method for preventing or reducing cytokine release syndrome (CRS) in a human subject in need thereof, wherein the pharmaceutical composition is administered to the subject in an effective amount to prevent or reduce CRS in the subject; and after administration, the subject does not exhibit symptoms of cytokine release syndrome, or at least one symptom of CRS is less severe compared to a human subject in which at least a part of an equivalent T cell population generated by expanding and activating T cells by contacting a plurality of T cells with a substance that binds to CD3ε is administered, and CRS is prevented or reduced in the subject.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the T cells are autologous or allogeneic to the subject.

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