Compositions and methods related to genetically engineered γδ-T cells and non-genetically engineered γδ-T cells for treating hematological malignancies

Abstract

Aspects of the present invention include compositions and methods for treating hematological tumors using genetically engineered or non-genetically engineered γδ T cells. In some embodiments, the γδ T cells comprise a chimeric antigen receptor (CAR) construct. The CAR construct can include an anti-CD20 binding domain or an anti-B-cell maturation antigen (BCMA) binding domain, a CD8 hinge and transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, a combination thereof, or all of these. The CAR construct can also include a domain encoding a secreted common gamma chain cytokine, such as a sIL15 domain. [Selection diagram] None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 739,822, filed Oct. 1, 2018, the content of which is incorporated herein in its entirety for all purposes.

[0002] Sequence Listing This application includes a sequence listing that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above - mentioned ASCII copy, created on Dec. 17, 2019, is named ADC - 0005 - PCT_SL.txt and is 147,616 bytes in size.

Background Art

[0003] Adoptive immunotherapy has been continuously repeated for more than 30 years, from the early days focusing on basic lymphokine activation and / or tumor infiltration to the most recent strategies of genetically engineering these immune cells to express genetically engineered antigen receptors such as chimeric antigen receptors (CARs). Among them, some hints and signs have been seen regarding the curative potential of these approaches, but there are still many things to be done. In particular, the success of tumor eradication by CAR - T lymphocytes depends on the persistence and effector function of CAR - T cells, but if either of them becomes excessive, the patient's graft - versus - host effect may be induced. Therefore, in the art, numerous co - stimulation strategies are being tested for both T cells and NK cells, especially αβ T cells, with the aim of balancing efficacy and safety. Notably, considering the current lack of understanding of the co - stimulation requirements of γδ T cells compared to αβ T cells, any practical translation of these various approaches to allogeneic γδ T cells is at best uncertain. See, for example, Ribot et al., “Searching for ‘signal 2’: costimulation requirements of γδ T cells”, Cell. Mol. Life Sci. (2011) 68:2345 - 2355.

[0004] Thus, in order to improve cell specificity or selectivity, for example, to improve cell safety by reducing or avoiding the graft-versus-host (GVH) effect, in order to improve cell effectiveness by, for example, avoiding suppression of effector function, and to improve cell activity and / or survival upon administration to a subject, there is still a need to improve the strategy. Provided are methods, cells, compositions, kits and systems that meet such needs. SUMMARY OF THE INVENTION

[0005] Aspects of the invention include an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of hematopoietic tumor cells, the binding domain being, for example, the hinge domain of CD8α, for example, the transmembrane domain of CD8α, a co-stimulatory signaling region, etc., wherein the co-stimulatory signaling region is optionally selected from a 4-1BB (CD137) co-stimulatory signaling region, a CD27 co-stimulatory signaling region, and a CD3ζ signaling domain.

[0006] Aspects of the invention further include γδ T cells that have not been genetically engineered as described herein, and γδ T cells that contain a nucleic acid encoding a CAR construct as described herein, wherein the γδ T cells functionally express the nucleic acid encoding the CAR on the surface of the γδ T cells. Aspects of the invention further include a plurality of genetically engineered or non-genetically engineered γδ T cells as described herein. Aspects of the invention further include a method of making the γδ T cells or plurality of γδ T cells described herein, wherein the method includes transfecting the γδ T cell(s) with the construct described herein. Aspects of the invention further include a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the γδ T cell or plurality of γδ T cells as described herein. Aspects of the invention further include contacting the γδ T cells, in an effective amount for tumor cell killing, with hematologic tumor cells as described herein.

[0007] In one aspect, the invention provides an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of hematologic tumor cells; (b) a hinge domain such as a CD8α hinge domain; (c) a transmembrane domain such as a CD8α transmembrane domain; (d) a co-stimulatory signaling region or combination of co-stimulatory signaling regions, optionally wherein the co-stimulatory signaling region is a co-stimulatory signaling region selected from a 4-1BB (CD137) co-stimulatory signaling region and a CD27 co-stimulatory signaling region; and (e) a signaling domain such as a CD3ζ signaling domain. In some embodiments, the foregoing elements (a)-(e) are encoded in the 5' to 3' order on the sense strand of the isolated nucleic acid.

[0008] In some embodiments, the binding domain specifically binds to CD20. In some embodiments, the binding domain selectively binds to an epitope in CD20 to which an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, and 9C11, preferably 3H7, binds, or competes to bind with that anti-CD20 antibody. In some embodiments, the binding domain comprises the complementarity-determining regions of an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, and 9C11, preferably 3H7.

[0009] In some embodiments, the isolated nucleic acid encodes a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence. For example, wherein the HCVR sequence and the LCVR sequence are SEQ ID NO: 99 and 107, respectively, the heavy chain complementarity determining region 1, 2, and 3 sequences of SEQ ID NO: 101, 103, and 105, respectively, the light chain complementarity determining region 1, 2, and 3 sequences of SEQ ID NO: 109, 111, and 113, respectively, the heavy chain complementarity determining region 3 (HCDR3) and the light chain CDR3 (LCDR3), wherein the HCDR3 and the LCDR3 are HCDR3 and LCDR3 selected from the group consisting of SEQ ID NO: 345 and 353, 201 and 209, and 249 and 257, the heavy chain variable region (HCVR) sequence and the light chain variable region (LCVR) sequence, wherein the HCVR sequence and the LCVR sequence are HCVR sequences and LCVR sequences selected from the group consisting of SEQ ID NO: 339 and 347, 195 and 203, and 243 and 251, and / or the heavy chain complementarity determining region 3 (HCDR3) domain and the light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain comprises the amino acid sequence of the formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19, wherein X1 = A, V or T, X2 = K, X3 = D, X4 = P, F or G, X5 = S or H, X6 = Y, X7 = G, X8 = S or H, X9 = G or F, X10 = S or Y, X11 = Y, N or S, X12 = Y, G or H, X13 = G, L or S, X14 = Y, M or D, X15 = Y, D or V, X16 = G, V or absent, X17 = M or absent, X18 = D or absent, X19 = V or absent (SEQ ID NO: 369), and the LCDR3 domain comprises the amino acid sequence of X1-X2-X3-X4-X5-X6-X7-X8-X9, wherein X1 = Q, X2 = Q, X3 = R or S, X4 = N, Y or F, X5 = N, D, or Y, X6 = W, X7 = P, X8 = L, X9 = T (SEQ ID NO: 370).

[0010] In some embodiments, the isolated nucleic acid encodes a binding domain that specifically binds to CD19 or BCMA. In some embodiments, the binding domain specifically binds to BCMA. In some embodiments, the binding domain selectively binds to an epitope of BCMA that binds to an anti-BCMA binding region having a sequence selected from the group consisting of SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, and SEQ ID NOs: 31 and 32, or competes to bind to its anti-BCMA binding region. In some embodiments, the binding domain comprises a complementarity-determining region of an anti-BCMA binding region having a sequence selected from the group consisting of SEQ ID NOs: 27 and 28, SEQ ID NOs: 29 and 30, and SEQ ID NOs: 31 and 32.

[0011] In any one of the foregoing or in some embodiments as described herein, the CAR comprises a CD8α hinge domain comprising SEQ ID NO: 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). In any one of the foregoing or in some embodiments as described herein, the CAR comprises a CD8α transmembrane domain comprising SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC).

[0012] In any one of the foregoing or in some embodiments as described herein, the CAR comprises a CD3ζ signaling domain comprising SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0013] In any one of the foregoing or in some embodiments as described herein, the CAR comprises a 4-1BB co-stimulatory signaling region comprising SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL) or a CD27 co-stimulatory signaling region comprising SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP). In any one of the foregoing or in some embodiments as described herein, the isolated nucleic acid encodes a 4-1BB co-stimulatory signaling region comprising SEQ ID NO: 6 and a CD27 co-stimulatory signaling region comprising SEQ ID NO: 7.

[0014] In any one of the foregoing or in some embodiments as described herein, the isolated nucleic acid encodes a secreted cytokine, or a secreted common gamma chain interleukin, or a secreted common gamma chain interleukin such as IL-15, and preferably, this secreted common gamma chain interleukin such as IL-15 comprises an interleukin polypeptide sequence operably linked to a secretion signal sequence (e.g., the secretion signal of SEQ ID NO: 33 or 49). In some embodiments, the isolated nucleic acid encodes secreted IL-15, and preferably, wherein IL-15 comprises the sequence of SEQ ID NO: 34, and more preferably, wherein IL-15 comprises the sequence of 34 operably linked to the secretion signal sequence of SEQ ID NO: 33, or wherein IL-15 comprises the sequence of SEQ ID NO: 34 operably linked to the secretion signal sequence of SEQ ID NO: 49. Optionally, the secreted cytokine, common gamma chain interleukin and / or IL-15 has its carboxy terminus encoded in a binding region, hinge and transmembrane domain, signaling domain and / or co-stimulatory end domain. Optionally, the secreted cytokine, common gamma chain interleukin and / or IL-15 is encoded 3' of the sense strand of a binding region, hinge and transmembrane domain, signaling domain and / or co-stimulatory end domain.

[0015] In some embodiments, the nucleic acid encodes a multi-cistronic linker region configured to facilitate translation of CAR and a secreted cytokine, a common gamma chain cytokine, or IL-15 as a separate polypeptide. In some embodiments, the multi-cistronic linker region encodes a self-cleaving sequence and / or a cleavage polypeptide sequence. Optionally, the self-cleaving sequence is a P2A, F2A, T2A, or E2A self-cleaving sequence. Optionally, the cleavage sequence is a furin cleavage sequence. Optionally, the cleavage sequence (e.g., the furin cleavage sequence) is at the amino terminus of the self-cleaving sequence. In some embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. In some embodiments, the nucleic acid encodes an interleukin or cytokine or an interleukin or cytokine secretion signal at the amino terminus of the multi-cistronic linker region, and preferably, wherein the multi-cistronic linker region comprises any one of the sequences of SEQ ID NOs: 43-45, 47 or 52-55 or combinations thereof, or encodes an internal ribosome entry site such as SEQ ID NO: 56 or 60.

[0016] In some embodiments, the binding domain specifically binds to CD20 and the nucleic acid encodes SEQ ID NOs: 8, 9, 10, 11, 12, 46, 48, or 57 and 58. In some embodiments, the nucleic acid comprises the sequences of SEQ ID NOs: 13, 14, 15, 16, 17, 50, 51 or 59. In some embodiments, the binding domain specifically binds to BCMA and the nucleic acid encodes SEQ ID NOs: 35, 36, 37 or 38. In some embodiments, the nucleic acid comprises the sequences of SEQ ID NOs: 39, 40, 41 or 42.

[0017] In some embodiments, the present invention provides any one of the aforementioned isolated nucleic acids, or a polypeptide or polypeptides encoded as described herein. In some embodiments, the present invention provides T cells, such as γδ T cells, comprising any one of the aforementioned polypeptides or polypeptides. In some embodiments, the T cells express a functional binding domain on the surface of the T cells as described herein. In some embodiments, the T cells secrete cytokines such as common gamma chain interleukins such as IL-15.

[0018] In some embodiments, the T cells exhibit in vitro and / or in vivo cytotoxic activity against hematological tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the hematological tumor cell cytotoxic activity of these, for example, γδ, T cells is greater than the native level of in vitro and / or in vivo hematological tumor cell cytotoxic activity in a control that does not contain a CAR construct such as, for example, γδ, T cells. In some embodiments, for example, γδ, T cells are HLA class I + The cytotoxic activity against hematological tumor cells is increased. In some embodiments, the cytotoxic activity against hematological tumor cells or the increase in cytotoxic activity against hematological tumor cells persists for about, at least, or at least about 6 days to 180 days after the initial contact with the hematological tumor cells.

[0019] In some embodiments, for example, γδ, T cells proliferate in response to contact with hematological tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, compared to a control such as γδ, T cells that do not functionally express a nucleic acid encoding a CAR on the surface, for example, γδ, T cells have increased proliferation in response to contact with hematological tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, for example, γδ, T cells proliferate in a host organism comprising hematological tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA).

[0020] In some embodiments, the cell proliferation of γδ, T cells, etc., or the increase in the cell proliferation of γδ, T cells persists for about, at least, or at least about 6 days to 180 days after the initial contact with hematological tumor cells. In some embodiments, for example, γδ, T cells express pro-inflammatory cytokine(s) such as tumor necrosis factor alpha and / or interferon gamma after contact with hematological tumor cells. In some embodiments, for example, γδ, T cells express pro-inflammatory cytokine(s) such as tumor necrosis factor alpha and / or interferon gamma in a greater amount than T cells that are a control that do not functionally express a nucleic acid encoding a CAR on the cell surface after contact with hematological tumor cells.

[0021] In some embodiments, compared to the graft-versus-host response exhibited by allogeneic host-administered αβ T cells, when an allogeneic host is introduced, for example, the graft-versus-host response exhibited by γδ, T cells is reduced, substantially reduced, substantially absent, or absent. In some embodiments, the T cell is a γ T cell. In some embodiments, the T cell is a δ T cell. In some embodiments, the T cell is a γδ T cell. In some embodiments, the T cell is a δ1, δ2, δ3, or δ4 T cell, preferably δ2 - δ T cell, more preferably a δ1δ T cell. In some embodiments, the T cell is a δ1, δ2, δ3, or δ4 γδ T cell, preferably δ2 - γδ T cell, more preferably a δ1γδ T cell.

[0022] In another aspect, the present invention provides a plurality of any one of the aforementioned γδ, T cells, etc., or a plurality of γδ, T cells, etc. as described herein. In some embodiments, the plurality is at least about 10 8 for example, γδ, T cells, preferably about 10 8 for example, γδ, T cells, about 10 11For example, it includes γδ T cells. In some embodiments, the plurality is at least 60%, 80%, or about 60% or 80% to about 90% or 95% of δ1, δ2, δ3, or δ4 γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2 - γδ T cells, and most preferably a composition comprising δ1 γδ T cells.

[0023] In some embodiments, the present invention provides a method of making, for example, γδ T cells as described herein, or a plurality of γδ T cells as described herein, wherein the method comprises transfecting T cell(s) having a construct comprising an isolated nucleic acid sequence as described herein. Optionally, the method comprises, for example, gamma retroviral transduction. Optionally, the method comprises ex vivo expansion of the T cell(s), wherein the ex vivo expansion is performed before and / or after transfection of the isolated nucleic acid sequence. Optionally, the method comprises ex vivo expansion of the T cell(s), wherein the ex vivo expansion is performed before and after transfection of the isolated nucleic acid sequence. Optionally, the method comprises ex vivo expansion of the T cell(s), wherein the ex vivo expansion is performed after transfection of the isolated nucleic acid sequence. In some embodiments, the method is capable of functionally expressing the CAR described herein within about 30 days of transfection, from about 10 8 For example, from γδ T cells, about 10 11 For example, making γδ T cells.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and γδ T cells as described herein.

[0025] In another aspect, the present invention provides a method of killing hematological tumor cells, the method comprising contacting the hematological tumor cells with a therapeutically effective amount of, for example, γδ T cells, multiple such cells, and / or a pharmaceutical composition comprising such cells as described herein. In some embodiments, the method comprises introducing a therapeutically effective amount of, for example, γδ T cells or a pharmaceutical composition into a host organism comprising hematological tumor cells. In some embodiments, the method comprises introducing into a host organism comprising hematological tumor cells a therapeutically effective amount of, for example, γδ T cells or a pharmaceutical composition, and simultaneously or sequentially, one or more methods of increasing a common gamma chain cytokine(s).

[0026] In some embodiments, one or more administration methods of increasing a common gamma chain cytokine(s) are, for example, by continuously administering an effective amount of the common gamma chain cytokine(s) simultaneously with the introduction of γδ T cells or sequentially, to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδ T cells, preferably, wherein the method comprises administering IL-2, and more preferably, wherein the method comprises administering IL-15. In some embodiments, one or more methods of increasing a common gamma chain cytokine(s) are by administering an effective amount of the common gamma chain cytokine(s) to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced T cells before and / or after introducing the T cells. In some embodiments, one or more methods of increasing a common gamma chain cytokine(s) comprise lymphodepletion prior to introducing the T cells.

[0027] In some embodiments, one or more methods of increasing one or more common gamma chain cytokines include secretion of one or more common gamma chain cytokines from introduced T cell(s). In some embodiments, this method reduces the in vivo tumor burden of a host organism and / or increases the mean survival time of the host organism as compared to a control organism, where the control organism is not treated with T cell(s) or a pharmaceutical composition. In some embodiments, this method is a method of treating cancer in a subject in need of treatment. In some embodiments, the present invention provides a pharmaceutical composition described herein for use in treating, for example, γδ, T cells, a plurality of, for example, γδ, T cell(s), or hematological tumor cells of a subject in need of treatment.

[0028] In one aspect, the present invention provides a method of treating cancer by administering a therapeutically effective amount of γδ T cells, wherein the cancer is a hematological tumor cell exhibiting cell surface expression of CD20, or provides a method of treating cancer by administering a therapeutically effective amount of γδ T cells, wherein the cancer includes hematological tumor cells exhibiting cell surface expression of BCMA. In some embodiments, this method includes administering one or more methods of increasing one or more common gamma chain cytokines simultaneously with or sequentially to the administration of γδ T cells. In some embodiments, this method includes performing multiple administrations of γδ T cells, wherein the interval between the multiple administrations is at least about 1 week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, and / or less than once every 6 months or 12 months. In some embodiments, the present invention provides a pharmaceutical composition for use in any one of the aforementioned treatment methods.

[0029] Incorporation by reference All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0030]

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Modes for Carrying Out the Invention

[0031] Definition: For the purpose of interpreting this specification, the following definitions apply. When appropriate, terms used in the singular include their plurals and vice versa. If any definition described conflicts with any document incorporated herein by reference, the definition described below shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] As used herein, the term "about" when referring to a measurable value such as an amount, a time duration, etc. is intended to encompass variations of ±20%, or ±10%, more preferably ±5%, even more preferably ±1% and still even more preferably ±0.1% from a particular value, such that the variations are appropriate for carrying out the disclosed method.

[0033] As used herein, the term "γδ T cell (gamma delta T cell)" refers to a subset of T cells that express on their surface a different T cell receptor (TCR), namely a γδ TCR, which is composed of one γ chain and one δ chain. The term "γδ T cell" includes all subsets of γδ T cells, including but not limited to, in particular, Vδ1 and Vδ2, Vδ3 γδ T cells, as well as naive, effector memory, central memory and terminally differentiated γδ T cells. As a further example, the term "γδ T cell" includes Vδ4, Vδ5, Vδ7 and Vδ8 γδ T cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10 and Vγ11 γδ T cells. In some embodiments, the γδ T cell is Vδ1 - , Vδ2 - or Vδ1 - and Vδ2 -There are compositions and methods for producing and using genetically engineered and non-genetically engineered γδ T cells and / or their subtypes, including but not limited to those described in US2016 / 0175358, WO2017 / 197347, US9499788, US2018 / 0169147, US9907820, US2018 / 0125889, and US2017 / 0196910. The content of each of these is incorporated by reference for all purposes, such as compositions and methods for producing and using these genetically engineered and non-genetically engineered γδ T cells and / or their subtypes. This application further contemplates T cells that express one γ chain or one δ chain, optionally in combination with a second polypeptide to form a functional TCR, or other genetically engineered leukocytes or lymphocytes. Such genetically engineered leukocytes or lymphocytes that express one γ chain or one δ chain may be used in the methods or may be present in the compositions described herein.

[0034] As used herein, the term "T lymphocyte" or "T cell" refers to immune cells that express or have expressed CD3 (CD3+) and the T cell receptor (TCR+). T cells play a central role in cellular immunity. T cells that have "expressed" CD3 and TCR have been genetically engineered to exclude expression on the cell surface of CD3 and / or TCR.

[0035] As used herein, the term "TCR" or "T cell receptor" refers to a dimeric heterologous cell surface signaling protein that forms an alpha-beta or gamma-delta receptor or a combination thereof. The αβ TCR recognizes antigens presented by MHC molecules, while the γδ TCR is capable of recognizing antigens independently of MHC presentation.

[0036] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encode antigen-presenting proteins on the cell surface. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC or HLA are used interchangeably.

[0037] As used herein, "activation" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers, among other things, to a T cell that is undergoing cell division.

[0038] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an untreated immunoglobulin derived from a natural or recombinant source, and can further be an immunoreactive portion of an untreated immunoglobulin. Typically, an antibody is a tetramer of immunoglobulin molecules. The antibodies of the present invention can exist in various forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) 2 , as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0039] The term "antibody fragment" refers to a part of an intact antibody and to the variable antigen - determining region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2 and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0040] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation.

[0041] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation. κ and λ light chains refer to the two major antibody light - chain isotypes.

[0042] As used herein, the term "synthetic antibody" means an antibody made using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage as described herein. Further, this term should be interpreted to mean an antibody made by synthesizing a DNA molecule encoding the antibody, where the DNA molecule expresses an antibody protein or an amino - acid sequence that specifies an antibody, and where the DNA or amino - acid sequence is available and obtained using synthetic DNA or amino - acid sequence techniques well - known in the art.

[0043] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production or activation of specific immunocompetent cells. One of ordinary skill in the art will understand that any macromolecule, including proteins or peptides, can serve as an antigen. Further, an antigen can be derived from recombinant or genomic DNA. Thus, one of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of one or more genes, and further that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded at all by a "gene". It is readily apparent that an antigen can be produced, synthesized, or be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0044] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant that is capable of specifically binding to an immunoglobulin or a T cell receptor. An epitope determinant usually consists of the active surface groups of a molecule such as an amino acid, lipid or sugar side chain, and usually has specific three-dimensional structural characteristics as well as specific charge characteristics. When the equilibrium dissociation constant (K D ) is in the range of 10 -6 ~10 -12 M, the antibody is said to specifically bind to the antigen.

[0045] Antibodies 3B9, 9C11, 3H7, 2B7, and 10F2 represent exemplary embodiments of antibodies that specifically recognize CD20. These antibodies, their fragments, and their complementarity-determining regions are also described in US2009 / 0035322 and are referred to as 3B9-10, 9C11-14, 3H7-6, 2B7-7, and 10F2-13, respectively. As described herein, these antibodies, their fragments, and their complementarity-determining regions are useful in generating anti-CD20 chimeric antigen receptor (CAR) constructs and in genetically engineering and using CAR-T cells for treating blood tumors that express CD20.

[0046] Binding domains 21587N, 16747P, 16711P, and 16716P represent exemplary embodiments of binding domains that specifically recognize BCMA. These antibodies, their fragments, and their complementarity-determining regions are also described in US16 / 516028, filed on July 18, 2019, the content of which is hereby incorporated by reference in its entirety for all purposes, particularly for binding domains, antibodies, antibody fragments, complementarity-determining regions, polypeptides containing this complementarity-determining region, nucleic acids encoding this complementarity-determining region, and epitope specificities and assays for determining the epitope specificities described herein. Optionally, 21587N, 16747P, 16711P, and 16716P are referred to as H2aM21587N, H1H16747P, H1H16711P, and H1H16716P, respectively. As described herein, these antibodies, their fragments, and their complementarity-determining regions are useful in generating anti-BCMA chimeric antigen receptor (CAR) constructs and in genetically engineering and using CAR-T cells for treating blood tumors that express BCMA.

[0047] As used herein, the term "chimeric antigen receptor (CAR)" may refer to, for example, an artificial T cell receptor, a T body, a single-chain immunoreceptor, a chimeric T cell receptor, or a chimeric immunoreceptor, and encompasses a genetically engineered receptor that imparts artificial specificity to a particular immune effector cell. A CAR may be used to confer the specificity of a monoclonal antibody on a T cell, thereby making it possible to produce a large number of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, the CAR directs, for example, the specificity of a cell for a tumor-associated antigen. In some embodiments, the CAR comprises an intracellular activation domain (which enables activation of a T cell upon binding of a target cell, such as a target tumor cell, to a target moiety), a transmembrane domain, and an extracellular domain of variable length, and further comprises, for example, a tumor-antigen binding region associated with a disease or disorder. In certain aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane domain and an endodomain. The specificity of other CAR designs may be derived from a ligand of the receptor (such as a peptide donor) or a pattern recognition receptor such as dectin. In certain cases, it is possible to vary the spacing of the antigen recognition domains to reduce activation-induced cell death. In certain cases, the CAR comprises domains for adding co-stimulatory signaling such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, ICOS, TLR (such as TLR2). In some cases, the molecule may be co-expressed with a CAR along with co-stimulatory molecules, reporter genes for imaging (such as positron emission tomography), gene products that conditionally excise T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. Furthermore, those skilled in the art will understand that the co-stimulatory domain need not be encoded only by the full-length nucleotide sequence of the gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of one or more genes, and further that these nucleotide sequences are arranged in various combinations to elicit a desired immune response.

[0048] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in the average lifespan, or an improvement in various physiological symptoms associated with the cancer condition. Further, the "anti-tumor effect" can be demonstrated by the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent the occurrence of tumors in the first place.

[0049] As used herein, the term "self-antigen" means, in the context of the present invention, any self-antigen that is erroneously recognized as foreign by the immune system. Self-antigens include, but are not limited to, cellular proteins, phosphorylated proteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins including cell surface receptors.

[0050] As used herein, the term "self-derived" is intended to refer to any material derived from the same individual as the individual into which it is later reintroduced.

[0051] As used herein, the term "allogeneic" is intended to refer to any material derived from an animal that is later introduced into another animal of the same species.

[0052] The term "therapeutically effective amount" refers to the amount of a composition that elicits a biological or medical response of a tissue, system or subject as required by a researcher, veterinarian, physician or other clinician. The term "therapeutically effective amount" includes, when administered, an amount of the composition sufficient to prevent the onset of, or to alleviate to some extent, one or more of the signs or symptoms of a disease or disorder being treated (e.g., blood cancer). The therapeutically effective amount will vary depending on the composition, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0053] As used herein, to "treat" a disease means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0054] Administration "in combination with" one or more therapeutic agents includes simultaneous (co - administration) and sequential administration in any order.

[0055] As used herein, the term "pharmaceutically acceptable" refers to, but is not limited to, materials such as salts, carriers, or diluents that do not inhibit the biological activity or properties of a compound and are relatively non - toxic. In other words, this material can be administered to an individual without causing undesirable biological effects or acting in a harmful manner with any of the components of the composition in which it is included.

[0056] "Encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA that serves as a template for synthesizing other polymers and macromolecules by a biological method having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, when transcription and translation of the mRNA corresponding to that gene produce a protein in a cell or other biological system, that gene encodes the protein. It is possible to say that both the coding strand, which is identical to the sequence of the mRNA and is usually the nucleotide sequence provided in the sequence listing, and the non - coding strand used as a template for transcription of the gene or cDNA encode the protein or other product of that gene or cDNA.

[0057] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its co - existing substances in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non - natural environment such as a host cell.

[0058] Unless otherwise specified, the "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate versions and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0059] Terms such as "patient", "subject", "individual", etc. are used interchangeably herein and refer to any animal compliant with the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0060] With respect to an antibody, the term "specifically binds" as used herein means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen obtained from one species may also bind to that antigen obtained from one or more species. However, such cross-reactivity per se does not change the classification of the antibody according to its properties. In another example, an antibody that specifically binds to an antigen may bind to various alleles of the antigen. However, such cross-reactivity per se does not change the classification of the antibody according to its properties. In some cases, the terms "specific binding" or "specifically binding" may be used with respect to the interaction between an antibody, protein or peptide and a second chemical species, where this interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species. For example, an antibody means recognizing and binding to a particular protein structure rather than a protein in general. When an antibody is specific for an epitope "A", in a reaction containing labeled "A" and the antibody, the amount of labeled A that binds to the antibody decreases if a molecule containing epitope A (or free unlabeled A) is present.

[0061] In some embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10 -8 M or less (e.g., K DThe smaller it is, the stronger the binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. Furthermore, a multispecific antibody that binds to a first antigen and one or more additional antigens, or a bispecific antibody that binds to two different regions of an antigen, is nevertheless considered an antibody that "specifically binds" as used herein.

[0062] Blood cancer is cancer that occurs in the blood or bone marrow. Examples of blood (or hematogenous) cancers include acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (low grade and high grade), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and leukemias such as myelodysplasia. In a preferred embodiment, the blood cancer expresses or overexpresses CD20. In a preferred embodiment, the blood cancer expresses or overexpresses B cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17).

[0063] "Expression cassette" refers to a nucleic acid comprising an expression control sequence operably linked to a nucleic acid encoding a transcript or polypeptide to be expressed. The expression cassette contains cis-acting elements sufficient for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. The expression cassette can be a component of a vector such as a cosmid, plasmid (e.g., naked or liposome-encapsulated in liposomes), or virus (e.g., lentivirus, retrovirus, adenovirus and adeno-associated virus). The expression cassette can be present in a host cell such as a γδ T cell.

[0064] Scope: Throughout the present disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, a description of a range should be considered to specifically disclose not only the individual numerical values within that range but also all possible sub-ranges. For example, a description of a range such as from 1 to 6 should be considered to specifically disclose sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, as well as individual numbers within the range such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0065] Chimeric antigen receptor construct: Aspects of the invention include nucleic acids encoding a CAR, as well as constructs and vectors containing such nucleic acids. Optionally, the nucleic acid is, for example, a component of a heterologous expression cassette. In some embodiments, the nucleic acid is, for example, a component of a heterologous retroviral vector. In some embodiments, the nucleic acid is, for example, a component of a heterologous αβ or γδ T cell, preferably a γδ T cell. In some embodiments, the nucleic acid is, for example, a component of a heterologous γ + T cell and / or δ + T cell. In some embodiments, the nucleic acid is, for example, a component of a heterologous α - T cell and / or β - T cell.

[0066] Described herein is a nucleic acid encoding a CAR binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of hematological tumor cells. Exemplary TAAs include CD19, CD20, and BCMA. In some embodiments, the binding domain is a CD19 binding domain such as the CD19 binding domain described in U.S. Patent No. 9,540,445, the content of which is incorporated herein by reference in its entirety and for all purposes, particularly the binding domain, antibody, antibody fragment, complementarity-determining region, polypeptide comprising this complementarity-determining region, nucleic acid encoding this complementarity-determining region, and the epitope specificities and assays for determining the epitope specificities described herein. In some embodiments, the binding domain is a CD20 binding domain such as the CD20 binding domain described in U.S. Patent Application No. 2009 / 0035322, the content of which is incorporated herein by reference in its entirety and for all purposes, particularly the binding domain, antibody, antibody fragment, complementarity-determining region, polypeptide comprising this complementarity-determining region, nucleic acid encoding this complementarity-determining region, and the epitope specificities and assays for determining the epitope specificities described herein. In some embodiments, the binding domain is a BCMA binding domain such as the BCMA binding domain described in WO2018 / 133877 or the BCMA binding domain described in US16 / 516028 filed on July 18, 2019, the content of each of which is incorporated herein by reference in its entirety and for all purposes, particularly the binding domain, antibody, antibody fragment, complementarity-determining region, polypeptide comprising this complementarity-determining region, nucleic acid encoding this complementarity-determining region, and the epitope specificities and assays for determining the epitope specificities described herein. Typically, the region encoding the binding domain is 5' of a linker region (e.g., the region encoding the CD8α hinge domain).

[0067] In some embodiments, the binding domain binds to an antigen such that it is expressed as a full-length functional polypeptide on the surface of a cell. In some embodiments, the binding domain binds to an antigen such that it is presented in an MHC:antigen complex. In some embodiments, the binding domain binds to an antigen in an HLA-restricted manner. Binding domains that are specific for an MHC:antigen complex are described, for example, in WO / 2016 / 199140 and WO / 2016 / 199141, the contents of each of which are incorporated herein by reference in their entirety and for all purposes, particularly for binding domains, antibodies, antibody fragments, complementarity determining regions, polypeptides comprising such complementarity determining regions, nucleic acids encoding such complementarity determining regions, and epitope specificities and assays for determining the epitope specificities described herein.

[0068] Exemplary CD20 binding domains include, but are not limited to, 3B9, 3H7, 2B7, 9C11 or 10F2, or a binding domain that selectively binds to an epitope within CD20 that binds 3B9, 3H7, 2B7, 9C11 or 10F2, or 3B9, 3H7, 2B7, 9C11 or 3H7, or that competes for binding to 3B9, 3H7, 2B7, 9C11 or 10F2, or 3B9, 3H7, 2B7, 9C11 or 3H7. Additionally or alternatively, the CD20 binding domain can include a complementarity determining region of an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, 9C11 and 10F2, selected from the group consisting of 3B9, 3H7, 2B7 and 9C11, or selected from the group consisting of 3H7. The present disclosure also contemplates CD20, CD19 and BCMA binding domains that compete for binding to the sequences provided herein.

[0069] By using known methods, it is possible to determine whether a CD20 binding domain binds to the same epitope as a reference antibody or binding domain, or competes to bind to that reference antibody or binding domain. For example, to determine whether a test antibody binds to the same epitope as a reference binding domain, it can be achieved by binding the reference binding domain to CD20 under saturation conditions. Next, it is possible to evaluate the ability of a test binding domain to bind to the CD20 molecule. If this test binding domain is able to bind to CD20 following saturation binding with the reference binding domain, it is concluded that this test binding domain binds to an epitope different from the reference binding domain. On the other hand, if the test binding domain is unable to bind to CD20 following saturation binding with the reference binding domain, this test binding domain may bind to the same epitope as the epitope bound by the reference binding domain.

[0070] If the binding domain competes to bind to the reference binding domain, the binding methodology described above is carried out in two directions. In the first direction, the reference binding domain is bound to CD20 under saturation conditions, followed by evaluating the binding of the test binding domain to the CD20 molecule. In the second direction, the test binding domain is bound to the CD20 molecule under saturation conditions, followed by evaluating the binding of the reference binding domain to the CD20 molecule. In both directions, if only the first (saturated) binding domain is capable of binding to the CD20 molecule, it is concluded that the test binding domain and the reference binding domain compete for binding to CD20. As will be understood by those skilled in the art, a binding domain that competes to bind to a reference binding domain does not necessarily bind to the same epitope as the reference binding domain, but can sterically block the binding of the reference binding domain by binding to overlapping or adjacent epitopes. The methods described above for determining competition and epitope binding with an anti-CD20 binding domain can similarly be applied to anti-CD19 binding domains and anti-BCMA binding domains.

[0071] When two binding domains competitively inhibit (block) each other's binding to an antigen, they bind to the same or overlapping epitopes. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one binding domain inhibits the other's binding by at least 50%, e.g., 75%, 90%, or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if essentially all amino acid mutations of the antigen that reduce or eliminate the binding of one binding domain also reduce or eliminate the binding of the other, the two binding domains have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one binding domain also reduce or eliminate the binding of the other, the two binding domains have overlapping epitopes.

[0072] Furthermore, by performing routine experiments (e.g., peptide mutagenesis and binding assays), it is possible to determine whether the observed lack of binding of the test binding domain is actually due to binding to the same epitope as the reference binding domain or whether steric hindrance (or other phenomenon) is the cause of the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative binding assay available in the art.

[0073] The present disclosure provides antibodies and CARs that have "substantial identity" or "substantial similarity" to the sequences provided herein in the CDR or framework regions. The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, when optimally aligned with another nucleic acid (or the complementary strand of another nucleic acid), as measured by any known algorithm for sequence identity, such as FASTA, BLAST, or GAP, as described below, the nucleotide sequence identity is, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of nucleotide bases. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0074] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that when optimally aligned by programs such as GAP or BESTFIT using the default gap weights, the two peptide sequences share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity. In some embodiments, the positions of non-identical residues differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of identity may be adjusted upward to account for the conservative nature of the substitution. Methods for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative substitution is any change to the PAM250 log-likelihood matrix that has a positive value as disclosed in Gonnet et al. (1992) Science 256:1443-45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0075] The sequence identity and / or similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software is used to match similar sequences when measuring the similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT that can use default parameters to measure sequence homology or sequence identity between homologous polypeptides of various species of living organisms, between polypeptides of closely related species, or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, i.e., using the program FASTA in GCG Version 6.1. FASTA (such as FASTA2 and FASTA3) provides the alignment of the optimal overlap region between the query sequence and the search sequence and the percent sequence identity (Pearson (2000) supra). Sequences can be compared using the Smith-Waterman homology search algorithm with an affine gap search where the gap start penalty is 12, the gap extension penalty is 2, and the BLOSUM matrix is 62. When comparing a database containing a large number of sequences obtained from various living organisms with the sequences disclosed herein, another preferred algorithm is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402. Each of these disclosures is incorporated herein by reference.

[0076] Provided herein are anti-CD20, anti-BCMA, or anti-CD19 CARs comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more substitutions (e.g., conservative substitutions). For example, the disclosure provides an anti-CD20 CAR having an HCVR, LCVR, and / or CDR amino acid sequence with, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitution as compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, the anti-CD20 CAR can include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (such as conservative amino acid substitutions) as compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0077] Similarly, the present disclosure includes an anti-BCMA CAR having an HCVR, LCVR, and / or CDR amino acid sequence with, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitutions as compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, the anti-BCMA CAR may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (such as conservative amino acid substitutions) as compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0078] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain complementarity determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 and LCDR3 are selected from the group consisting of SEQ ID NOs: 345 (AKDPSYGSGSYHSYYGMDV) and 353 (QQRFNWPLT), 201 (VKDFHYGSGSNYGMDV) and 209 (QQSNDWPLT), and 249 (TKDGSYGHFYSGLDV) and 257 (QQRYYWPLT).

[0079] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR sequence and the LCVR sequence are SEQ ID NO: 339 (EEQLVESGGDLVQPGRSLRLSCAASGFTFHDYTMH WVRQAPGKGLEWVSGISWNSGSLGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDPSYGSGSYHSYYGMDVWGQGTTVTVSS), 347 (EIVLTQSPATLSLSPGE RATLSCWASQSISRYLVWYQQKCGQAPRLLIYEASKRATGIPVRFSGSGSGTDFTLTISSLESEDFAVYYCQQRFNWPLTFGGGTKVEIK), 195 (EVQLAESGGDLVQSGRSLRLSCAAS GITFHDYAMHWVRQPPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKKSLYLQMNSLRPDDTALYYCVKDFHYGSGSNYGMDVWGQGTTVTVSP), and 203 (EIVMTQSPATL SMSPGERATLSCRASQSVSRNLAWYQQKVGQAPRLLISGASTRATGIPARFSGSGSGTEFTLTINSLQSEDFAVYYCQQSNDWPLTFGQGTRLEIK), 243 (VQLVESGGGLVQPGR SLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSDTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCTKDGSYGHFYSGLDVWGQGTTVTVSS), and 251 (EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYVASNRATGIPARFSGSGSGTDFTLTISSLEPDDFAVYYCQQRYYWPLTFGGGTKVEIK) selected from the group consisting of.

[0080] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain complementarity determining region 3 (HCDR3) domain and a light chain CDR3 (LCDR3) domain, wherein the HCDR3 domain comprises the amino acid sequence of formula X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19, where X1 = A, V or T, X2 = K, X3 = D, X4 = P, F or G, X5 = S or H, X6 = Y, X7 = G, X8 = S or H, X9 = G or F, X10 = S or Y, X11 = Y, N or S, X12 = Y, G or H, X13 = G, L or S, X14 = Y, M or D, X15 = Y, D or V, X16 = G, V or absent, X17 = M or absent, X18 = D or absent, X19 = V or absent (SEQ ID NO: 369), and the LCDR3 domain comprises the amino acid sequence of X1-X2-X3-X4-X5-X6-X7-X8-X9, where X1 = Q, X2 = Q, X3 = R or S, X4 = N, Y or F, X5 = N, D, or Y, X6 = W, X7 = P, X8 = L, X9 = T (SEQ ID NO: 370).

[0081] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR sequence and the LCVR sequence are SEQ ID NO: 99 (EVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSS) and 107 (EIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIK), respectively.

[0082] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to the same epitope as, competes with, or is the anti-CD20 binding domain itself of an anti-CD20 binding domain having a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR), wherein the HCDR and LCDR sequences are the HCDR sequence of SEQ ID NO: 99 and the LCDR sequence of SEQ ID NO: 107, respectively.

[0083] In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to the same epitope as, competes with, or is the anti-CD20 binding domain itself that has an HCDR1 that is or includes SEQ ID NO: 101 (GFTFYDYA), an HCDR2 that is or includes SEQ ID NO: 103 (ISWNSGYI), and / or an HCDR3 that is or includes SEQ ID NO: 105 (AKDNSYGKFYYGLDV). In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to the same epitope as, competes with, or is the anti-CD20 binding domain itself that has an LCDR1 that is or includes SEQ ID NO: 109 (QSVSSN), an LCDR2 that is or includes SEQ ID NO: 111 (GAS), and / or an LCDR3 that is or includes SEQ ID NO: 113 (QQYNNWPIT). In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that binds to the same epitope as, competes with, or is the anti-CD20 binding domain itself that has an HCDR1 that is or includes SEQ ID NO: 101, an HCDR2 that is or includes SEQ ID NO: 103, an HCDR3 that is or includes SEQ ID NO: 105, an LCDR1 that is or includes SEQ ID NO: 109, an LCDR2 that is or includes SEQ ID NO: 111, and an LCDR3 that is or includes SEQ ID NO: 113. In some embodiments, the isolated nucleic acid encodes an anti-CD20 binding domain that has an HCDR1 comprising SEQ ID NO: 101, an HCDR2 comprising SEQ ID NO: 103, an HCDR3 comprising SEQ ID NO: 105, an LCDR1 comprising SEQ ID NO: 109, an LCDR2 comprising SEQ ID NO: 111, and an LCDR3 comprising SEQ ID NO: 113.

[0084] Exemplary BCMA binding domains include, but are not limited to, binding domains that bind to the BCMA binding domains described in WO2018 / 133877 or the BCMA binding domains described in US16 / 516028 filed on July 18, 2019, or that selectively bind to an epitope within BCMA that competes for binding with those domains. Alternatively or additionally, the BCMA binding domain may comprise complementarity determining regions of an anti-BCMA antibody selected from the group consisting of the anti-BCMA antibodies or chimeric antigen receptors described in WO2018 / 133877 and the anti-BCMA antibodies or chimeric antigen receptors described in US16 / 516028 filed on July 18, 2019.

[0085] Exemplary BMCA binding domains include, but are not limited to, binding domains that selectively bind to an epitope within BCMA that binds by anti-BCMA-CAR16716P, anti-BCMA-CAR16747P and / or anti-BCMA-CAR21587N or that competes for binding with them. Alternatively or additionally, the BCMA binding domain may comprise complementarity determining regions of an anti-BCMA CAR selected from the group consisting of anti-BCMA-CAR16716P, anti-BCMA-CAR16747P and anti-BCMA-CAR21587N.

[0086] In some embodiments, the isolated nucleic acid encodes an anti-BCMA binding domain having a heavy chain complementarity determining region 3 (HCDR3) and a light chain CDR3 (LCDR3), wherein the HCDR3 and LCDR3 are selected from the group consisting of SEQ ID NO: 21 (RAGDNWNWFDP) and SEQ ID NO: 22 (QQAKSVPFT), SEQ ID NO: 23 (EGGNYGMDV) and SEQ ID NO: 24 (QQANSFPPT), and SEQ ID NO: 25 (FAEYCGGNICYYYGMDV) and SEQ ID NO: 26 (QQCGGSPWT).

[0087] In some embodiments, the isolated nucleic acid encodes an anti-BCMA binding domain having a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence, wherein the HCVR sequence and the LCVR sequence are the 16716P binding domain HCVR SEQ ID NO: 27 (MSVPTQVLGLLLLWLTDARCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSAIIGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRAGDNWNWFDPWGQGTLVTV) and the 16716P binding domain LCVR SEQ ID NO: 28 (DIQMTQSPSSVSASLGDRVTITCRASQGISSWLAWYQRKPGKAPKLLIYAASSLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQAKSVPFTFGPGTKVDIK), and the 16747P binding domain HCVR SEQ ID NO: 29 (MSVPTQVLGLLLLWLTDARCQVQLVESGGGLV KPGGSLRLSCAASGFTFSDYYISWIRQAPGKGLEWVSYISSSGSSIKYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGNYGMDVWGQGTTVTV), and the 16747P binding domain LCVR SEQ ID NO: 30 (DIQMTQSPSSVSASVGDRVTITCRASQGINNWIt is selected from the group consisting of: LVWYQQKPGKAPKLLIYAATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIK), 21587N binding domain HCVR SEQ ID NO: 31 (MSVPTQVLGLLLLWLTDARCQVQLQESGPGLVKPSETLSLTCTVSGGSINYYYWNWIRQPPGKGLEWIGYISYSGNTNYNPSLKSRVTISVATSRNQFSLTLSSVTAADTAVYYCARFAEYCGGNICYYYGMDVWGQGTTVTV), and 21587N binding domain LCVR SEQ ID NO: 32 (EIVLTQSPGTLSLSPGERATFSCRASQSVGSSFLAWYQQKPGQAPRRLMYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQCGGSPWTFGQGTKVEIK).

[0088] This specification provides an anti - BCMA CAR comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more substitutions (such as conservative substitutions). For example, the present disclosure provides an anti - BCMA CAR having an HCVR, LCVR, and / or CDR amino acid sequence with, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitution as compared to any of the HCVR, LCVR, and / or CDR (such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, the anti - BCMA CAR may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (such as conservative amino acid substitutions) as compared to any of the HCVR, LCVR, and / or CDR (such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0089] Exemplary binding domains described herein typically include a heavy chain region followed by a light chain region (VH-VL) in order from the amino terminus to the carboxy terminus. If the specific order of the VH and VL regions in the binding domain is explicitly or implicitly described, the present disclosure is also understood to describe alternative embodiments in which the order of the VH and VL regions is reversed, for example, in a CAR comprising an scFV or scFv binding domain. Thus, a description of the VH-VL order also describes, for example, an alternative VL-VH order in a CAR comprising an scFV or scFv binding domain. Further, a description of the VL-VH order also describes, for example, an alternative VH-VL order in a CAR comprising an scFV or scFv binding domain.

[0090] Generally, nucleic acids encoding the CARs described herein include an extracellular linker portion encoding a peptide linker that links the binding domain to the transmembrane domain. Exemplary linker portions include, but are not limited to, linker portions encoding the CD8α hinge domain, such as SEQ ID NO: 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). Typically, the region encoding the peptide linker (e.g., the CD8α hinge domain) is 3' of the region encoding the binding domain and 5' of the region encoding the transmembrane domain.

[0091] The nucleic acid encoding the CAR described herein includes a transmembrane domain. The transmembrane domain can link an extracellular antigen-binding domain, such as a hinge, to one or more intracellular signaling components. For example, the transmembrane domain can link an antigen-binding domain, such as a hinge, to a CD3ζ signaling domain and optionally one or two co-stimulatory endodomains. Exemplary transmembrane domains include, but are not limited to, the CD8α transmembrane domain, such as SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain (e.g., the CD8α transmembrane domain) is 3' to the region encoding a peptide linker (e.g., the CD8α hinge domain) and 5' to the region encoding one or more cytoplasmic domains.

[0092] In some embodiments, the isolated nucleic acid encodes a cytoplasmic region that includes one or more cytoplasmic domains. The region encoding the cytoplasmic region is typically 3' of the region encoding the transmembrane domain. The cytoplasmic domain is typically a signaling domain that provides an activating signal for γδ T cell proliferation, cytotoxic activity, and / or inflammatory cytokine expression (e.g., TNF-α or IFNγ). An exemplary cytoplasmic domain is the CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is or comprises SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the CD3ζ signaling domain is or comprises SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the cytoplasmic region includes multiple (e.g., 2, 3, 4, 5, or 6) signaling domains, such as multiple (e.g., 2, 3, 4, 5, or 6) CD3ζ signaling domains, independently selected, for example, from SEQ ID NOs: 4 and 5. In some embodiments, the cytoplasmic region includes multiple (e.g., 2, 3, 4, 5, or 6) non-CD3ζ signaling domains and a CD3ζ signaling domain. In some embodiments, the cytoplasmic region includes a non-CD3ζ signaling domain and multiple (e.g., 2, 3, 4, 5, or 6) CD3ζ signaling domains.

[0093] The cytoplasmic region may include one or more co-stimulatory endodomains. The region encoding one or more co-stimulatory endodomains may be 5' or 3' of the region encoding the signaling domain. In some embodiments, the region encoding one or more co-stimulatory endodomains may be 5' of the region encoding the signaling domain. In some embodiments, the region encoding one or more co-stimulatory endodomains is 5' of the region of the signaling domain, and an additional region encoding one or more co-stimulatory endodomains is 3' of the signaling domain. Exemplary co-stimulatory endodomains include, but are not limited to, CD28, CD137 (4-1BB), CD278 (ICOS), CD27, CD134 (OX40), Dap10, Dap12, DNAm-1, 2B4, SLAM domain, and TLR2 co-stimulatory endodomain, and combinations thereof.

[0094] In some embodiments, the construct encodes at least one 4-1BB co-stimulatory endodomain and optionally a second co-stimulatory endodomain selected from the 4-1BB, 2B4, ICOS, CD28, and CD27 co-stimulatory endodomains. In some embodiments, the construct encodes at least two 4-1BB co-stimulatory endodomains or two 4-1BB co-stimulatory endodomains combined with 1, 2, 3, or 4 or more co-stimulatory endodomains selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB co-stimulatory endodomain includes SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

[0095] In some embodiments, the construct encodes one CD27 co-stimulatory endodomain and, optionally, a second co-stimulatory endodomain selected from 4-1BB, ICOS, CD28, and the CD27 co-stimulatory endodomain. In some embodiments, the construct encodes a CD27 co-stimulatory endodomain and a 4-1BB co-stimulatory endodomain. In some embodiments, the construct encodes two CD27 co-stimulatory endodomains. In some embodiments, the CD27 co-stimulatory endodomain comprises SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[0096] In some embodiments, the construct encodes a secretion signal, such as SEQ ID NO: 33 (MALPVTALLLPLALLLHAARP), operably linked to promote the secretion of a C-terminal polypeptide, such as a cytokine, that supports the activation, cytotoxicity, and / or persistence of T cells (such as CAR-T cells). In some embodiments, the construct encodes a secretion signal, such as SEQ ID NO: 33, operably linked to promote the secretion of a common gamma chain cytokine, such as IL-15, or an active fragment thereof, such as SEQ ID NO: 34 (NWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Exemplary common gamma chain cytokines include IL-2 and IL-15. In some embodiments, the common gamma chain cytokine is selected from IL-2, IL-7, and IL-15. In some embodiments, the common gamma chain cytokine is IL-15. An IL-15 sequence comprising a codon-optimized nucleic acid sequence encoding sIL15 is disclosed herein and in WO2007 / 037780.

[0097] In some embodiments, the construct encodes one or more multicistronic linker regions between, for example, a signaling domain and / or a co-stimulatory endodomain and a secretion signal operably linked to promote secretion of the cytokine. A multicistronic linker region is a region of a polypeptide or RNA sequence that facilitates the production of multiple distinct polypeptides from a single transcript. In some embodiments, the multicistronic linker region encodes a cleavage sequence. Suitable cleavage sequences include self-cleaving sequences such as the P2A, F2A, E2A, or T2A cleavage sequences and / or sequences that are cleaved by endogenous proteases such as furin.

[0098] In some embodiments, the cleavage sequence is the P2A cleavage sequence. In some embodiments, the cleavage sequence is the furin cleavage sequence. In some embodiments, the cleavage sequence is the P2A and furin cleavage sequences. In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 43 (SGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is the furin cleavage sequence of SEQ ID NO: 44 (RAKR). In some embodiments, the cleavage sequence is the P2A + furin cleavage sequence of SEQ ID NO: 45 (RAKRSGSGATNFSLLKQAG DVEENPGP).

[0099] In some embodiments, the cleavage sequence is or comprises the P2A cleavage sequence of SEQ ID NO: 52 (ATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is or comprises the F2A cleavage sequence of SEQ ID NO: 53 (VKQTLNNFDLLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises the E2A cleavage sequence of SEQ ID NO: 54 (QCTNYALLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or comprises the T2A cleavage sequence of SEQ ID NO: 55 (EGRSLLTCGDVEENPGP). In one aspect, the plurality of self-cleaving sequences can encode the carboxy terminus of the signaling and / or co-stimulatory domain, and the amino terminus of the encoded secreted cytokine (e.g., a common gamma chain cytokine such as IL-15), wherein the plurality of self-cleaving sequences are preferably independently selected from the group consisting of P2A cleavage sequences, T2A cleavage sequences, E2A cleavage sequences, and F2A cleavage sequences. In one aspect, one or more self-cleaving sequences and one or more sequences cleaved by an endogenous protease are encoded by the constructs described herein. In certain embodiments, the endogenous protease recognition site is encoded at the amino terminus of the self-cleaving sequence.

[0100] In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by SEQ ID NO: 56 (CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATA).

[0101] Another exemplary internal ribosome entry site is encoded by SEQ ID NO: 60 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC).

[0102] Further preferred internal ribosome entry sites include, but are not limited to, those described in Nucleic Acids Res. 2010 Jan; 38 (Database issue): D131-6. doi: 10.1093 / nar / gkp981. Epub Nov 16, 2009, those described in iresite.org, those described in WO2018 / 215787, the sequences described in GenBank accession No. KP019382.1 and the IRES elements disclosed in GenBank accession No. LT727339.1.

[0103] Additional multicistronic linker regions such as cleavage self-cleavage and IRES elements are disclosed in US2018 / 0360992 and US8865467.

[0104] In some embodiments, the isolated nucleic acid encodes a 3H7-CD8-CD27z polypeptide comprising, in order, the following domains: the 3H7 binding domain, the CD8α hinge and transmembrane domain, the CD27 co-stimulatory end domain, and the CD3ζ signaling domain of SEQ ID NO: 8 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0105] In some embodiments, the isolated nucleic acid encodes a 3B9-CD8-BBz polypeptide comprising, in order, SEQ ID NO: 9 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGTSTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPLTFGGGTKVEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), the following domains: a 3B9 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory end domain, and a CD3ζ signaling domain.

[0106] In some embodiments, the isolated nucleic acid encodes a 3H7-CD8-BBz polypeptide comprising, in order, the following domains: a 3H7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory end domain, and a CD3ζ signaling domain, having the sequence set forth in SEQ ID NO: 10 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0107] In some embodiments, the isolated nucleic acid encodes a 2B7-CD8-BBz polypeptide comprising, in order, the following domains: a 2B7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory end domain, and a CD3ζ signaling domain, having the sequence SEQ ID NO: 11 (MSVPTQVLGLLLLWLTDARCEIVLTQSPATLSLSPGERAALSCRASQSVSNYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIRGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFRDYTMHWVRQGPGKGLEWVSGISWNSDYIGYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCAKLSGTYRDYFYGVDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0108] In some embodiments, the isolated nucleic acid encodes a 9C11-CD8-BBz polypeptide comprising, in order, the following domains: a 9C11 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory end domain, and a CD3ζ signaling domain, having the sequence SEQ ID NO: 12 (MSVPTQVLGLLLLWLTDARCEIVVTQSPATLSLSPGERATLSCRTSQTTTSYLAWYRQKPGQAPRLLIYDASNRAAGIPARFSGSGSGTDFTLTINSLEPEDFAVYYCQLRTNWITFGQGTRLEIKGGGGSGGGGSGGGGQVQLVESGGDSVKPGGSLRLSCAASGFTFSDSYMTWIRQAPGKGLEWVSFISSSGSTIYYADSVKGRFTISRDNVKKSLYLQMNRLRAEDTAVYYCAREEPGNYVYYGMDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0109] In some embodiments, the isolated nucleic acid encodes a 3H7-CD3z polypeptide comprising, in order, the following domains: the 3H7 binding domain, the CD8α hinge and transmembrane domain, and the CD3ζ signaling domain, having the sequence set forth in SEQ ID NO: 20 (MSVP TQVLLLLWLTDARC EIVMTQS PATLSVS PGERTTL SCRASQS VSSSNL AWYLQKP GQAPRLL IYGASTRA TGIPARFS GSGSGSTE FILTISSL QSEDFAVY YCQQYNNW PITFGQGT RL EIKGGGG SGGGG SGGGG EVQLVESG GGLVQPGR SLRLSCAA SGFTFYDY AMHWRQAP GKGLEWVS GISWN SGYIGYAD SVKG RFTISRD NA KN SLYLQMN SLRAEDTA LYYCAKD NSYGKFYY GLDVWGQG TTVTVSS TTTPPAPR PPT PAPTIASQ PLSLRPEA CRPAAGGG AVHTRGLD FACDIYIW APLAGTCG VLLLLSLV ITLYCRVK FSRSADAP AYQQGQNQ LYNELNLG RR EE YDVL DKRRGRDP EMGGKPQR RKN PQEGLYN ELQKD KMAE AYS EIGMKGER RR GKGH DG LYQGLSTA TKDTYDA LH MQALPPR).

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] In some embodiments, the isolated nucleic acid comprises a codon-optimized sequence encoding the CD8α hinge region. Exemplary codon-optimized CD8α hinge region nucleic acid sequences include, but are not limited to, SEQ ID NO: 18 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCA CAGCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8α hinge region is encoded by SEQ ID NO: 19 (ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT).

[0116] In some embodiments, the isolated nucleic acid encodes a 3B9 binding domain and comprises the following sequence encoding SEQ ID NO: 18 of the CD8α hinge domain. In some embodiments, the isolated nucleic acid encodes a 2B7 binding domain and comprises the following sequence encoding SEQ ID NO: 18 of the CD8α hinge domain. In some embodiments, the isolated nucleic acid encodes a 9C11 binding domain and comprises the following sequence encoding SEQ ID NO: 18 of the CD8α hinge domain. In some embodiments, the isolated nucleic acid encodes a 3H7 binding domain and comprises the following sequence encoding SEQ ID NO: 19 of the CD8α hinge domain.

[0117] In some embodiments, the isolated nucleic acid encodes an anti-BCMA-CAR polypeptide comprising, in order, the following domains: a 16716P binding domain, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory end domain, and a CD3ζ signaling domain, having the sequence set forth in SEQ ID NO: 35 (MSVPTQVLGLLLLWLTDARCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSAIIGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKRAGDNWNWFDPWGQGTLVTVSSGGGGSGGGGSGGGGDIQMTQSPSSVSASLGDRVTITCRASQGISSWLAWYQRKPGKAPKLLIYAASSLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQAKSVPFTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0118] In some embodiments, the isolated nucleic acid encodes an anti-BCMA-CAR polypeptide comprising, in order, the 16747P binding domain, the CD8α hinge and transmembrane domain, the 4-1BB co-stimulatory end domain, and the CD3ζ signaling domain of SEQ ID NO: 36 (MSVPTQVLGLLLLWLTDARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYISWIRQAPGKGLEWVSYISSSGSSIKYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGNYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGDIQMTQSPSSVSASVGDRVTITCRASQGINNWLVWYQQKPGKAPKLLIYAATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0119] In some embodiments, the isolated nucleic acid encodes an anti-BCMA-CAR polypeptide comprising, in order, the following domains: the 21587N binding domain, the CD8α hinge and transmembrane domain, the 4-1BB co-stimulatory end domain, and the CD3ζ signaling domain of SEQ ID NO: 37 (MSVPTQVLGLLLLWLTDARCQVQLQESGPGLVKPSETLSLTCTVSGGSINYYYWNWIRQPPGKGLEWIGYISYSGNTNYNPSLKSRVTISVATSRNQFSLTLSSVTAADTAVYYCARFAEYCGGNICYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGEIVLTQSPGTLSLSPGERATFSCRASQSVGSSFLAWYQQKPGQAPRRLMYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQCGGSPWTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0120] In some embodiments, the isolated nucleic acid encodes an anti-BCMA-CAR polypeptide comprising, in order, SEQ ID NO: 38 (MSVPTQVLGLLLLWLTDARCQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYISWIRQAPGKGLEWVSYISSSGSSIKYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGNYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGDIQMTQSPSSVSASVGDRVTITCRASQGINNWLVWYQQKPGKAPKLLIYAATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), the following domains: 16747P binding domain, CD8α hinge and transmembrane domain, 4-1BB co-stimulatory end domain, CD3ζ signaling domain, furin + P2A cleavage domain, secretion signal and sIL15 domain.

[0121]

[0122]

[0123]

[0124]

[0125] In some embodiments, the isolated nucleic acid encodes an anti-CD20 CAR polypeptide comprising, in order, the following domains: 3H7 binding domain, CD8α hinge and transmembrane domain, 4-1BB co-stimulatory end domain, CD3ζ signaling domain, P2A cleavage domain (GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 47), a secretion signal, and the sIL15 domain, having the sequence number 46 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*).

[0126] In some embodiments, the isolated nucleic acid encodes an anti-CD20 CAR polypeptide comprising, in order, SEQ ID NO: 48 (MSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERTTLSCRASQSVSSNLAWYLQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFILTISSLQSEDFAVYYCQQYNNWPITFGQGTRLEIKGGGGSGGGGSGGGGEVQLVESGGGLVQPGRSLRLSCAASGFTFYDYAMHWVRQAPGKGLEWVSGISWNSGYIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDNSYGKFYYGLDVWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), the following domains: 3H7 binding domain, CD8α hinge and transmembrane domain, 4-1BB co-stimulatory end domain, CD3ζ signaling domain, P2A cleavage domain of SEQ ID NO: 47, secretion signal of SEQ ID NO: 49 (MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA) and sIL15 domain.

[0127]

[0128]

[0129] In some embodiments, the isolated nucleic acid encodes an anti-CD20 CAR polypeptide that sequentially includes the following domains: a 3H7 binding domain, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory end domain, and a CD3ζ signaling domain, but through an internal ribosome entry site (e.g., the site encoded by SEQ ID NO: 56) in the region encoding SEQ ID NO: 57, this isolated nucleic acid further encodes SEQ ID NO: 58 (MALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), a secretion signal of SEQ ID NO: 33 and an sIL15 domain.

[0130]

[0131] In some embodiments, the isolated nucleic acid is a linear nucleic acid. In some embodiments, the isolated nucleic acid is a circular nucleic acid. In some embodiments, the isolated nucleic acid is a vector such as a plasmid vector, an adenovirus vector, an adeno-associated virus vector, a viral vector, a retrovirus vector, or a lentivirus vector. In some embodiments, the isolated nucleic acid, or a continuous portion of the isolated nucleic acid, such as, for example, a binding domain transmembrane domain and one or more signaling and / or co-stimulatory endodomains, is integrated into the genome of a host cell such as a host γδ T cell. In exemplary embodiments, the isolated nucleic acid is a retrovirus vector.

[0132] γδ T cells: Aspects of the invention include γδ T cells that functionally express the isolated nucleic acids described herein, thereby expressing a CAR on the surface of the γδ T cells.

[0133] Aspects of the invention may alternatively or additionally include γδ T cells having in vitro or in vivo cytotoxic activity against hematological tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some cases, the cytotoxic activity is a native activity. In some cases, the cytotoxicity is at least partially, significantly (> about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to a TAA expressed on the surface of the hematological tumor cells. In some cases, in this γδ T cell, it is shown that the hematological tumor cell killing activity of the γδ T cell is greater than the native level of in vitro and / or in vivo hematological tumor cell killing activity in control γδ T cells. In some cases, the control γδ T cells do not contain a CAR construct. In some cases, the control γδ T cells contain a CAR construct lacking a binding domain, a hinge region, a transmembrane domain, a signaling domain, and / or a co-stimulatory endodomain as described herein.

[0134] In some cases, the cytotoxicity is at least partially, significantly (> about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to CD20 or an epitope within CD20. In some cases, γδ T cells functionally express a CD20-specific CAR encoded by the isolated nucleic acids described herein.

[0135] In some embodiments, the γδ T cells described herein can exhibit HLA-restricted (e.g., HLA class I-restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all of the cytotoxic activity is not restricted by HLA (e.g., HLA class I restriction). HLA-restricted cytotoxic activity can be evaluated by comparing in vitro cytotoxicity against HLA (e.g., HLA class I) (null) tumor cell lines to in vitro cytotoxicity against HLA+ (e.g., HLA class I+) tumor cell lines. In some embodiments, HLA-restricted cytotoxic activity is provided at least partially, significantly (>25%), or completely by the use of a T cell receptor-like binding domain. A T cell receptor, such as a binding domain, is a binding domain that specifically recognizes an antigen when presented on the surface of a cell in complex with an MHC molecule. T cell receptor-like binding domains are further described, for example, in WO2016 / 199141.

[0136] The γδ T cells described herein are capable of exhibiting potent and / or sustained hematological tumor cell killing activity. In some cases, the hematological tumor cell killing activity can persist for at least about 6 days to 120 days, or at least about 6 days to 180 days from the first contact with the hematological tumor cells. In some cases, the hematological tumor cell killing activity of the γδ T cells or their progeny described herein can persist for at least about 6 days to 120 days, or at least about 6 days to 180 days from the first contact with the hematological tumor cells or from the administration of the γδ T cells described herein. This sustained hematological tumor cell killing activity can be demonstrated in vitro, in vivo, or both in vitro and in vivo.

[0137] Aspects of the invention may alternatively or additionally include γδ T cells that proliferate in response to contact with cells that exhibit cell surface expression or overexpression of a tumor-associated antigen (TAA). The cells that exhibit cell surface expression of a tumor-associated antigen (TAA) may be normal blood cells such as normal B cells. The cells that exhibit cell surface expression or overexpression of a tumor-associated antigen (TAA) may be hematological tumor cells. In some cases, the proliferation is an innate activity. In some cases, the proliferation is at least partially, significantly (> about 20% or > about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to a TAA expressed on the surface of blood cells or hematological tumor cells. In some cases, the γδ T cells exhibit a higher level of proliferation in vitro and / or in vivo compared to control γδ T cells. In some cases, the control γδ T cells do not contain a CAR construct. In some cases, the control γδ T cells contain a CAR construct lacking the binding domain described herein, the hinge region described herein, the transmembrane domain described herein, the signaling domain described herein, and / or the co-stimulatory endodomain described herein.

[0138] In some cases, proliferation is at least partially, significantly (> about 20% or > about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to CD20 or an epitope within CD20. In some cases, γδ T cells that exhibit proliferation in response to contact with blood cells or hematological tumor cells that show cell surface expression of CD20 functionally express a CD20-specific CAR encoded by the isolated nucleic acid described herein.

[0139] The γδ T cells described herein are capable of showing robust and / or sustained proliferation in a host organism comprising blood cells or hematological tumor cells that show cell surface expression or overexpression of a tumor-associated antigen (TAA). In some cases, proliferation can persist for at least about 6 days to 120 days, or at least about 6 days to 180 days, from the first contact with hematological tumor cells or from the date of administration of the γδ T cells to the host organism. In some cases, the proliferation of the γδ T cells or their progeny described herein in a host organism comprising blood cells or hematological tumor cells that show cell surface expression or overexpression of a tumor-associated antigen (TAA) can persist for at least about 6 days to 120 days, or at least about 6 days to 180 days, from the first contact with blood cells or hematological tumor cells or from the first date of administration of the γδ T cells to the host organism. In some cases, proliferation in the host organism is at least partially, significantly (> about 20% or > about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to CD20 or an epitope within CD20. In some cases, γδ T cells that exhibit proliferation in a host organism comprising blood cells or hematological tumor cells that show cell surface expression of CD20 functionally express a CD20-specific CAR encoded by the isolated nucleic acid described herein.

[0140] In some embodiments, the γδ T cells described herein express, or continuously express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with blood cells or blood tumor cells. In some embodiments, the γδ T cells described herein or their progeny express, or continuously express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with blood cells or blood tumor cells, such as in a host organism containing blood cells or blood tumor cells.

[0141] In some embodiments, γδ T cells or a pharmaceutical composition containing γδ T cells, when introduced into an allogeneic host, do not substantially exhibit, or do not exhibit, a graft-versus-host response. In some embodiments, γδ T cells or a pharmaceutical composition containing γδ T cells, when introduced into an allogeneic host, exhibit a graft-versus-host response at a clinically acceptable level. In some embodiments, a clinically acceptable level is the amount of graft-versus-host response that does not require discontinuation of γδ T cell therapy to achieve a therapeutically effective treatment. In some embodiments, a clinically acceptable level of graft-versus-host response (GvHD) is an acute response with a severity lower than grade C according to the applicable IBMTR assessment scale. The severity of acute graft-versus-host reaction is determined by evaluating the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of lesions in individual organs are combined to create an overall grade with prognostic significance. Grade I (A) GvHD is a mild disease, grade II (B) GvHD is moderate, grade III (C) is severe, and grade IV (D) is considered life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997;97:855): ● Grade A - No lesions in the liver or gastrointestinal tract, only stage 1 skin lesions (maculopapular rash in <25% of the body) ● Grade B - Stage 2 skin lesions, stage 1 to 2 intestinal or liver lesions ● Grade C - Stage 3 lesions of any organ system (generalized erythroderma, bilirubin 6.1 - 15.0 mg / dL, diarrhea 1500 - 2000 mL / day) ● Grade D - Stage 4 lesions of any organ system (generalized erythroderma with blister formation, bilirubin > 15 mg / dL, diarrhea > 2000 mL / day or pain or ileus). See also Tables 1 and 2 of Schoemans et al., Bone Marrow Transplantation volume 53, pages 1401 - 1415 (2018). This also discloses criteria for evaluating and grading acute GvHD.

[0142] In some embodiments, the γδ T cells, or a pharmaceutical composition comprising γδ T cells, when introduced into an allogeneic host, exhibit a decreased or substantially decreased graft - versus - host response as compared to the graft - versus - host response exhibited by control αβ T cells or a control pharmaceutical composition comprising control αβ T cells administered to the allogeneic host. In some cases, the control αβ T cells are allogeneic, unmanipulated control αβ T cells. In some cases, the control αβ T cells do not contain a CAR or do not contain the same CAR as the reference γδ T cells.

[0143] The γδ T cells described herein can be δ1, δ2, δ3 or δ4 γδ T cells, or combinations thereof. In some cases, the γδ T cells are mostly (> 50%), substantially (> 90%), essentially all, or completely δ2 - γδ T cells. In some cases, the γδ T cells are mostly (> 50%), substantially (> 90%), essentially all, or completely δ1 γδ T cells.

[0144] γδ T cells are obtainable from allogeneic or autologous donors. γδ T cells can be partially or fully purified or unpurified and amplified ex vivo. Methods and compositions for ex vivo amplification include, but are not limited to, those described in WO2017 / 197347. Amplification can be performed before, after, or before and after the CAR construct is introduced into the γδ T cell(s).

[0145] The γδ T cells described herein can be stored, for example, by cryopreservation, for use in adoptive cell transfer.

[0146] Methods of inhibiting or killing tumor cells One or more unmanipulated γδT cell populations, manipulated γδT cell populations, and / or mixtures thereof having cytotoxic activity against hematological tumor cells can be administered to a subject in any order or simultaneously. When administered simultaneously, the multiple unmanipulated γδT cell populations, manipulated γδT cell populations, and / or mixtures thereof of the present invention can be provided in a single unified form such as intravenous injection, or in multiple forms such as multiple intravenous infusions, subcutaneous injections, or tablets. The unmanipulated γδT cell populations, manipulated γδT cell populations, and / or mixtures thereof of the present invention can be packaged together or separately in a single package or multiple packages. One or all of the unmanipulated γδT cell populations, manipulated γδT cell populations, and / or mixtures thereof of the present invention can be administered in multiple doses. When not administered simultaneously, the timing between multiple administrations can vary from about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the unmanipulated enriched γδT cell populations, manipulated enriched γδT cell populations, and / or mixtures thereof of the present invention can proliferate in vivo, in the subject's body, after administration to the subject. It is possible to freeze one or more unmanipulated γδT cell populations, one or more manipulated γδT cell populations, and / or mixtures thereof to provide cells for multiple treatments with the same cell preparation. One or more unmanipulated γδT cell populations, one or more manipulated γδT cell populations, and / or mixtures thereof of the present disclosure, and pharmaceutical compositions containing the same, can be packaged as a kit. The kit can include instructions (such as written instructions) regarding the use of the unmanipulated γδT cell populations, manipulated γδT cell populations, and / or mixtures thereof, and the compositions containing the same.

[0147] In some cases, a method of treating blood cancer involves administering to a subject a therapeutically effective amount of an unengineered γδ T cell population, an engineered γδ T cell population, and / or a mixture thereof, wherein administering treats the blood cancer. In some embodiments, the therapeutically effective amount of the unengineered γδ T cell population, the engineered γδ T cell population, and / or the mixture thereof is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of the unengineered γδ T cell population, the engineered γδ T cell population, and / or the mixture thereof is administered for at least 1 week. In some embodiments, the therapeutically effective amount of the unengineered γδ T cell population, the engineered γδ T cell population, and / or the mixture thereof is administered for at least 2 weeks.

[0148] The non-gene-manipulated γδT cell population, gene-manipulated γδT cell population, and / or mixtures thereof described herein can be administered before, during, or after the onset of a disease or symptom, and the timing of administration of the pharmaceutical composition containing the γδT cell population can vary. For example, the γδT cell population can be used as a prophylactic agent and can be continuously administered to a subject with a tendency for a symptom or disease to reduce the likelihood of the occurrence of the disease or symptom. The first administration can be carried out via any practical route, such as by any of the routes described herein using any of the formulations described herein. In some embodiments, the administration of the γδT cell population of the present disclosure is intravenous administration. The single or multiple doses of the γδT cell population can be administered as soon as practicable after the onset of blood cancer, and, for example, for the time required to treat an immune disease for about 24 hours to about 48 hours, about 48 hours to about 1 week, about 1 week to about 2 weeks, about 2 weeks to about 1 month, about 1 month to about 3 months. In some embodiments, the single or multiple doses of the γδT cell population can be administered for years after the onset of cancer and before and after other treatments.

[0149] In some embodiments, the γδ T cell population is administered simultaneously or sequentially in one or more ways that increase common gamma chain cytokine(s). As used herein, "one or more ways that increase common gamma chain cytokine(s)" refers to a method or combination of methods that change the physiological state of a subject such that the level of at least one common gamma chain cytokine is increased in the subject. In some embodiments, the method increases the level of one or more common gamma chain cytokine(s) selected from the group consisting of IL-2, IL-7, and IL-15, and preferably, in which case, the method increases the level of IL-15 in the subject. In some embodiments, the method includes lymphodepletion. In some embodiments, the method includes administering to the subject one or more common gamma chain cytokine(s). Optionally, IL-2, IL-7, and / or IL-15, preferably IL-15, is administered. In some embodiments, the method includes secreting common gamma chain cytokine(s) from the administered γδ T cells and the like. Optionally, IL-2, IL-7, and / or IL-15, preferably IL-15, is secreted.

[0150] In some embodiments, one or more dosing methods for increasing common gamma chain cytokine(s) include lymphodepletion prior to introduction of γδ T cell(s). In some embodiments, one or more dosing methods for increasing common gamma chain cytokine(s) include, concurrent with the introduction of γδ T cell(s) or by continuously administering an effective amount of common gamma chain cytokine(s), increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδ T cell(s), preferably wherein the method includes administering IL-2 or one or more mimetics thereof, more preferably wherein the method includes administering IL-15 or one or more mimetics thereof. The dosage of common gamma chain cytokine(s) can increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδ T cell(s) before and / or after introduction of the γδ T cell(s). Exemplary amounts of IL-15 include, but are not limited to, between 0.01 and 10 μg / kg / dose every 24 hours for IL-15. Exemplary amounts of IL-2 include, but are not limited to, between about 3×10 6 and about 22×10 6 units, but are not limited thereto. For example, the dosing schedule of IL2 in RCC is intravenous injection of 600,000 international units / kg (0.037 mg / kg) over 15 minutes for up to 14 administrations, every 8 hours.

[0151] In some embodiments, one or more dosing methods for increasing common gamma chain cytokine(s) include lymphodepletion prior to administration of γδ T cell(s), prior to concurrent administration with the introduction of γδ T cell(s), or prior to continuous administration of common gamma chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδ T cell(s).

Example

[0152] Example 1 1x106 Human PBMCs at / mL were activated in the culture medium of modified cells on pre-coated anti-Vδ1 antibodies D1-08 or D1-35 in a 24-well plate (Costar) in the presence of IL-2 (100 U / mL) for 5 days (see WO2017 / 197347). On the 5th day, in the presence of RetroNectin, the cell culture was transduced with a γ-retroviral construct encoding chimeric antigen receptors (2B7-5.1, SEQ ID NO: 11, 3B9-5.1, SEQ ID NO: 9, 3H7-5.1, SEQ ID NO: 10, 9C11-5.1, SEQ ID NO: 12). On the 6th day, the cells were returned to the culture medium of the modified cells and further expanded by feeding and replenishing IL-2 as needed. Cells were harvested on days 17, 18, or 19, and the remaining αβ T cells were depleted using an AutoMACS® kit (Miltenyi Biotec). The purity and transduction efficiency of the γδ cell population were evaluated by FACS. In parallel, non-transduced cell cultures were grown in the same manner without adding the supernatant of the retrovirus. As shown in Figure 3, the non-transduced expanded Vδ1 cells obtained from multiple donors did not show cytotoxicity against normal B cells of allogeneic donors. Introduction of CD20CAR into Vδ1 cells conferred strong cytotoxicity to these cells against normal B cells. Cytotoxicity was measured by flow cytometry as % annexin V+ cells in a 4-hour assay.

[0153] Example 2 Vδ1 cells were activated, transduced, and expanded in the same manner as described above. The 3H7 CAR construct, SEQ ID NO: 10, was used to demonstrate cytotoxicity against two CD20+ cell lines, Daudi and Raji. As shown in Figure 4, introduction of the CAR increased the innate cytotoxicity of unmanipulated Vδ1 cells.

[0154] Example 3 Vδ1 cells were activated, transduced, and proliferated in the same manner as described above. Four different constructs (SEQ ID NOs: 9, 10, 11, 12) were introduced into Vδ1 cells during proliferation and tested against Raji-Luc cells. Cytotoxicity was measured by total luminescence measurement after adding the luminescent substrate D-luciferin (Perkin Elmer) after co-culturing for 18 hours at various E / T ratios. As shown in FIG. 5, the anti-CD20 CAR cells containing the 4-1BB co-stimulatory endodomain described herein exhibited potent cytotoxic activity against Raji cells.

[0155] Example 4 CAR constructs were made with several different domains and CD20 binding domains (3H7-CD3z, SEQ ID NO: 20, 3H7-5.1, SEQ ID NO: 10, 3H7-CD27z, SEQ ID NO: 8). As described above, the CAR constructs were introduced and cytotoxicity was tested against Raji-Luc cells as described in the previous example (18-hour cytotoxicity at various E / T ratios). FIG. 6 shows potent cytotoxic activity against Raji cells having various signaling and / or co-stimulatory endodomain(s).

[0156] Example 5 Various CAR constructs were introduced into proliferated Vδ1 cells and tested in a long-term cytotoxicity assay (serial killing) by readministration of target cells using an IncuCyte® instrument. Briefly, Raji cells were labeled with a NucRed reagent and the total fluorescence of the cells was recorded over time. Without adding any cytokines, the cells were co-cultured in growth medium at an E / T ratio of 3 for 72 hours. After 72 hours, the culture was readministered with another dose of Raji cells and monitored for death. This procedure was repeated up to 144 hours in cultures from which Raji cells had been removed. In FIG. 7, 3H7-ICOSz is a construct in which the 4-1BB co-stimulatory endodomain has been replaced with the ICOS endodomain (WLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 354)).

[0157] Example 6 Raji cells were subcutaneously transplanted into NSG mice (Jackson Labs). When the tumor reached approximately 100 mm 3 in size, the animals were treated with 5×10 6 Vδ1CD20CAR+ cells to compare the effectiveness of various co-stimulatory co-stimulatory end domains (“co-stim” or “costim”). The animals were co-administered IL-2 (60,000 U / dose) three times a week until the end of the study. As shown in Figure 8, the constructs tested showed strong in vivo efficacy in treating hematological tumors in NSG mice. Without being bound by theory, it is hypothesized that the optimized CAR constructs of 3H7-5.1, 3H7-CD3z and 3H7-CD27z have superior in vivo tumor control, proliferation, activation, persistence and / or cytotoxicity compared to the non-optimized CAR constructs.

[0158] Example 7 Raji cells were subcutaneously transplanted into NSG mice (Jackson Labs). When the tumor reached approximately 100 mm 3 in size, the animals were treated with 5×10 6 Vδ1CD20CAR+ cells pre-labeled with CellTrace Violet. On days 2 and 6, the tumors and various other organs were extracted, digested, and the resulting cell suspensions were analyzed for (A) the presence of γδ T cells and Raji cells (Figure 9) and (B) the proliferation of γδ cells as demonstrated by CellTrace Violet dye dilution (Figure 10). The animals were co-administered IL-2 (60,000 U / dose) three times a week until day 6. As shown in Figure 9, the introduced γδ T cells strongly increased in the tumor microenvironment and promoted a significant decrease in the ratio of tumor cells to γδ T cells from day 2 to day 6. As shown in Figure 10, the γδ T cells proliferated strongly and preferentially in the intratumoral space.

[0159] Example 8 NSG mice were inoculated with Raji-Luc cells (0.5 ml / animal). On day 4, the animals were treated with 8.7x10 6 Vδ1CAR+ cells (SEQ ID NO: 10) or 6.8x10 transduced with the same construct5 Treated with αβ T cells. The survival of the animals was monitored over 140 days. All animals received three administrations of IL-2 (60,000 / dose) on days 0, 1, and 2 (Figure 11). As shown in Figure 11, administration of the γδ T cells described herein increased the survival time of subjects with blood cancer.

[0160] Example 9 SRG-15 mice expressing human IL-15 (Herndler-Brandstetter et al., PNAS, 2017) were inoculated with Raji-Luc cells (0.5 x 10 6 / animal). On day 4, the animals were treated with 20.2 x 10 6 Vδ1 CAR+ cells (SEQ ID NO: 10) or 1.9 x 10 6 αβ T cells transduced with the same construct. The survival of the animals was monitored over 70 days. (Figure 12). As shown in Figure 12, the introduced γδ T cells did not induce a GvHD response. In contrast, the introduced αβ T cells induced a GvHD response.

[0161] Example 10 NSG mice were subcutaneously inoculated with Raji cells (1 x 10 6 / animal) in the right hind flank. When the tumor volume reached approximately 100 mm 3 , the mice were randomized and treated with CD20 CAR or 5 x 10 6 Vδ1 CAR T cells encoding CARCD20 and soluble IL-15. The animals were co-administered IL-2 (60,000 U / dose, Peprotech, three times a week) until the end of the study. On day 62, four animals in the CD20 + sIL15CAR T group with no measurable tumors were re-administered 1 x 10 6 Raji cells subcutaneously in the opposite (left) flank. A control group of animals was also included to demonstrate the tumor growth kinetics. The results are shown in Figure 14. As shown in Figure 14, administration of γδ CAR-T cells having a nucleic acid construct encoding the heterologous soluble IL-15 resulted in a sustained anti-tumor effect lasting beyond 60 days (e.g., 60 to 110 days).

[0162] Example 11 In a 24-well plate (Costar) pre-coated with anti-Vδ1 antibody D1-08 or D1-35 for 5 days in the presence of IL-2 (100 U / mL), 1x10 6 / mL of human PBMCs in the growth medium were activated. On day 5, the cell cultures were transduced with a γ-retroviral construct encoding the BCMA chimeric antigen receptor (SEQ ID NOs: 35-38) in the presence of Retronectin. On day 6, the cells were returned to the growth medium and further expanded by feeding and replenishment of IL-2 as needed. Cells were harvested on days 17, 18, or 19, and the remaining αβ T cells were depleted using an AutoMACS® kit (Miltenyi Biotec). The purity and transduction efficiency of the γδ cell population were evaluated by FACS (Figure 15). Briefly, CAR-T cells were stained by incubating the cells with 1 μg / mL of soluble recombinant biotinylated BCMA (Acro Biosystems). Detection of binding was performed using streptavidin-BV421 at a dilution of 1:500 recommended by the manufacturer. In parallel, non-transduced cell cultures were grown in the same manner without adding the retroviral supernatant. The expanded cells were tested in an in vitro cytotoxicity assay against BCMA-positive cell lines. As shown in Figures 16 and 17, non-transduced expanded Vδ1 cells induced a certain degree of cytotoxicity against multiple myeloma and Burkitt lymphoma cell lines, which are known to express BCMA to varying degrees. This cytotoxicity was enhanced by introducing the BCMA CAR construct. Cytotoxicity was measured by total luminescence measurement in a 96-well plate after adding the luminescent substrate D-luciferin (Perkin Elmer) after co-culture for 18 hours at the indicated E / T ratio. The BCMA-negative SCABER cell line was used as a control.

[0163] Example 12 NCI-H929 multiple myeloma cells (1x10 6 / animal) were subcutaneously transplanted into NSG mice (Jackson Labs). When the tumors reached approximately 200 mm 3Once the animals reached the appropriate size, they were treated with 5×10 6 Vδ1 BCMA CAR+ cells to compare the in vivo efficacy of the CAR constructs derived from 16716P and 16747P scFv. The animals were co-administered with IL-2 (13,000 IU / dose, Proleukin®) three times a week until the end of the study. The results are shown in Figure 18. As shown in Figure 19, the anti-BCMA CAR+ cells demonstrated potent in vivo tumor burden control.

[0164] The foregoing merely illustrates the principles of the present invention. It will be appreciated by those skilled in the art that, although not explicitly described or illustrated herein, various configurations that embody the principles of the present invention and fall within its spirit and scope can be devised. Further, all examples and conditional language recited herein are principally intended to aid the reader's understanding of the concepts contributed by the inventors to promote the present invention and its technology, and are not to be construed as being limited to such specifically recited examples and conditions. Moreover, all descriptions in this specification listing the principles, aspects, and specific examples of the present invention are intended to encompass both structural equivalents and functional equivalents thereof. Further, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function regardless of structure. Accordingly, the scope of the present invention is not intended to be limited to the exemplary aspects shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims.

Claims

**Claim 1** A δ1γδ T cell, comprising: a. a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (i) a binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of hematological tumor cells; (ii) a CD8α hinge domain; (iii) a CD8α transmembrane domain; (iv) a co-stimulatory signaling region; and (v) a CD3ζ signaling domain; or b. a polypeptide comprising a CAR comprising the amino acid sequence encoded by the nucleic acid of a. above, wherein the δ1γδ T cell functionally expresses the binding domain of the polypeptide or the CAR encoded by the nucleic acid on the surface of the δ1γδ T cell. **Claim 2** The δ1γδ T cell according to claim 1, wherein the co-stimulatory signaling region is selected from a 4-1BB (CD137) co-stimulatory signaling region and a CD27 co-stimulatory signaling region. **Claim 3** The δ1γδ T cell according to claim 1 or 2, wherein (i)-(v) are in the order from 5' to 3'. **Claim 4** The δ1γδ T cell according to any one of claims 1 to 3, wherein the binding domain specifically binds to CD20. **Claim 5** The δ1γδ T cell according to claim 4, wherein the binding domain comprises complementarity-determining regions of an anti-CD20 antibody selected from the group consisting of 3B9, 3H7, 2B7, and 9C11. **Claim 6** The δ1γδ T cell according to claim 5, wherein the anti-CD20 antibody is 3H7. **Claim 7** The δ1γδ T cell according to any one of claims 1 to 6, wherein the binding domain comprises: a. the heavy chain variable region (HCVR) and light chain variable region (LCVR) sequences having SEQ ID NOs: 99 and 107, respectively; b. the heavy chain complementarity-determining regions 1, 2, and 3 sequences of SEQ ID NOs: 101, 103, and 105, respectively, and the light chain complementarity-determining regions 1, 2, and 3 sequences of SEQ ID NOs: 109, 111, and 113, respectively, wherein the binding domain specifically binds to CD20; and / or c. heavy chain variable region (HCVR) sequences and light chain variable region (LCVR) sequences selected from the group consisting of SEQ ID NOs: 339 and 347, 195 and 203, and 243 and 251. **Claim 8** The δ1γδ T cell according to any one of claims 1 to 3, wherein the binding domain specifically binds to CD19 or BCMA. **Claim 9** ​ ​ The δ1γδ T cell according to claim 8, wherein the binding domain specifically binds to BCMA.

10. The δ1γδ T cell according to claim 9, wherein the binding domain comprises complementarity-determining regions of an anti-BCMA binding region having a sequence selected from the group consisting of SEQ ID NO: 27 and 28, SEQ ID NO: 29 and 30, and SEQ ID NO: 31 and 32.

11. The CAR is a. a CD8α hinge domain comprising SEQ ID NO: 1 (PTPAPTIA SQPLSLRP EACRPAAGG AVHTRGLDF ACDIY) or SEQ ID NO: 2 (TTTPAPRPP TPAPTIA SQPLSLRP EACRPAAGG AVHTRGLDF ACDIY); b. a CD8α transmembrane domain comprising SEQ ID NO: 3 (IWAPLAGTC GVLLLLSLV ITLYC) and / or c. (i) SEQ ID NO: 4 (RVKFSSRSA DAPAYQQGQ NQLYNE LNLGRREEY DVLDKRRGR DPEMGGKPR RKNPQEGLY NELQKD KMAEAYS EIGMKGERRR GKGHDG LYQG LSTATKDTY DALHMQALPPR) or (ii) a CD3ζ signal transduction domain comprising SEQ ID NO: 5 (RVKFSSRSA DAPAYQQGQ NQLYNE LNLGRREEY DVLDKRRGR DPEMGGKPR RKNPQEGLY NELQKD KMAEAYS EIGMKGERRR GKGHDG LYQG LSTATKDTY DALHMQALPPR) The δ1γδ T cell according to any one of claims 1 to 10, comprising.

12. The CAR is a. a 4-1BB co-stimulatory signal transduction region comprising SEQ ID NO: 6 (KRGRKKLLY IFKQPFMRP VQTTEEDGC SCRFPEEEEE GGCE) or b. a CD27 co-stimulatory signal transduction region comprising SEQ ID NO: 7 (QRRKYRSNK GESPVEP AEPC HYSCPREEEE GSTIPIQEDY RKPEPACS) comprising, or The δ1γδ T cell according to claim 11, wherein the nucleic acid encodes the 4-1BB co-stimulatory signal transduction region comprising SEQ ID NO: 6 and the CD27 co-stimulatory signal transduction region comprising SEQ ID NO:

7.

13. The nucleic acid is a. a secreted cytokine b. a secreted common gamma chain interleukin; c. a secreted IL-15; or d. A secreted common gamma chain interleukin and a multi-cistronic linker region at the amino terminus of the interleukin or interleukin secretion signal The δ1γδ T cell according to any one of claims 1 to 12, further encoding the same.

14. The IL-15 in c. is The sequence of SEQ ID NO: 34, The sequence of SEQ ID NO: 34 operably linked to the secretion signal sequence of SEQ ID NO: 33, or The sequence of SEQ ID NO: 34 operably linked to the secretion signal sequence of SEQ ID NO: 49 The δ1γδ T cell according to claim 13, comprising the same.

15. The secreted common gamma chain interleukin in d. is IL-15, and the multi-cistronic linker region is: Comprising any one sequence of SEQ ID NOs: 43-45, 47 or 52-55 or combinations thereof, or Encoding an internal ribosome entry site comprising the sequence described in SEQ ID NO: 56 or 60 The δ1γδ T cell according to claim 13.

16. The binding domain specifically binds to CD20, and the nucleic acid encodes the peptide described in SEQ ID NOs: 8, 9, 10, 11, 12, 20, 46, 48, 57, or 58. The δ1γδ T cell according to any one of claims 1 to 7 or 11 to 15.

17. The nucleic acid comprises the sequence of SEQ ID NOs: 13, 14, 15, 16, 17, 50, 51, or 59. The δ1γδ T cell according to claim 16.

18. The binding domain specifically binds to BCMA, and the nucleic acid encodes the peptide described in SEQ ID NOs: 35, 36, 37, or 38. The δ1γδ T cell according to any one of claims 1 to 3 or 8 to 15.

19. The nucleic acid comprises the sequence of SEQ ID NOs: 39, 40, 41, or 42. The δ1γδ T cell according to claim 18.

20. The δ1γδ T cell proliferates in response to contact with a cell showing cell surface expression or overexpression of a tumor-associated antigen (TAA), and the cell showing cell surface expression of the tumor-associated antigen (TAA) is a normal blood cell. The δ1γδ T cell according to claim 1.

21. The δ1γδ T cell proliferates in response to contact with a cell showing cell surface expression or overexpression of a tumor-associated antigen (TAA), and the cell showing cell surface expression or overexpression of the tumor-associated antigen (TAA) is a hematological tumor cell. The δ1γδ T cell according to any one of claims 1 to 20.

22. A cell population comprising more than 50% of the δ1γδ T cells according to any one of claims 1 to 21.

23. At least 10 ８ The cell population according to claim 22, comprising δ1γδ T cells.

24. 10 ８ δ1γδ T cells of 10 １１ The cell population according to claim 22, comprising δ1γδ T cells of 10

25. A cell population according to any one of claims 22 to 24, comprising at least 60% δ1γδ T cells.

26. A cell population according to claim 25, comprising 60% to 95% δ1γδ T cells.

27. A method for producing the δ1γδ T cells according to any one of claims 1 to 21 or the cell population according to any one of claims 22 to 25, said method comprising transfecting δ1γδ T cell(s) with a construct comprising the nucleic acid according to any one of claims 1 to 21.

28. The method according to claim 27, wherein said method comprises retroviral transduction.

29. The method according to claim 27 or 28, comprising ex vivo expansion of said δ1γδ T cell(s), said ex vivo expansion being performed before and / or after transfection of said nucleic acid.

30. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the δ1γδ T cells according to any one of claims 1 to 21, or the cell population according to any one of claims 22 to 25.

31. A medicament for killing hematological tumor cells, comprising the δ1γδ T cells according to any one of claims 1 to 21, which is in an amount effective for killing tumor cells, the cell population according to any one of claims 22 to 25, or the pharmaceutical composition according to claim 30, wherein said hematological tumor cells are contacted with the δ1γδ T cells, the cell population, or the pharmaceutical composition, which is in an amount effective for killing tumor cells.

32. The medicament according to claim 31, wherein the δ1γδ T cell(s) or the pharmaceutical composition, which is in a therapeutically effective amount, is introduced into a host organism comprising said hematological tumor cells.

33. The medicament according to claim 32, wherein the δ1γδ T cell(s) or the pharmaceutical composition, which is in a therapeutically effective amount, is introduced into a host organism comprising said hematological tumor cells, and simultaneously or sequentially, one or more agents that increase common gamma chain cytokine(s) are administered.

34. Administering one or more agents that increase the common gamma chain cytokine(s) comprises administering an effective amount of the common gamma chain cytokine(s) simultaneously with or subsequent to the introduction of the delta1gamma delta T cell(s), thereby increasing the proliferation, cytotoxic activity, persistence, or combination thereof of the introduced delta1gamma delta T cell(s). The pharmaceutical according to claim 33.

35. The pharmaceutical according to claim 34, wherein the common gamma chain cytokine(s) is / are IL-2 and / or IL-15.

36. The pharmaceutical according to claim 34 or 35, wherein one or more agents that increase the common gamma chain cytokine(s) comprise an effective amount of the common gamma chain cytokine(s) to increase the proliferation, cytotoxic activity, persistence, or combination thereof of the introduced delta1gamma delta T cell(s) before and / or after introducing the delta1gamma delta T cell(s).

37. The pharmaceutical according to any one of claims 33 to 36, wherein one or more agents that increase the common gamma chain cytokine(s) deplete lymphocytes before introducing the delta1gamma delta T cell(s).

38. The pharmaceutical according to any one of claims 33 to 36, wherein one or more agents that increase the common gamma chain cytokine(s) induce secretion of one or more common gamma chain cytokine(s) from the introduced delta1gamma delta T cell(s).

39. The pharmaceutical according to any one of claims 32 to 38, wherein the pharmaceutical decreases the in vivo tumor burden of the host organism and / or increases the mean survival time of the host organism as compared to a control organism, wherein the control organism is not treated with the delta1gamma delta T cell(s) or the pharmaceutical composition.

40. The pharmaceutical according to any one of claims 31 to 39, wherein the pharmaceutical is for treating cancer in a subject in need of treatment.

41. Use in the manufacture of a pharmaceutical for treating hematological tumor cell cancer in a subject in need of treatment of a delta1gamma delta T cell according to any one of claims 1 to 21, a cell population according to any one of claims 22 to 25, or a pharmaceutical composition according to claim 30, wherein the amount is effective for killing tumor cells.

42. A pharmaceutical composition for treating cancer in a subject in need of treatment, a. A therapeutically effective amount of the δ1γδ T cells according to any one of claims 1 to 7, 11 to 17, 20, or 21, wherein the cancer comprises hematological tumor cells that exhibit cell surface expression of CD20; or, b. A therapeutically effective amount of the δ1γδ T cells according to any one of claims 1 to 3, 8 to 10, 11 to 15, or 18 to 21, wherein the cancer comprises hematological tumor cells that exhibit cell surface expression of BCMA, comprising a pharmaceutical composition.

43. The pharmaceutical composition according to claim 42, wherein one or more agents that increase common gamma chain cytokine(s) are administered simultaneously with, or consecutively with, the administration of the δ1γδ T cells.

44. The pharmaceutical composition according to claim 42 or 43, wherein multiple administrations of the δ1γδ T cells are performed, and the interval between the multiple administrations is at least one week.

45. The pharmaceutical composition according to claim 43 or 44, wherein multiple administrations of the δ1γδ T cells are performed no more than once every six months.

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