Compositions and methods relating to genetically engineered and non-genetically engineered gamma delta T cells for treating solid tumors

Abstract

Aspects of the present invention include compositions and methods for treating solid 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-TryD binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, a combination thereof, or all of these. The CAR construct can include an anti-GPC3 binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a CD3ζ signaling domain, a combination thereof, or all of these. The CAR construct can include a domain encoding a secreted general gamma chain cytokine, such as a sIL15 domain.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 739,826, filed on October 1, 2018, the content of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The above - mentioned ASCII copy, created on January 8, 2020, is named ADC - 0006 - PCT_SL.txt and is 48406 bytes in size.

Background Art

[0003] Adoptive immunotherapy has been continuously repeated for over 30 years, from its early focus on basic lymphokine activation and / or tumor infiltration to more recent strategies of genetically engineering these immune cells to express genetically engineered antigen receptors such as chimeric antigen receptors (CARs). Among them, although there have been some hints and signs about the curative potential of these approaches, 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, it may induce graft-versus-host effects in patients. Furthermore, especially in solid tissues, there is a problem that there is no available positive stimulus and there is an inhibitory environment. Therefore, in this technical field, numerous co-stimulation strategies have been tested for both T cells and NK cells, especially αβ T cells, with the aim of balancing efficacy and safety. Notably, given 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 γδ T cells is uncertain at best. 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] Therefore, improved strategies are still needed to improve the specificity or selectivity of cells, for example, to improve the safety of cells by reducing or avoiding graft-versus-host (GVH) effects, to avoid suppression of effector function, to improve the efficacy against solid tumor cells, and to improve the activity and / or survival of cells upon administration to a subject. Provided are methods, cells, compositions, kits, and systems that meet such needs. SUMMARY OF THE INVENTION

[0005] Aspects of the invention are isolated nucleic acid sequences encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a binding domain that specifically binds to a protein-peptide complex comprising a tumor-associated antigen (TAA) peptide and an MHC protein, wherein the complex is expressed on the surface of solid tumor cells, and optionally, wherein the binding domain binds to the complex in an HLA-restricted manner, and an isolated nucleic acid sequence encoding a CD8α hinge domain, a CD8α transmembrane domain, a co-stimulatory signaling region selected from a 4-1BB co-stimulatory signaling region and a CD27 co-stimulatory signaling region, and a CD3ζ signaling domain. Aspects of the invention further comprise non-genetically engineered γδT cells as described herein, and genetically engineered γδT cells comprising a nucleic acid encoding the CAR construct described herein, wherein the γδT cells functionally express the nucleic acid encoding the CAR on the surface of the γδT cells.

[0006] Aspects of the invention further comprise a plurality of γδT cells as described herein. Aspects of the invention further comprise a method of making the γδT cells or plurality of γδT cells described herein. Aspects of the invention further comprise a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the γδT cells or plurality of γδT cells described herein. Aspects of the invention further comprise contacting solid tumor cells with an amount of γδT cells or a plurality of γδT cells effective for tumor cell injury as described herein.

[0007] In one aspect, the present 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 protein-peptide complex comprising a tumor-associated antigen (TAA) peptide and an MHC protein, wherein the complex is expressed on the surface of solid tumor cells and optionally, wherein the binding domain binds to the complex with HLA restriction; (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 a combination of co-stimulatory signaling regions, optionally, wherein the co-stimulatory signaling region(s) is / are a co-stimulatory signaling region(s) selected from the 4-1BB (CD137) co-stimulatory signaling region and the CD27 co-stimulatory signaling region; and (e) a signaling domain such as a CD3ζ signaling domain. In some embodiments, the aforementioned 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 TAA comprises the adjacent region of TyrD. In some embodiments, the adjacent region of TyrD comprises at least 4, or at least about 4 and up to 12, or up to about 12 adjacent amino acids of TyrD, preferably 7, 8 or 9 or preferably about 7, 8 or 9 adjacent amino acids of TyrD. In some embodiments, the adjacent region of TyrD is TyrD 369-377 itself. In some embodiments, the binding domain that specifically binds to the TAA peptide-MHC complex specifically binds to HLA-A2 / TyrD 369-377

[0009] ​In some embodiments, the binding domain specifically binds to, or competes with, an epitope bound to an antibody comprising CDRH1 comprising TSGMGVS (SEQ ID NO: 33), CDRH2 comprising HIYWDDKRYNPSLKS (SEQ ID NO: 34), CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35), CDRL1 comprising KASQDIHNYIA (SEQ ID NO: 36), CDRL1 comprising YTSTLQP (SEQ ID NO: 37), and CDRL2 comprising LQYDNLWT (SEQ ID NO: 38).

[0010] In another aspect, the binding domain specifically binds to a tumor-associated antigen (TAA) expressed on the surface of solid tumor cells, and optionally, wherein the antigen is a protein-peptide complex, wherein the protein is an MHC protein, wherein the binding domain binds to the protein-peptide complex restricted by HLA, and further, the CAR encoded by the isolated nucleic acid sequence comprises (b) a hinge domain such as the CD8α hinge domain, (c) a transmembrane domain such as the CD8α transmembrane domain, (d) a co-stimulatory signaling region or a combination of co-stimulatory signaling regions, optionally, wherein the co-stimulatory signaling region is selected from the 4-1BB (CD137) co-stimulatory signaling region and the CD27 co-stimulatory signaling region, a co-stimulatory signaling region or a combination of co-stimulatory signaling regions, and (e) a signaling domain such as the CD3ζ signaling domain. In some embodiments, the aforementioned elements (a)-(e) are encoded in the 5' to 3' order on the sense strand of the isolated nucleic acid.

[0011] In some embodiments, the binding domain specifically binds to an epitope within GPC3 that is expressed on the surface of solid tumor cells. In some embodiments, the binding domain binds to the same GPC3 epitope as an antibody comprising the following complementarity determining regions (CDRs), and / or the following CDRs: CDRH1 comprising the sequence DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)), CDRH2 comprising the sequence ALDPKTGDTAYSQKFKG (SEQ ID NO: 41), CDRH3 comprising the sequence FYSYTY (SEQ ID NO: 42), CDRL1 comprising the sequence RSSQSLVHSNRNTYLH (SEQ ID NO: 43), CDRL2 comprising the sequence KVSNRFS (SEQ ID NO: 44), and / or CDRL3 comprising the sequence SQNTHVPPT (SEQ ID NO: 45), and competes with an antibody comprising the same for binding to the GPC3 epitope.

[0012] In some embodiments of any one of the foregoing aspects or embodiments of the CAR-encoding nucleic acids described herein or any thereof, the encoded CAR comprises a CD8α hinge domain comprising SEQ ID NO: 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY), and / or a CD8α transmembrane domain comprising SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC), and / or a CD3ζ signaling domain. Optionally, the CD3ζ signaling domain comprises the sequence of SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0013] In some embodiments, 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), or a nucleic acid sequence encoding 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: 12 or 26). In some embodiments, the isolated nucleic acid encodes secreted IL-15, and preferably, wherein IL-15 comprises the sequence of SEQ ID NO: 14, and more preferably, wherein IL-15 comprises 14 sequences operably linked to the secretion signal sequence of SEQ ID NO: 12, or wherein IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to the secretion signal sequence of SEQ ID NO: 26. 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 the 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: 15-17, 25 or 27-30 or a combination thereof, or encodes an internal ribosome entry site such as SEQ ID NO: 31 or 32.

[0016] In some embodiments, the secretion signal comprises the sequence of SEQ ID NO: 12 or SEQ ID NO: 26, preferably SEQ ID NO: 12, and / or the sIL15 domain comprises the sequence of SEQ ID NO: 14, and / or the P2A cleavage sequence comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 25, and / or the furin cleavage sequence comprises the sequence of SEQ ID NO: 16, and / or the CAR comprises the sequences of SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 14 in amino to carboxy order.

[0017] In some embodiments, the binding domain is HLA-A2 / TyrD 369-377Specifically binds to, and the nucleic acid encodes SEQ ID NO: 8 or SEQ ID NO: 18. In some embodiments, the binding domain specifically binds to GPC3, and the nucleic acid encodes SEQ ID NO: 20 or SEQ ID NO: 22. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 21, 23 or 24.

[0018] In another aspect, the present invention provides a polypeptide comprising a CAR binding domain, such as one of the polypeptides encoded by any one of the aforementioned nucleic acids, or a polypeptide described herein.

[0019] In another aspect, the present invention provides a γδ, T cell comprising, for example, a nucleic acid encoding a CAR as described herein or comprising a polypeptide as described above, wherein the cell functionally expresses the binding domain of the polypeptide or the nucleic acid encoding the CAR on the surface of the cell. In some embodiments, the cell exhibits in vitro and / or in vivo cytotoxic activity against solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the solid tumor cell cytotoxic activity of these cells is greater than the native level of in vitro and / or in vivo solid tumor cell cytotoxic activity in control cells that do not contain the CAR construct. In some embodiments, the cell is HLA class I + The solid tumor cell cytotoxic activity is increased against solid tumor cells. In some embodiments, the solid tumor cell cytotoxic activity or the increase in solid tumor cell cytotoxic activity persists for about, at least, or at least about 6 days to 180 days after the first contact with the solid tumor cells.

[0020] In some embodiments, the cells proliferate in response to contact with solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the cells exhibit increased proliferation in response to contact with solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA), as compared to control cells that do not functionally express a nucleic acid encoding a CAR on their surface. In some embodiments, the cells proliferate in a host organism that includes solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). In some embodiments, the cell proliferation or increased cell proliferation persists for about, at least, or at least about 6 days to 180 days after the initial contact with the solid tumor cells. In some embodiments, the cells express one or more pro-inflammatory cytokines, and optionally, wherein the one or more pro-inflammatory cytokines include a greater amount of tumor necrosis factor alpha and / or interferon gamma after contact with the solid tumor cells, preferably as compared to cells that are controls that do not functionally express a nucleic acid encoding a CAR on their cell surface.

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

[0022] In another aspect, the present invention provides a plurality of any one of the aforementioned cells such as, for example, γδ, T cells, or a plurality of cells such as, for example, γδ, T cells as described herein. In some embodiments, the plurality is, for example, at least about 10 8 γδ, T cells, etc., preferably about 10 8 cells, preferably about 10 8 cells, for example, from about 10 11 cells, for example, including about 10 - 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 cells such as, for example, γδT cells, preferably δ1 or δ2 γδT cells, more preferably δ2

[0023] γδT cells, most preferably a composition comprising δ1 γδT cells.

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

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and cells or a plurality of cells as described herein, such as γδ, T cells (plural), etc., as described herein.

[0025] In another aspect, the present invention provides a method for killing solid tumor cells, the method comprising contacting a solid tumor cell with an amount of the above-described cell or plurality of cells or pharmaceutical composition that is effective for tumor cell killing, or a cell or plurality of cells or pharmaceutical composition as described herein. In some cases, the cell or plurality of cells is, for example, γδ, T cells (plural).

[0026] In some embodiments, the method comprises introducing into a host organism containing solid tumor cells a therapeutically effective amount of a cell or pharmaceutical composition, such as γδ, T cells (plural), etc. In some embodiments, the method comprises introducing into a host organism containing solid tumor cells a therapeutically effective amount of a cell or its pharmaceutical composition, such as γδ, T cells (plural), etc., and simultaneously or sequentially administering one or more methods for increasing a common gamma chain cytokine (plural).

[0027] In some embodiments, one or more methods of raising a common gamma chain cytokine(s) increase the proliferation, cytotoxic activity, persistence, or combination thereof of the introduced cell(s) by administering, simultaneously with the introduction of the cell(s), or continuously administering an effective amount of the common gamma chain cytokine(s), 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 raising a common gamma chain cytokine(s) increase the proliferation, cytotoxic activity, persistence, or combination thereof of the introduced cell(s) before and / or after introducing the cell(s) by administering an effective amount of the common gamma chain cytokine(s).

[0028] In some embodiments, one or more methods of raising a common gamma chain cytokine(s) include lymphodepletion prior to introducing γδ T cell(s). In some embodiments, one or more methods of raising a common gamma chain cytokine(s) include secretion of one or more common gamma chain cytokine(s) from the introduced cell(s). In some embodiments, the method 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 cell(s) or pharmaceutical composition. In some embodiments, the method is a method of treating cancer in a subject in need of treatment.

[0029] In another aspect, the present invention provides the use of any one of the above-described cells, or cells as described herein (e.g., γδ T cells, etc.), a plurality of such cells, or such cells in the manufacture of a medicament for treating solid tumor cell cancer in a subject in need of treatment, in an amount effective for killing tumor cells. In another aspect, the present invention provides a method for treating cancer in a subject in need of treatment, the method comprising administering a therapeutically effective amount of cells, wherein the cancer comprises solid tumor cells that exhibit cell surface expression of TyrD or GPC3.

[0030] In some embodiments, the method comprises administering one or more methods for elevating common gamma chain cytokine(s) simultaneously with, or sequentially to, the administration of the cells. In some embodiments, the method comprises performing multiple administrations of the 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.

[0031] In another embodiment, the present invention provides a pharmaceutical composition for use in any one of the aforementioned methods or the methods described herein.

[0032] Incorporation by reference All publications, patents and patent applications mentioned herein are hereby incorporated by reference 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

[0033]

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Mode for Carrying Out the Invention

[0034] Definition: For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular include their plurals and vice versa. If any definition set forth conflicts with any document incorporated herein by reference, the definitions set forth below shall control. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] As used herein, the term "about," when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass variations of ±20%, ±10%, more preferably ±5%, still more preferably ±1%, and even still more preferably ±0.1% from a particular value as appropriate for practicing the disclosed method.

[0036] 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 -is. Compositions and methods for generating and using genetically engineered and non-genetically engineered γδ T cells and / or their subtypes include, but are 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 generating and using such 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.

[0037] As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses or has expressed CD3 (CD3+) and a 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 of CD3 and / or TCR from the cell surface.

[0038] 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. αβ TCR recognizes antigens presented by MHC molecules, while γδ TCR is capable of recognizing antigens independently of MHC presentation.

[0039] 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.

[0040] 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 functions. The term "activated T cell" refers, among other things, to a T cell that is undergoing cell division.

[0041] 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 also be the 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).

[0042] The term "antibody fragment" refers to a part of an intact antibody and to the variable antigen - determining regions 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.

[0043] 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.

[0044] 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.

[0045] 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 construed 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 wherein the DNA or amino - acid sequence is obtainable using available and well - known synthetic DNA or amino - acid sequence techniques in the art.

[0046] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can include either or both antibody production and 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 by a "gene" at all. It is readily apparent that an antigen can be produced, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0047] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant capable of specifically binding to an immunoglobulin or T cell receptor. Epitope determinants usually consist of the active surface groups of molecules such as amino acids, lipids or sugar side chains, and usually have 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.

[0048] 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 confers artificial specificity on 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 includes an intracellular activation domain (which can activate a T cell upon binding of a target cell, such as a target tumor cell, to the target moiety), a transmembrane domain, and an extracellular domain of variable length, and further includes, for example, a tumor-antigen binding region associated with a disease or disorder. In certain aspects, the CAR includes 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) 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 includes domains for adding co-stimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, ICOS, TLR (e.g., TLR2). In some cases, the molecule may be co-expressed with a CAR, such as a co-stimulatory molecule, a reporter gene for imaging (e.g., positron emission tomography), a gene product that conditionally excises a T cell when adding a prodrug, a homing receptor, a chemokine, a chemokine receptor, a cytokine, and a cytokine receptor. 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 induce a desired immune response.

[0049] 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 mean survival time, 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.

[0050] As used herein, the term "self-antigen" means any self-antigen that is erroneously recognized as foreign by the immune system in the context of the present invention. 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.

[0051] 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 re-introduced.

[0052] 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.

[0053] 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 desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of the composition that, when administered, is sufficient to prevent the onset of, or to alleviate to some extent, one or more symptoms or signs of a disease or disorder being treated (e.g., solid cancer). A therapeutically effective amount will vary depending on the composition, the disease and its severity, and the age, weight, etc. of the subject being treated.

[0054] As used herein, to "treat" a disease means to reduce the frequency or severity of at least one symptom or sign of the disease or disorder experienced by a subject.

[0055] Administration "in combination with" one or more therapeutic agents includes simultaneous (co - administration) and sequential administration in any order.

[0056] 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 undesired biological effects or acting in a harmful manner with any of the components of the composition in which it is included.

[0057] "Encoding" refers to the unique property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA, or mRNA, which serves as a template for 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.

[0058] "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.

[0059] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate versions and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns.

[0060] 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.

[0061] 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 by itself 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 by itself 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 of an antibody, protein or peptide with a second chemical species, where the interaction depends on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, an antibody is meant to recognize and bind to a particular protein structure rather than a protein in general. If an antibody is specific for epitope "A", in a reaction containing labeled "A" and the antibody, the amount of labeled A that binds to the antibody will decrease if a molecule containing epitope A (or free, unlabeled A) is present.

[0062] 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, and the like. Further, 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 nonetheless considered an antibody that "specifically binds" as used herein.

[0063] A solid tumor is a tumor that contains a tumor mass of at least about 10 or at least about 100 tumor cells. Solid tumors include soft tissue tumors, primary solid tumors, or metastatic lesions.

[0064] Examples of solid tumors include sarcomas, adenocarcinomas, and carcinomas of various organ systems, such as those affecting the liver, lung, breast, lymph, gastrointestinal tract (such as the colon), urogenital tract (kidney, urothelial cells, etc.), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. In one embodiment, the cancer is a melanoma such as advanced melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the present invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenocortical cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney cancer or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, spinal cord axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epithelioid carcinoma, squamous cell carcinoma, cancers caused by the environment including those caused by asbestos, and combinations of these cancers. In a preferred embodiment, the solid tumor cells express, or overexpress, TyrD or a fragment thereof. In some embodiments, the solid tumor cells express or overexpress an HLA:peptide complex comprising a TyrD fragment. In some embodiments, the TyrD fragment is TyrD 369-377It is. In some embodiments, the HLA is a class I HLA such as HLA-A2. In some embodiments, the solid tumor cells are HLA-A2 / TyrD 369-377 express or overexpress.

[0065] In some embodiments, the solid tumor cells express or overexpress glypican 3 (GPC3). In some embodiments, the solid tumor cells express or overexpress an epitope of GPC3 that is specifically bound by an anti-GPC3 antibody, T cell receptor, or chimeric antigen receptor as described in US7919086, WO2014 / 180306, WO2018 / 019772, WO2016 / 049459, WO2003 / 000883, WO2006 / 046751, WO2007 / 047291, WO2016 / 086813, WO2016 / 047722, WO2016 / 036973, Cancer Res. 2008;68:9832-9838, Proc Natl Acad Sci USA. 2013 Mar 19;110(12):E1083-91, the entire contents of each of which are incorporated by reference for all purposes, particularly the binding domain, antibody, antibody fragment, complementarity-determining region, polypeptide containing this complementarity-determining region, nucleic acid encoding this complementarity-determining region, and epitope specificity, as well as the assays for determining the epitope specificity described therein. In some embodiments, the solid tumor cells express or overexpress an epitope of glypican 3 that is specifically bound by the anti-GPC3 antibody GC33. In some embodiments, the solid tumor expresses or overexpresses an HLA:peptide complex containing a GPC3 fragment. In some embodiments, the HLA is a class I HLA such as HLA-A2. In some embodiments, the solid tumor is GPC3 144-152 expresses or overexpresses an HLA:peptide complex containing a peptide. In some embodiments, the solid tumor is GPC3 298-306 expresses or overexpresses an HLA:peptide complex containing a peptide. See Oncoimmunology. 2012 Nov 1;1(8):1448-1450.

[0066] An "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 provided 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-containing 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.

[0067] Range: Throughout this disclosure, various aspects of the invention may be presented in 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 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., 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.

[0068] 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 +It is a component of T cells. In some embodiments, the nucleic acid is, for example, heterologous, α - T cells and / or β - It is a component of T cells.

[0069] 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 solid tumor cells. Exemplary TAAs are tyrosinase (TyrD) or peptide fragments thereof. Optionally, the TAA is glypican 3 or a peptide fragment thereof. Optionally, the TAA is a peptide bound to an HLA molecule such as a class I HLA molecule. The tyrosinase peptide that binds to a class I HLA molecule (also interchangeably referred to herein as HLA-restricted tyrosinase epitope, HLA-restricted tyrosinase epitope, and MHC-restricted tyrosinase antigen) is derived from the tyrosinase enzyme (Genebank Accession No: NP_000363.1), is typically 8-10 amino acids in length, and binds to the groove of the heavy chain α1-α2 via two or three anchor residues that interact with the corresponding binding pocket in the HLA molecule.

[0070] Tyrosinase is a membrane-bound N-linked glycoprotein and an important enzyme in melanin synthesis. It is expressed in all normal melanocytes and almost all melanoma tumor samples (H. Takeuchi, et al., 2003; S. Reinke, et al., 2005). Peptides derived from this enzyme are presented by MHC class I molecules and recognized by autologous cytolytic T lymphocytes in melanoma patients [T. Wolfel, et al., 1994; Brichard, et al., 1993; Renkvist et al, Cancer immunology immunotherapy 2001 50:3-15; Novellino L, et al., March 2004 update. Cancer Immunol Immunotherapy. 54:187-207, 2005]. Another tumor tyrosinase HLA-restricted peptide derived from a tumor-associated antigen (TAA) can be identified on the website of the Istituto Nazionale per lo Studio e la Cura dei Tumori (www.istitutotumori.mi.it).

[0071] Non-limiting examples of antigenic peptides of MHC class I-restricted tyrosinase are provided in WO2008 / 120202, and the whole of which, such as Table 139 of WO2008 / 120202, is incorporated herein by reference. According to some embodiments of the present invention, the tyrosinase antigen peptide is a TyrD 369-377 peptide. Binding domains that specifically bind to TyrD, which is an epitope within TyrD, include but are not limited to those that bind restricted by HLA (such as HLA of class I), and those described in WO2016 / 199140, WO2016 / 199141, US9688739, PCT / IB2017 / 053539 which is co-pending. Each of these contents is incorporated by reference for all purposes, including but not limited to compositions and methods for identifying, generating and using binding domains that specifically bind to TyrD or an epitope within TyrD, whether HLA-restricted or HLA-independent.

[0072] The GPC3 peptides that bind to class I HLA molecules (also interchangeably referred to herein as HLA-restricted GPC3 epitopes, HLA-restricted GPC3 epitopes, and MHC-restricted GPC3 antigens) are derived from the glypican 3 protein (Genebank Accession No: NM_001164617.2), are typically 8-10 amino acids in length, and bind to the groove of the heavy chain α1-α2 via two or three anchor residues that interact with the corresponding binding pockets in the HLA molecule.

[0073] As used herein, binding domains, CARs, or CAR T cells that specifically bind to TyrD and / or specifically bind to an epitope within TyrD include, but are not limited to, binding domains, CARs, or CAR T cells that specifically bind to a TyrD peptide fragment. The binding domains, CARs, or CAR T cells that specifically bind to a TyrD peptide fragment are capable of specifically binding to the reference TyrD peptide fragment under HLA restriction. Similarly, as used herein, cells that express TyrD on their surface include cells that express or overexpress a TyrD peptide fragment on their surface, such as a peptide:HLA complex.

[0074] As used herein, binding domains, CARs, or CAR T cells that specifically bind to GPC3 and / or specifically bind to an epitope within GPC3 include, but are not limited to, binding domains, CARs, or CAR T cells that specifically bind to a GPC3 peptide fragment. The binding domains, CARs, or CAR T cells that specifically bind to a GPC3 peptide fragment are capable of specifically binding to the reference GPC3 peptide fragment under HLA restriction. Similarly, as used herein, cells that express TyrD on their surface include cells that express or overexpress a GPC3 peptide fragment on their surface, such as a peptide:HLA complex.

[0075] In some embodiments, the binding domain binds to an antigen so as to be expressed as a full-length functional polypeptide on the surface of a cell. In some embodiments, the binding domain binds to an antigen so as to be presented in an MHC:antigen complex. In some embodiments, the binding domain binds to an antigen restricted by HLA. Binding domains that are specific for an MHC:antigen complex are described, for example, in WO / 2016 / 199140 and WO / 2016 / 199141.

[0076] In some embodiments, the isolated nucleic acid encodes an anti-TyrD binding domain having CDRH1 comprising TSGMGVS (SEQ ID NO: 33), CDRH2 comprising HIYWDDKRYNPSLKS (SEQ ID NO: 34), CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO: 35), CDRL1 comprising KASQDIHNYIA (SEQ ID NO: 36), CDRL1 comprising YTSTLQP (SEQ ID NO: 37), and / or CDRL2 comprising LQYDNLWT (SEQ ID NO: 38).

[0077] In some embodiments, the isolated nucleic acid encodes an anti-GPC3 binding domain having CDRH1 comprising DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)), CDRH2 comprising ALDPKTGDTAYSQKFKG (SEQ ID NO: 41), CDRH3 comprising FYSYTY (SEQ ID NO: 42), CDRL1 comprising RSSQSLVHSNRNTYLH (SEQ ID NO: 43), CDRL2 comprising KVSNRFS (SEQ ID NO: 44), and / or CDRL3 comprising SQNTHVPPT (SEQ ID NO: 45).

[0078] The present disclosure also contemplates anti-TyrD binding domains or anti-GPC3 binding domains that compete to bind to the sequences provided herein. By using known methods, it is possible to determine whether an anti-TyrD 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 is made possible by binding the reference binding domain to TyrD under saturation conditions. Next, it is possible to evaluate the ability of a test binding domain to bind to the TyrD molecule. If this test binding domain is able to bind to TyrD following saturation binding with the reference binding domain, it is concluded that this test binding domain binds to an epitope different from that of the reference binding domain. On the other hand, if the test binding domain is unable to bind to TyrD 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.

[0079] If the binding domains compete 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 TyrD under saturation conditions, followed by evaluation of the binding of the test binding domain to the TyrD molecule. In the second direction, the test binding domain is bound to the TyrD molecule under saturation conditions, followed by evaluation of the binding of the reference binding domain to the TyrD molecule. In both directions, if only the first (saturating) binding domain is capable of binding to the TyrD molecule, it is concluded that the test binding domain and the reference binding domain compete for binding to TyrD. As will be appreciated by those skilled in the art, binding domains that compete to bind to a reference binding domain do not necessarily bind to the same epitope as the reference binding domain, but may 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-TyrD binding domain may similarly be applied to an anti-TyrD binding domain.

[0080] When two binding domains bind to the same or overlapping epitopes such that each competitively inhibits (blocks) the binding of the other to the antigen, i.e., a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one binding domain inhibits the binding of the other 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, two binding domains have the same epitope 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. Two binding domains have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one binding domain also reduce or eliminate the binding of the other.

[0081] Furthermore, performing routine experiments (e.g., peptide mutagenesis and binding analysis) can confirm whether the lack of binding of an observed test binding domain is 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.

[0082] 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 a 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 identity of the nucleotide sequence 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.

[0083] When applied to polypeptides, the terms "substantially similar" or "substantially similarity" mean that when optimally aligned using a program such as GAP or BESTFIT with the default gap weight, 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.

[0084] 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 polypeptides that are related species, such as homologous polypeptides of various species of living organisms, 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., the programs of GCG Version 6.1. FASTA (such as FASTA2 and FASTA3) provides an alignment of the optimal overlap region between a query sequence and a 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.

[0085] This specification provides anti-TyrD CAR or anti-GPC3 CAR comprising variants 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-TyrD 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-TyrD 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.

[0086] Similarly, the present disclosure provides an anti-GPC3 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-GPC3 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.

[0087] 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 where the order of the VH and VL regions is reversed, for example, in a CAR comprising a 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 a 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 a scFV or scFv binding domain.

[0088] Generally, the 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 (TTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). 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.

[0089] 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 end domains. 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' of the region encoding a peptide linker (e.g., the CD8α hinge domain) and 5' of the region encoding one or more cytoplasmic domains.

[0090] 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 activation signals for γδ T cell proliferation, cytotoxic activity, and / or pro-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 a plurality (e.g., 2, 3, 4, 5, or 6) of signaling domains, such as a plurality (e.g., 2, 3, 4, 5, or 6) of CD3ζ signaling domains, independently selected, for example, from SEQ ID NOs: 4 and 5. In some embodiments, the cytoplasmic region includes a plurality (e.g., 2, 3, 4, 5, or 6) of non-CD3ζ signaling domains and a CD3ζ signaling domain. In some embodiments, the cytoplasmic region includes a non-CD3ζ signaling domain and a plurality (e.g., 2, 3, 4, 5, or 6) of CD3ζ signaling domains. Substitute or additional signaling domains are included, but not limited to, these.

[0091] 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), and TLR2 co-stimulatory endodomains and combinations thereof.

[0092] In some embodiments, it is possible to include additional signaling modalities to increase the proliferation, persistence, and / or cytotoxic activity of the γδ-T cells described herein. For example, in some embodiments, the CAR construct can encode a soluble common gamma chain cytokine at the 3' end of the isolated nucleic acid. The common gamma chain cytokine coding region can be linked to the 5' portion of the CAR construct via a T2A linker coding region, such that the common gamma chain cytokine is cleaved from the CAR polypeptide and secreted by the cell.

[0093] 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, 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 one, two, three, or four or more co-stimulatory endodomains selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB co-stimulatory endodomain comprises SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

[0094] In some embodiments, the construct encodes one CD27 co-stimulatory endodomain and optionally a second co-stimulatory endodomain selected from the 4-1BB, ICOS, CD28, and CD27 co-stimulatory endodomains. In some embodiments, the construct encodes the CD27 co-stimulatory endodomain and the 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).

[0095] In some embodiments, the construct is operably linked to a secretion signal, such as a C-terminal polypeptide that promotes the secretion of cytokines that support the activation, cytotoxicity, and / or persistence of T cells (such as CAR-T cells), for example, encoding SEQ ID NO: 12 (MALPVTALLLPLALLLHAARP). In some embodiments, the secretion signal is the secretion signal of SEQ ID NO: 26 (MRISKPHLRSISIQKYLCLLNSHFLTEAGIHVFILGCFSAGLPKTEA). In some embodiments, the construct is operably linked to a secretion signal, such as SEQ ID NO: 12, that promotes the secretion of a common gamma chain cytokine, such as IL-15, or an active fragment thereof, for example, SEQ ID NO: 14 (NWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Other IL-15 sequences, including codon-optimized nucleic acid sequences encoding sIL15, are disclosed in WO2007 / 037780. 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.

[0096] In some embodiments, the construct encodes one or more multicistronic linker regions, for example, between a signaling domain and / or a co-stimulatory endodomain and a secretion signal operably linked to promote the secretion of a cytokine. A multicistronic linker region is a region of a polypeptide sequence 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 an endogenous protease such as furin.

[0097] In some embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a furin cleavage sequence. In some embodiments, the cleavage sequence is a P2A and furin cleavage sequence. In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 15 (SGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is the furin cleavage sequence of SEQ ID NO: 16 (RAKR). In some embodiments, the cleavage sequence is the P2A+furin cleavage sequence of SEQ ID NO: 17 (RAKRSGSGATNFSLLKQAGDVEENP GP). In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 25 (GSGATNFSLLKQAGDVEENPGP).

[0098] In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 27 (ATNFSLLKQAGDVEENPGP) or comprises its P2A cleavage sequence. In some embodiments, the cleavage sequence is the F2A cleavage sequence of SEQ ID NO: 28 (VKQTLNNFDLLKLAGDVESNPGP) or comprises its F2A cleavage sequence. In some embodiments, the cleavage sequence is the E2A cleavage sequence of SEQ ID NO: 29 (QCTNYALLKLAGDVESNPGP) or comprises its E2A cleavage sequence. In some embodiments, the cleavage sequence is the T2A cleavage sequence of SEQ ID NO: 30 (EGRSLLTCGDVEENPGP) or comprises its T2A cleavage sequence. 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.

[0099] 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: 31 (CTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATA).

[0100] Another exemplary internal ribosome entry site is encoded by SEQ ID NO: 32 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC).

[0101] 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 2009 Nov 16, those described in iresite.org, those described in WO2018 / 215787, the sequences described in GenBank accession No. KP019382.1 and the IRES elements described in GenBank accession No. LT727339.1. The contents of these are hereby incorporated by reference in their entirety for all purposes, particularly the internal ribosome entry sites and their use described herein.

[0102] Additional multicistronic linker regions such as cleavage self-cleavage and IRES elements are disclosed in US2018 / 0360992 and US8865467.

[0103] In some embodiments, the isolated nucleic acid encodes an hD11-CD8-BBz polypeptide comprising SEQ ID NO: 8 (MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCKASQDIHNYIAWYQQKPGKAPKLLIHYTSTLQPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVEIKRGGGGSGGGGSGGGGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARKDYGSSFYAMHYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), the hD11 anti-TyrD binding domain (anti-TyrD 369-377 ), the CD8α hinge and transmembrane region, the 4-1BB co-stimulatory end domain, and the CD3ζ signaling domain.

[0104] In some embodiments, anti-TyrD 369-377

[0105] 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: 10 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCACA GCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8α hinge region is encoded by SEQ ID NO: 11 (ACCACGACGCCAGCG CCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT) below.

[0106] In some embodiments, the isolated nucleic acid encodes an hD11-CD8-BBz-sIL15 polypeptide operably linked to a secretion signal, an anti-TyrD hD11 (anti-TyrD 369-377 ) binding domain, a CD8α hinge and transmembrane region, a 4-1BB co-stimulatory end domain, a CD3ζ signaling domain, a furin-P2A cleavage sequence, and an IL-15 domain.

[0107]

[0108] In some embodiments, the isolated nucleic acid encodes a polypeptide comprising SEQ ID NO: 20 (MSVPTQVLGLLLLWLTDARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*), the GC33 anti-GPC3 binding domain, the CD8α hinge and transmembrane region, the 4-1BB co-stimulatory end domain, and the CD3ζ signaling domain.

[0109]

[0110] In some embodiments, the isolated nucleic acid encodes a polypeptide comprising SEQ ID NO: 22 (MSVPTQVLGLLLLWLTDARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMALPVTALLLPLALLLHAARPNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*), a GC33 anti-GPC3 binding domain, a CD8α hinge and transmembrane region, a 4-1BB co-stimulatory end domain, a CD3ζ signaling domain, a furin and P2A cleavage region, and a secretion signal operably linked to the IL15 domain.

[0111]

[0112]

[0113] In some embodiments, the isolated nucleic acid is a linear 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 (such as a gamma-retrovirus vector), or a lentivirus vector. In some embodiments, the isolated nucleic acid, or a continuous portion of the isolated nucleic acid, such as including a binding domain, a 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 gamma-delta T cell. In exemplary embodiments, the isolated nucleic acid is a retrovirus vector.

[0114] Gamma-delta T cells: Aspects of the invention include gamma-delta T cells that functionally express the isolated nucleic acids described herein, thereby expressing a CAR on the surface of the gamma-delta T cells.

[0115] Aspects of the invention may alternatively or additionally include gamma-delta T cells having in vitro or in vivo cytotoxic activity against solid tumor cells that exhibit cell surface expression of a tumor-associated antigen (TAA). Optionally, the cytotoxic activity is a native activity. Optionally, 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 solid tumor cells. Optionally, in this gamma-delta T cell, it is shown that the solid tumor cell killing activity of the gamma-delta T cell is greater than the native level of in vitro and / or in vivo solid tumor cell killing activity in control gamma-delta T cells. Optionally, the control gamma-delta T cells do not contain a CAR construct. Optionally, the control gamma-delta 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.

[0116] In some cases, 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 an epitope within TyrD or TyrD 369-377 such as. In some cases, 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 an epitope within TyrD or TyrD, subject to HLA restriction (e.g., restricted to class I HLA). 369-377 such as. In some cases, 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 an epitope within TyrD or TyrD. In some cases, cytotoxicity is due to HLA-A2 / TyrD 369-377 at least partially, significantly (> about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to. In some cases, γδ T cells functionally express a CAR encoded by the isolated nucleic acids described herein that specifically binds to TyrD or a peptide fragment thereof.

[0117] In some embodiments, the γδ T cells described herein can exhibit HLA-restricted (e.g., restricted to HLA class I) cytotoxicity. In other embodiments, most (> 50%), substantially all (> 90%), or all of the cytotoxic activity is not restricted by HLA (e.g., restricted to HLA class I). HLA-restricted cytotoxic activity can be evaluated by comparing the in vitro cytotoxicity against HLA (e.g., HLA class I) (null) tumor cell lines to the in vitro cytotoxicity against HLA+ (e.g., HLA class I+) tumor cell lines. In some embodiments, HLA-restricted cytotoxic activity is at least partially, significantly (> 25%), or completely provided 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 within a complex with an MHC molecule. T cell receptor-like binding domains are further described, for example, in WO2016 / 199141.

[0118] The γδ T cells described herein are capable of exhibiting potent and / or sustained solid tumor cell cytotoxic activity. In some cases, the solid tumor cell cytotoxic 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 solid tumor cells. In some cases, the solid tumor cell cytotoxic activity of the γδ T cells described herein or their progeny 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 solid tumor cells or from the administration of the γδ T cells described herein. This sustained solid tumor cell cytotoxic activity can be demonstrated in vitro, in vivo, or both in vitro and in vivo.

[0119] Aspects of the invention can 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) can be normal cells such as normal endothelial cells. The cells that exhibit cell surface expression or overexpression of a tumor-associated antigen (TAA) can be solid 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 the TAA expressed on the surface of the cell. 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.

[0120] 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 TyrD or an epitope within TyrD. In some cases, γδ T cells that exhibit proliferation in response to contact with cells showing cell surface expression of TyrD functionally express a TyrD-specific CAR encoded by the isolated nucleic acid described herein.

[0121] The γδ T cells described herein are capable of exhibiting robust and / or sustained proliferation in a host organism comprising cells showing 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 cells showing cell surface expression or overexpression of a tumor-associated antigen (TAA), or from the day of administration of the γδ T cells to the host organism. In some cases, the proliferation of the γδ T cells described herein, or their progeny, in a host organism comprising cells showing 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 the cells, or from the first day 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 TyrD or an epitope within TyrD. In some cases, γδ T cells that exhibit proliferation in a host organism comprising cells showing cell surface expression of TyrD functionally express a TyrD-specific CAR encoded by the isolated nucleic acid described herein.

[0122] 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 cells that express or overexpress TyrD or a peptide fragment thereof on the cell surface. 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 cells that express or overexpress TyrD or a peptide fragment thereof on the cell surface, such as in a host organism that includes cells that express or overexpress TyrD or a peptide fragment thereof on the cell surface.

[0123] In some embodiments, γδ T cells or a pharmaceutical composition comprising γδ T cells do not substantially exhibit, or do not exhibit, a graft-versus-host response when introduced into an allogeneic host. In some embodiments, γδ T cells or a pharmaceutical composition comprising γδ T cells exhibit a graft-versus-host response at a clinically acceptable level when introduced into an allogeneic host. 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 that is less severe than grade C according to the applicable IBMTR assessment scale. The severity of acute graft-versus-host reaction is determined by assessing 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 - Skin lesions at stage 2, intestinal or liver lesions at stage 1 to 2 ● Grade C - Stage 3 lesions in any organ system (generalized erythroderma, bilirubin 6.1 - 15.0 mg / dL, diarrhea 1500 - 2000 mL / day) ● Grade D - Stage 4 lesions in 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.

[0124] In some embodiments, the γδ T cells, or a pharmaceutical composition comprising γδ T cells, when introduced into an allogeneic host, exhibit a reduced or substantially reduced 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 non - genetically engineered 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.

[0125] 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.

[0126] γδ T cells are available from allogeneic or autologous donors. γδ T cells can be partially or fully purified or unpurified and expanded ex vivo. Methods and compositions for ex vivo expansion include, but are not limited to, those described in WO2017 / 197347. This expansion can be performed before, after, or before and after the CAR construct is introduced into the γδ T cell(s).

[0127] The γδ T cells described herein can be stored, for example, by cryopreservation for use in adoptive cell transfer.

[0128] Method for inhibiting or killing tumor cells One or more unmanipulated γδT cell populations, manipulated γδT cell populations, and / or mixtures thereof having cytotoxic activity against solid 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, within 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 compositions containing the same.

[0129] In some cases, a method of treating solid cancer involves administering a therapeutically effective amount of an ungenetically engineered γδT cell population, a genetically engineered γδT cell population, and / or a mixture thereof to a subject, wherein administering treats the solid cancer. In some embodiments, the therapeutically effective amount of the ungenetically engineered γδT cell population, the genetically engineered γδT 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 ungenetically engineered γδT cell population, the genetically engineered γδT cell population, and / or the mixture thereof is administered for at least 1 week. In some embodiments, the therapeutically effective amount of the ungenetically engineered γδT cell population, the genetically engineered γδT cell population, and / or the mixture thereof is administered for at least 2 weeks.

[0130] The non-gene-manipulated γδT cell population, gene-manipulated γδT cell population, and / or their mixture described in this specification 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 showing a tendency of 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 via any route described herein using any formulation 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 solid cancer, 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.

[0131] 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, wherein 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 administered γδ T cells and the like. Optionally, IL-2, IL-7, and / or IL-15, preferably IL-15, is secreted.

[0132] In some embodiments, one or more dosing methods for raising common gamma chain cytokine(s) include lymphodepletion prior to introduction of γδ T cell(s). In some embodiments, one or more dosing methods for raising common gamma chain cytokine(s) include, at the time of 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 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. Exemplary amounts of IL-2 include, but are not limited to, about 3×10 6 and about 22×10 6 units. 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.

[0133] In some embodiments, one or more dosing methods for raising common gamma chain cytokine(s) include lymphodepletion prior to administration of γδ T cell(s), prior to administration at the time of 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

[0134] Example 1 1x106 Human PBMCs per mL were pre-coated in 24-well plates (Costar) with pre-coated anti-Vδ1 antibodies D1-08 or D1-35 in the presence of IL-2 (100 U / mL) for 5 days and activated with modified culture medium. On day 5, the cell cultures were transduced with a γ-retroviral construct encoding an anti-TyrD chimeric antigen receptor (SEQ ID NO: 8) in the presence of RetroNectin. On day 6, the cells were returned to the modified culture 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. In parallel, non-transduced cell cultures were expanded in the same manner without adding the retroviral supernatant. As shown in Figure 2, non-transduced proliferating Vδ1 cells express tyrosine kinase and induce a certain degree of cytotoxicity against melanoma cell lines 526 and WM266.1-Luc that are known to present Tyr 369-377 peptide. This cytotoxicity was enhanced by the introduction of anti-TyrD CAR. 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.

[0135] Example 2 WM266.4-Luc cells (4x10 per animal 6 ) were subcutaneously implanted into NSG mice (Jackson Labs). When the tumors reached a size of 100 - 200 mm 3 , the animals were treated with 6x10 6 anti-TyrD CAR+Vδ1 cells. The animals were co-administered IL-2 (60,000 U / dose) three times a week until the end of the study. The results are shown in Figure 3. As shown in Figure 3, the animals administered anti-TyrD CAR+Vδ1 cells showed strong control of tumor burden.

[0136] Example 3 The Tyr CAR construct was introduced into Vδ1 T cells as described above, the cells were expanded, and tested in a cytotoxicity assay against WM266.4-Luc cells. A control, non-TyrD-targeted CAR construct was used as a control. The results are shown in Figure 5, and the cytotoxicity brought about by the anti-TyrD CAR construct is increased.

[0137] Example 4 Human PBMCs at 1 x 10 6 / mL in growth medium were activated in 24-well plates (Costar) pre-coated with anti-Vδ1 antibodies D1-08 or D1-35 for 5 days in the presence of IL-2 (100 U / mL). On day 5, the cell cultures were transduced with a γ-retroviral construct encoding an anti-GPC3 chimeric antigen receptor (SEQ ID NO: 20 (GC33CAR) or SEQ ID NO: 22 (GC33CAR + sIL15 and GC33CAR + CO sIL15)) in the presence of retronectin. GC33CAR is encoded by the nucleic acid sequence of SEQ ID NO: 21. GC33CAR + sIL15 is encoded by the nucleic acid sequence of SEQ ID NO: 23, and GC33CAR + CO sIL15 contains a codon-optimized sIL15 coding region and is encoded by the nucleic acid sequence of SEQ ID NO: 24. On day 6, the cells were returned to 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 6). Briefly, CAR-T cells were stained by incubating the cells with 1 μg / mL of soluble recombinant biotinylated GPC3 (R&D Systems). Detection of binding was performed using streptavidin-PE at a dilution of 1:500 as recommended by the manufacturer.

[0138] In parallel, untransduced cell cultures were grown in the same manner without the addition of retroviral supernatant. The grown cells were tested in an in vitro cytotoxicity assay for GPC3-positive (HepG2, Hep3B, PLC / PRF / 5) cells. As shown in Figure 7, untransduced proliferating Vδ1 cells induce a certain degree of cytotoxicity against liver cancer, which is known to express GPC3. This cytotoxicity is enhanced by the introduction of GPC3CAR, regardless of the presence or absence of the sIL15 cytokine engineered to be expressed in tandem. 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.

[0139] The foregoing merely illustrates the principles of the present invention. It will be understood by those skilled in the art that, although not explicitly described or illustrated herein, it is possible to embody the principles of the present invention and devise various configurations that are within its spirit and scope. Furthermore, 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 the technology, and are not to be construed as being limited to such specifically recited examples and conditions. Additionally, all descriptions in this specification listing the principles, aspects, and specific examples of the present invention are intended to encompass both their structural equivalents and functional equivalents. Moreover, 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

1. δ1 γδ T cells, (a) a nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising: (i) a binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a solid tumor cell; (ii) the CD8α hinge domain, (iii) the CD8α transmembrane domain, (iv) a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region, and (v) CD3ζ signaling domain or (b) a polypeptide comprising a CAR comprising an amino acid sequence encoded by the nucleic acid of (a). Including, said binding domain of said polypeptide or said CAR encoded by said nucleic acid is functionally expressed on the surface of said δ1γδ T-cells; δ1γδT cells.

2. The δ1 γδ T cell of claim 1, wherein the tumor-associated antigen is a protein-peptide complex, the protein is an MHC protein, and the binding domain binds to the complex in an HLA-restricted manner.

3. The δ1γδ T cell of claim 1 or 2, wherein (i) to (v) are in the order of 5' to 3'.

4. The δ1γδT cell of any one of claims 1 to 3, wherein the TAA comprises an adjacent region of TyrD.

5. The δ1 γδ T cell of claim 4, wherein the TyrD flanking region comprises at least 4 and no more than 12 flanking amino acids of TyrD.

6. The δ1 γδ T cell of claim 4, wherein the TyrD flanking region comprises 7, 8 or 9 TyrD flanking amino acids.

7. The adjacent region of TyrD is ３６９－３７７ The δ1 γδ T cell of claim 6,

8. The binding domain that specifically binds to the TAA peptide-MHC complex is HLA-A2 / TyrD ３６９－３７７ 8. The δ1 γδ T cell of any one of claims 1 to 7, which specifically binds to

9. The binding domain comprises: a) a CDRH1 comprising TSGMGVS (SEQ ID NO:33); and b) a CDRH2 comprising HIYWDDDKRYNPSLKS (SEQ ID NO:34); and c) a CDRH3 comprising KDYGSSFYAMHY (SEQ ID NO:35); and d) a CDRL1 comprising KASQDIHNYIA (SEQ ID NO:36); and e) a CDRL1 comprising YTSTLQP (SEQ ID NO:37); and f) CDRL2 comprising LQYDNLWT (SEQ ID NO:38); 9. A δ1 γδ T cell according to any one of claims 1 to 8, which specifically binds to an epitope bound by an antibody comprising:

10. The δ1 γδ T cell of claim 1, wherein the binding domain specifically binds to an epitope within GPC3 expressed on the surface of a solid tumor cell.

11. The binding domain comprises the following complementarity determining regions (CDRs): a) a CDRH1 comprising the sequence of DYEMH (SEQ ID NO: 39) (or GYTFTDYEMH (SEQ ID NO: 40)); b) a CDRH2 comprising the sequence ALDPKTGDTAYSQKFKG (SEQ ID NO: 41); c) a CDRH3 comprising the sequence FYSYTY (SEQ ID NO: 42); d) CDRL1 comprising the sequence RSSQSLVHSNRNTYLH (SEQ ID NO: 43); e) a CDRL2 comprising the sequence of KVSNRFS (SEQ ID NO:44); and f) CDRL3 comprising the sequence SQNTHVPPT (SEQ ID NO:45) binds to the same epitope of GPC3 as an antibody comprising the compound, and / or competes with said antibody for binding to the epitope of GPC3; The δ1 γδ T cell of claim 10 comprising said CDRs.

12. The CAR is a) a CD8α hinge domain comprising SEQ ID NO: 1 (PTPAPTIAQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRPPTPAPTIAQPLSLRPEACRPAAGGAVHTRGLDFACDIY); b) the CD8α transmembrane domain comprising SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC), and / or c) (i) SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or (ii) a CD3ζ signaling domain comprising SEQ ID NO:5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). The δ1γδ T cell of any one of claims 1 to 11, comprising:

13. The CAR is a) a 4-1BB costimulatory signaling region comprising SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), or b) comprising a CD27 costimulatory signaling region comprising SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP); or The δ1 γδ T cell of claim 12, wherein said nucleic acid encodes the 4-1BB costimulatory signaling region comprising SEQ ID NO:6 and the CD27 costimulatory signaling region comprising SEQ ID NO:

7.

14. The nucleic acid further comprises a) Secreted cytokines b) secreted common gamma-chain interleukins c) secreted IL-15, or d) a multicistronic linker region amino-terminal to the secreted common gamma chain interleukin and the interleukin or interleukin secretion signal 14. The δ1γδ T cell of any one of claims 1 to 13, encoding:

15. c) The secreted IL-15 in The sequence of SEQ ID NO: 14, The sequence of SEQ ID NO: 14 operably linked to the secretory signal sequence of SEQ ID NO: 12, or The sequence of SEQ ID NO:14 operably linked to the secretory signal sequence of SEQ ID NO:26 The δ1 γδ T cell of claim 14, comprising:

16. The δ1 γδ T-cell of claim 14, wherein the secreted common gamma chain interleukin in d) is IL-15 and the multicistronic linker region comprises the sequence of any one of SEQ ID NOs: 15-17, 25 or 27-30 or a combination thereof, or encodes an internal ribosome entry site.

17. A δ1γδT cell described in claim 14, wherein the multicistronic linker region comprises the sequence of SEQ ID NO: 31 or 32.

18. a) the secretion signal comprises the sequence of SEQ ID NO: 12 or SEQ ID NO: 26, b) the sIL15 domain comprises the sequence of SEQ ID NO: 14; c) the P2A cleavage sequence comprises the sequence of SEQ ID NO: 15 or SEQ ID NO: 25; d) the furin cleavage sequence comprises the sequence of SEQ ID NO: 16; and / or e) the CAR comprises the sequences of SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 14, in order from amino to carboxy; The δ1 γδ T cell of claim 14.

19. The δ1γδ T cell of claim 18, wherein the secretion signal in a) comprises the sequence of SEQ ID NO:

12.

20. a) the binding domain is HLA-A2 / TyrD ３６９－３７７ wherein the nucleic acid encodes a polypeptide as set forth in SEQ ID NO:8 or SEQ ID NO:18; or b) A δ1γδ T cell according to any one of claims 1 to 19, wherein the binding domain specifically binds to GPC3 and the nucleic acid encodes a polypeptide as set forth in SEQ ID NO: 20 or 22.

21. The δ1γδ T cell of claim 20, wherein the nucleic acid comprises the sequence of SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:21, 23 or 24.

22. A cell population comprising a plurality of δ1γδ T cells according to any one of claims 1 to 21.

23. At least 10 ８ The cell population of claim 22, comprising δ1 γδ T cells.

24. 10 ８ 10 from δ1γδT cells １１ The cell population of claim 22, comprising δ1 γδ T cells.

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

26. The cell population described in claim 25, comprising 60% to 95% δ1γδ T cells.

27. 27. A method for generating a δ1 γδ T-cell according to any one of claims 1 to 21 or a cell population according to any one of claims 22 to 26, said method comprising transfecting a δ1 γδ T-cell(s) with a nucleic acid according to any one of claims 1 to 21.

28. 28. The method of claim 27, wherein the method comprises retroviral transduction.

29. 29. The method of claim 28, wherein the retroviral transduction is gamma retroviral transduction.

30. 30. The method of any one of claims 27 to 29, comprising ex vivo expansion of the δ1 γδ T-cell(s), wherein the ex vivo expansion is performed before and / or after transfection of the nucleic acid.

31. 27. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a δ1γδ T cell according to any one of claims 1 to 21 or a cell population according to any one of claims 22 to 26.

32. 32. A medicament for killing solid tumor cells, comprising a δ1 γδ T-cell of any one of claims 1 to 21, a cell population of any one of claims 22 to 26, or a pharmaceutical composition of claim 31, in an amount effective for killing the tumor cells.

33. 33. The medicament of claim 32, wherein a therapeutically effective amount of the δ1 γδ T-cell(s) or the pharmaceutical composition is introduced into a host organism comprising the solid tumor cells.

34. 34. The medicament of claim 33, wherein a therapeutically effective amount of the δ1 γδ T-cell(s) or the pharmaceutical composition is introduced into a host organism comprising the solid tumor cells, and one or more agents that elevate general gamma chain cytokine(s) are administered simultaneously or sequentially.

35. 35. The medicament of claim 34, wherein administering one or more agents that increase the general gamma chain cytokine(s) comprises administering, simultaneously with or sequentially to the introduction of the δ1γδ T-cell(s), an amount of the general gamma chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced δ1γδ T-cell(s).

36. The method of claim 35, wherein the common gamma chain cytokine(s) is / are IL-2 and / or IL-15.

37. 36. The medicament of claim 35, wherein the one or more agents that increase general gamma chain cytokine(s) comprise an amount of general gamma chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof, of the introduced δ1γδ T-cell(s) before and / or after introduction of the δ1γδ T-cell(s).

38. 38. The medicament of any one of claims 32 to 37, wherein the one or more agents that elevate the general gamma chain cytokine(s) deplete lymphocytes prior to the introduction of the δ1γδ T-cell(s).

39. The medicament of any one of claims 32 to 38, wherein the one or more agents that increase general gamma chain cytokine(s) induce secretion of one or more general gamma chain cytokine(s) from the introduced δ1 γδ T-cell(s).

40. 38. The medicament of any one of claims 33 to 37, wherein the medicament reduces the in vivo tumor burden and / or increases the mean survival time of the host organism compared to a control organism, wherein the control organism is not treated with the δ1γδ T-cell(s) or pharmaceutical composition.

41. The medicament according to any one of claims 32 to 40, wherein the medicament is for treating cancer in a subject in need of treatment.

42. 32. A method for the manufacture of a medicament for the treatment of solid tumor cell cancer in a subject in need thereof, characterized in that a δ1 γδ T-cell of any one of claims 1 to 21, a cell population of any one of claims 22 to 26, or a pharmaceutical composition of claim 31 is used to kill solid tumor cells.

43. 22. A pharmaceutical composition for treating cancer in a subject in need of such treatment, comprising a therapeutically effective amount of a δ1γδ T-cell of any one of claims 1 to 21, wherein the cancer comprises solid tumor cells that exhibit cell surface expression of TyrD or GPC3.

44. 44. The pharmaceutical composition of claim 43, wherein the one or more agents that elevate general gamma chain cytokine(s) are administered simultaneously or sequentially with administration of δ1 γδ T-cells.

45. 45. The pharmaceutical composition of claim 43 or 44, wherein multiple administrations of the δ1 γδ T cells are administered, the interval between the multiple administrations being at least one week.

46. The pharmaceutical composition described in claim 43 or 44, wherein the multiple administration of the δ1γδ T cells does not exceed once every six months.

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