Composition and method relating to engineered and non-engineered γδ-T cells for the treatment of solid tumors

Abstract

Aspects of the present invention include compositions and methods for treating solid tumors with engineered or non-engineered γδ-T cells. In some embodiments, the γδ-T cells comprise a chimeric antigen receptor (CAR) construct. The CAR construct may contain an anti-TryD binding domain, a CD8 hinge and transmembrane domain, a co-stimulating domain, a CD3ζ signaling domain, a combination thereof, or all thereof. The CAR construct may contain an anti-GPC3 binding domain, a CD8α hinge and transmembrane domain, a co-stimulating domain, a CD3ζ signaling domain, a combination thereof, or all thereof. The CAR construct may contain a domain encoding for a secreted common gamma chain cytokine, such as a sIL15 domain.

Description

Technology Field

[0001] related Cross-applications -reference

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 739,826 filed on October 1, 2018, the contents of which are incorporated herein in their entirety for any and all purposes.

[0003] Sequence list

[0004] The present application is submitted electronically in ASCII format and contains a sequence list, the entirety of which is incorporated herein by reference. The said ASCII copy created on January 8, 2020, is named ADC-0006-PCT_SL.txt and has a size of 48,406 bytes. Background Technology

[0005] Adoptive cell therapy has undergone nearly constant iterations for over thirty (30) years, ranging from early approaches focused on primordial lymphokine activation and / or tumor invasion to more recent strategies involving the manipulation of these immune cells to express genetically engineered antigen receptors, such as chimeric antigen receptors (CARs). Depending on the method, there have been several hints and indications of the therapeutic potential of these approaches, but much work remains to be done. In particular, successful tumor eradication by CAR-T lymphocytes depends on CAR-T cell persistence and effector function, but an excess of either can lead to graft-versus-host disease in patients. Furthermore, solid tumors pose a problem, particularly due to the scarcity of available positive stimuli and the presence of a suppressive environment. As such, the industry is testing numerous co-stimulation strategies for both T cells and NK cells, particularly αβ T cells, with the aim of balancing efficacy and safety. Notably, the actual translation of any of these various approaches to γδ T cells is, at best, uncertain given the current lack of understanding regarding the co-stimulation requirements of γδ T cells compared to αβ T cells. For example, Ribot et al. "Searching for "signal 2": costimulation requirements of γδ T cells", Cell. Mol. Life Sci. Refer to (2011) 68:2345-2355.

[0006] Therefore, there is still a need for improved strategies to enhance cell specificity or selectivity, improve cell safety by, for example, reducing or avoiding graft-versus-host (GVH) effects, improve cell efficacy against solid tumor cells by, for example, avoiding inhibition of effector function, and improve cell activity and / or survival upon administration to a subject. Methods, cells, compositions, kits, and systems that meet these needs are provided.

[0007] Summary of the Invention

[0008] An aspect of the present invention comprises an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR is 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 a solid tumor cell, and optionally wherein the binding domain binds to the complex in an HLA-restrictive manner; a CD8α hinge domain; a CD8α transmembrane domain; a costimulatory signaling region selected from a 4-1BB costimulatory signaling region and a CD27 costimulatory signaling region; and a CD3ζ signaling domain. An aspect of the present invention further comprises a non-engineered γδ T cell as described herein and an engineered γδ T cell comprising a nucleic acid encoding the CAR construct as described herein, wherein the γδ T cell functionally expresses the nucleic acid encoding the CAR on the surface of the γδ T cell.

[0009] An aspect of the present invention further comprises a plurality of γδ T cells as described herein. An aspect of the present invention further comprises a method for producing γδ T cells or a plurality of γδ T cells as described herein. An aspect of the present invention further comprises a pharmaceutical composition comprising a pharmaceutically acceptable excipient and γδ T cells or a plurality of γδ T cells as described herein. An aspect of the present invention further comprises contacting solid tumor cells with an effective amount of γδ T cells or a plurality of γδ T cells as described herein to kill the tumor cells.

[0010] In one embodiment, 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 a solid tumor cell, and optionally, wherein the binding domain binds to the complex in an HLA-restrictive manner; (b) a hinge domain such as a CD8α hinge domain; (c) a transmembrane domain such as a CD8α transmembrane domain; (d) a costimulatory signaling domain or a combination of costimulatory signaling domains, wherein optionally, the costimulatory signaling domain(s) are selected from a 4-1BB (CD137) costimulatory signaling domain and a CD27 costimulatory signaling domain; 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.

[0011] In some embodiments, the TAA comprises an adjacent region of TyrD. In some embodiments, the adjacent region of TyrD comprises at least 4, or at least about 4 and 12 or fewer, or about 12 or fewer 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 is. In some embodiments, the binding domain that specifically binds to the TAA peptide MHC complex is HLA-A2 / TyrD 369-377 It binds specifically to.

[0012] In some embodiments, the binding domain specifically binds to or competes with an epitope that is bound to or competes with an antibody comprising CDRH1 containing TSGMGVS (SEQ No.: 33); CDRH2 containing HIYWDDDKRYNPSLKS (SEQ No.: 34); CDRH3 containing KDYGSSFYAMHY (SEQ No.: 35); CDRL1 containing KASQDIHNYIA (SEQ No.: 36); CDRL1 containing YTSTLQP (SEQ No.: 37); and CDRL2 containing LQYDNLWT (SEQ No.: 38).

[0013] In another embodiment, the binding domain specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a solid tumor cell, 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 in an HLA-restrictive manner, and the CAR encoded in the isolated nucleic acid sequence comprises (b) a hinge domain such as a CD8α hinge domain; (c) a transmembrane domain such as a CD8α transmembrane domain; (d) a co-stimulating signaling region or a combination of co-stimulating signaling regions, optionally the co-stimulating signaling region(s) are selected from the 4-1BB (CD137) co-stimulating signaling region and the CD27 co-stimulating 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.

[0014] In some embodiments, the binding domain specifically binds to an epitope within GPC3 expressed on the surface of solid tumor cells. In some embodiments, the binding domain comprises the following complementarity determining region (CDR) that binds to the same GPC3 epitope as an antibody containing the following CDR and / or competes for binding to the GPC3 epitope with an antibody containing the following CDR: a CDRH1 sequence containing DYEMH (SEQ No.: 39) (or GYTFTDYEMH (SEQ No.: 40)); a CDRH2 sequence containing ALDPKTGDTAYSQKFKG (SEQ No.: 41); a CDRH3 sequence containing FYSYTY (SEQ No.: 42); a CDRL1 sequence containing RSSQSLVHSNRNTYLH (SEQ No.: 43); a CDRL2 sequence containing KVSNRFS (SEQ No.: 44); CDRL3 containing the sequence of and / or SQNTHVPPT (Sequence No.: 45).

[0015] In any one of the aforementioned aspects or embodiments or in any embodiment of any CAR-encoding nucleic acid described herein, the encoded CAR comprises a CD8α hinge domain comprising SEQ No. 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ No. 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY); or a CD8α transmembrane domain comprising SEQ No. 3 (IWAPLAGTCGVLLLSLVITLYC); and / or a CD3ζ signaling domain. In some cases, the CD3ζ signaling domain comprises SEQ No. 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG GKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR); or includes sequence number: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0016] In some embodiments, the CAR comprises a 4-1BB co-stimulating signaling region comprising SEQ No. 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL); or a CD27 co-stimulating signaling region comprising SEQ No. 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or the isolated nucleic acid encodes a 4-1BB co-stimulating signaling region comprising SEQ No. 6 and a CD27 co-stimulating signaling region comprising SEQ No. 7.

[0017] In any one of the embodiments described above, or as described herein, the isolated nucleic acid further encodes a secreted cytokine; or a secreted common gamma-chain interleukin; or a secreted common gamma-chain interleukin such as IL-15, preferably wherein the secreted common gamma-chain interleukin such as IL-15 comprises an interleukin polypeptide sequence operably linked to a secreted signal sequence (e.g., the secreted signal of SEQ ID NO: 12 or 26). In some embodiments, the isolated nucleic acid encodes a secreted IL-15, preferably wherein IL-15 comprises the sequence of SEQ ID NO: 14, and more preferably wherein IL-15 comprises the sequence of 14 operably linked to the secreted signal sequence of SEQ ID NO: 12, or wherein IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to the secreted signal sequence of SEQ ID NO: 26. In some cases, the secreted cytokine, common gamma chain interleukin, and / or IL-15 is a carboxyl terminus encoded for the binding domain, hinge and transmembrane domain, signaling domain, and / or co-stimulating endodomain. In some cases, the secreted cytokine, common gamma chain interleukin, and / or IL-15 is encoded on the sense strand 3' of the region encoding the binding domain, hinge and transmembrane domain, signaling domain, and / or co-stimulating endodomain.

[0018] In some embodiments, the nucleic acid encodes a multi-cistron linker region configured to facilitate the translation of CAR and secreted cytokines, common gamma-chain cytokines, or IL-15 as separate polypeptides. In some embodiments, the multi-cistron linker region encodes a self-cleavage and / or cleavage polypeptide sequence. In some cases, the self-cleavage sequence is a P2A, F2A, T2A, or E2A self-cleavage sequence. In some cases, the cleavage sequence is a purine cleavage sequence. In some cases, the cleavage sequence (e.g., a purine cleavage sequence) is an amino terminus for the self-cleavage sequence. In some embodiments, the multi-cistron linker region encodes an internal ribosome entry site. In some embodiments, the nucleic acid encodes a multi-cistron linker region amino terminus for an interleukin or cytokine or interleukin or cytokine secretion signal, preferably wherein the multicistron linker region comprises any one of the sequences SEQ NOs: 15-17, 25, or 27-30, or a combination thereof, or encodes an internal ribosome entry site, e.g., SEQ NOs: 31 or 32.

[0019] In some embodiments, the secretion signal comprises the sequence of sequence number: 12 or sequence number: 26, preferably sequence number: 12; and / or the sIL15 domain comprises the sequence of sequence number: 14; and / or the P2A cleavage sequence comprises the sequence of sequence number: 15 or sequence number: 25; and / or the purine cleavage sequence comprises the sequence of sequence number: 16; and / or the CAR comprises the sequences of sequence number: 17, sequence number: 12, and sequence number: 14 in the amino-to-carboxyl order.

[0020] In some embodiments, the binding domain is HLA-A2 / TyrD 369 -377The nucleic acid specifically binds to and 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 22. In some embodiments, the nucleic acid comprises the sequences of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 21, 23, or 24.

[0021] 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 nucleic acids described above or the polypeptide described herein.

[0022] In another aspect, the present invention provides, for example, γδ, T cells comprising the aforementioned polypeptide or a nucleic acid encoding the CAR described herein, wherein the cell functionally expresses the nucleic acid encoding the CAR on the binding domain of the polypeptide or on the surface of the cell. In some embodiments, the cell is a solid tumor cell exhibiting cell surface expression of a tumor-associated antigen (TAA). In vitro and / or In vivo It exhibits apoptotic activity. In some embodiments, the solid tumor cell apoptotic activity of the cells is at an innate level compared to control cells not containing the CAR construct. In vitro and / or In vivo It is greater than the apoptotic activity of solid tumor cells. In some embodiments, the cells are HLA class I + It exhibits increased solid tumor cell death activity against solid tumor cells. In some embodiments, the solid tumor cell death activity or increased solid tumor cell death activity persists for 6 to 180 days, about 6 to 180 days, at least 6 to 180 days, or at least about 6 to 180 days after the first contact with solid tumor cells.

[0023] In some embodiments, cells proliferate in response to contact with solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAA). In some embodiments, cells exhibit increased proliferation in response to contact with solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAA) compared to control cells that do not functionally express nucleic acids encoding CAR on the cell surface. In some embodiments, cells proliferate in a host organism containing solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAA). In some embodiments, cell proliferation or increased cell proliferation persists for 6 to 180 days, about 6 to 180 days, at least 6 to 180 days, or at least about 6 to 180 days after the first contact with solid tumor cells. In some embodiments, the cell expresses one or more pro-inflammatory cytokines, optionally wherein one or more pro-inflammatory cytokines contain tumor necrosis factor alpha or interferon gamma in a larger amount than in control cells that do not functionally express nucleic acids encoding CAR on the surface of the cell after contact with solid tumor cells.

[0024] In some embodiments, the cells exhibit a reduced, substantially reduced, essentially absent, or no host response when introduced into a homologous host compared to the graft-versus-host response exhibited by αβ T cells administered to a homologous host. In some embodiments, for example, γδ T cells exhibit a reduced, substantially reduced, essentially absent, or no host response when introduced into a homologous host compared to the graft-versus-host response exhibited by αβ T cells administered to a homologous 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 δ1 δ T cells. In some embodiments, the T cells are δ1, δ2, δ3, or δ4 γδ T cells, preferably δ2 - γδ T cells, more preferably δ1 γδ T cells.

[0025] In another aspect, the present invention provides a plurality of cells 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 at least about 10 8 Cells, for example, 10 8 Cells, e.g., γδ, T cells, preferably about 10 8 Cells, e.g., γδ, T cells to about 10 11 The cells comprise, for example, γδ T cells. In some embodiments, the plurality comprises at least 60%, 80%, or about 60% or 80% to about 90% or 95% δ1, δ2, δ3, or δ4 cells, e.g., γδ T cells, preferably δ1 or δ2 γδ T cells, more preferably δ2- The composition comprises γδ T cells, most preferably δ1 γδ T cells.

[0026] In some embodiments, the present invention provides a method for producing, for example, γδ T cells as described herein, or a plurality of cells as described herein, for example, γδ T cells, wherein the method comprises transfecting T cell(s) with a construct comprising an isolated nucleic acid sequence as described herein. In some cases, the method comprises, for example, gamma retrovirus transduction. In some cases, the method comprises of T cell(s). In vitro Includes extension, where the above In vitro Expansion is performed before and / or after transfection of the isolated nucleic acid sequence. In some cases, the method is of the T cell(s). In vitro Includes extension, where the above In vitro Expansion is performed before and after transfection of the isolated nucleic acid sequence. In some cases, the method is of the T cell(s). In vitro Includes extension, where the above In vitro Extension is performed after transfection of the isolated nucleic acid sequence. In some embodiments, the method involves about 10 functionally expressing the CAR described herein within about 30 days of transfection. 8 Cells, e.g., γδ, T cells to about 10 11 This includes producing cells, for example, γδ and T cells.

[0027] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a cell or a plurality of cells described herein, e.g., γδ, T cell(s) described herein.

[0028] In another aspect, the present invention provides a method for killing solid tumor cells, the method comprising the step of contacting solid tumor cells with any one or a plurality of the aforementioned cells or a pharmaceutical composition, or with a cell or a plurality of cells or a pharmaceutical composition as described herein, in an amount effective for killing tumor cells. In some cases, the cell or a plurality of cells are, for example, γδ, T cell(s).

[0029] In some embodiments, the method comprises introducing a therapeutically effective amount of a cell, such as γδ, T cell(s), or a pharmaceutical composition into a host organism containing solid tumor cells. In some embodiments, the method comprises introducing a therapeutically effective amount of a cell, such as γδ, T cell(s), or a pharmaceutical composition thereof into a host organism containing solid tumor cells, for example, and administering one or more methods that simultaneously or sequentially elevate common gamma chain cytokine(s).

[0030] In some embodiments, administering one or more methods for increasing common gamma chain cytokine(s) comprises, for example, simultaneously with introducing cell(s) or sequentially administering an amount of common gamma chain cytokine(s) effective for increasing proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced cell(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 for increasing common gamma chain cytokine(s) comprise administering an amount of common gamma chain cytokine(s) effective for increasing proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced cell(s) before and / or after introducing cell(s).

[0031] In some embodiments, one or more methods for increasing common gamma chain cytokine(s) include lymphocyte removal before introducing γδ T cell(s). In some embodiments, one or more methods for increasing 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 in a host organism In vivo Reduces tumor burden and / or increases the average survival time of a host organism compared to a control organism, wherein said control organism is not treated with cell(s) or pharmaceutical composition. In some embodiments, the method is a method for treating cancer in a subject requiring cancer treatment.

[0032] In another aspect, the present invention provides a tumor cell apoptotic amount of any one of the aforementioned cells or the cells described herein (e.g., γδ T cells); and the use of a plurality of such cells or a pharmaceutical composition containing such cells in the manufacture of a medicine for the treatment of solid tumor cell cancer in a subject requiring treatment for solid tumor cell cancer. In another aspect, the present invention provides a method for treating cancer in a subject requiring treatment for cancer, the method comprising administering a therapeutically effective amount of cells, wherein the cancer comprises solid tumor cells exhibiting cell surface expression of TyrD or GPC3.

[0033] In some embodiments, the method comprises administering one or more methods that elevate common gamma-chain cytokine(s) simultaneously with or sequentially with the administration of cells. In some embodiments, the method comprises performing a plurality of administrations of cells, wherein the interval between the plurality of administrations is at least about 1 week, preferably at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks and / or no more than once every 6 or 12 months.

[0034] In another aspect, the present invention provides a pharmaceutical composition for use in any one of the methods described above or in the method described herein.

[0035] Integration by reference

[0036] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. Brief explanation of the drawing

[0037] Brief explanation of the drawing Fig. 1 is a schematic diagram of an embodiment of a chimeric antigen receptor (CAR) containing one co-stimulating signaling endodomain (left) or two co-stimulating signaling endodomains (right). As used herein, co-stimulating signaling endodomains are also referred to as co-stimulating endodomains or co-stimulating endodomains. Exemplary co-stimulating signaling endodomains useful for an exemplary CAR include, without limitation, CD28; CD137 (4-1BB); CD278 (ICOS); CD27; CD134 (OX40); TLR2 and combinations thereof. Fig. 2 is the engineered and non-engineered γδ T cells described herein for 526 and WM266.1-Luc melanoma cell lines In vitro It exemplifies cytotoxicity. Fig. 3 Silver γδ T cells as described herein in a subcutaneous WM266.4 cell NOD scid gamma (NSG) mouse model In vivo It exemplifies therapeutic efficacy. Fig. 4 This exemplifies a manufacturing process for the production of engineered γδ CAR-T cells and non-engineered γδ CAR-T cells for the treatment of solid tumors, for example. Fig. 5 This exemplifies the cytotoxic activity of Vδ1 T cells transduced with a control CAR construct or a construct targeting tyrosinase polypeptide. Fig. 6This exemplifies the transduction efficiency of Vδ1 cells with anti-Glypican 3 (GPC3) CAR constructs containing soluble IL-15 (sIL15) (sequence number: 14) and codon optimized (WO 2007 / 037780A2) sIL15. Fig. 7 This exemplifies the cytotoxic activity of Vδ1 T cells that are not transduced or are transduced with an anti-GPC3 CAR construct against a panel of liver cancer cell lines having different levels of GPC3 expression. Specific details for implementing the invention

[0038] definition:

[0039] To interpret this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any of the definitions presented conflict with any literature incorporated herein by reference, the definitions set forth below shall prevail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention pertains.

[0040] When referring to measurable values ​​such as quantities, time periods, etc., the term “about” as used herein means to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, because such variation is suitable for performing the disclosed method.

[0041] As used herein, the term “γδ T-cell (gamma delta T-cell)” refers to a subset of T-cells expressing a distinct T-cell receptor (TCR) consisting of one γ-chain and one δ-chain on its surface, namely the γδTCR. The term “γδ T-cell” specifically, without limitation, includes all subsets of γδ T-cells, including Vδ1, Vδ2, and 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, γδ T-cells are Vδ1 - , Vδ2 - , or Vδ1 - and Vδ2 - Compositions and methods for creating and using engineered and non-engineered γδ T cells and / or sub-types thereof are, without limitation, incorporated into US 2016 / 0175358; WO 2017 / 197347; US 9499788; US 2018 / 0169147; US 9907820; US 2018 / 0125889 and US 2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including said compositions and methods for creating and using engineered and non-engineered γδ T cells and / or sub-types thereof. The present application further considers T cells expressing one γ-chain or one δ-chain in combination with a second polypeptide to form a functional TCR, or other engineered leukocytes or lymphocytes. These engineered leukocytes or lymphocytes expressing one γ-chain or one δ-chain may be used in the methods described herein or may be present in the composition.

[0042] As used herein, the terms “T lymphocyte” or “T cell” refer to immune cells that express or have expressed CD3 (CD3+) and T cell receptor (TCR+). T cells play a central role in cell-mediated immunity. T cells “expressing” CD3 and TCR have been engineered to eliminate CD3 and / or TCR cell surface expression.

[0043] As used herein, the term “TCR” or “T cell receptor” refers to a dimeric heterogeneous cell surface signaling protein forming an alpha-beta or gamma-delta receptor or a combination thereof. While αβTCRs recognize antigens presented by MHC molecules, γδTCRs can recognize antigens independently of MHC presentation.

[0044] The term "MHC" (Major Histocompatibility Complex) refers to a subset of genes encoding cell-surface antigen-presenting proteins. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC and HLA are used interchangeably.

[0045] As used herein, “activation” refers to the state of T cells sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term “activated T cell” refers, among other things, to a T cell undergoing cell division.

[0046] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from a natural source or a recombinant source, or it may be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. In the present invention, antibodies may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as short-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, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0047] The term "antibody fragment" refers to a part of an intact antibody and refers to the antigenic variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0048] As used herein, “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in the antibody molecule in its naturally occurring stereotype.

[0049] As used herein, “antibody light chain” refers to the smaller of the two types of polypeptide chains present in an antibody molecule in their naturally occurring stereotypes. κ and λ light chains refer to two major antibody light chain isotypes.

[0050] As used herein, the term “synthetic antibody” means an antibody produced using recombinant DNA technology, for example, an antibody expressed by a bacteriophage described herein. The term should also be interpreted to mean an antibody or an amino acid sequence that specifies an antibody, which is produced by the synthesis of a DNA molecule encoding an antibody and in which the DNA molecule expresses an antibody protein, wherein the DNA or amino acid sequence is obtained using available and well-known synthetic DNA or amino acid sequence technology in the art.

[0051] As used herein, the term “antigen” or “Ag” is defined as a molecule that triggers an immune response. This immune response may include antibody production or the activation of specific immune-qualified cells, or both. A person skilled in the art will understand that any macromolecule, including proteins or peptides, may act as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. A person skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that triggers an immune response therefore encodes an “antigen” as the term is used herein. Furthermore, a person skilled in the art will understand that an antigen does not need to be encoded solely by the full nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to induce a desired immune response. Furthermore, a person skilled in the art will understand that an antigen does not need to be encoded by a “gene” at all. It is readily apparent that an antigen may be generated or synthesized, or may be derived from a biological sample. These biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0052] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specifically binding to immunoglobulin or T-cell receptors. The determinants of an epitope generally consist of active surface groupings of molecules, such as amino acids, lipids, or sugar side chains, and typically possess specific charge properties as well as specific three-dimensional structural characteristics. Antibodies have an equilibrium dissociation constant (K D ) is 10 -6 - 10 -12 It is said that it specifically binds to antigens when in the M range.

[0053] As used herein, the term “Chimeric Antigen Receptor (CAR)” may refer, for example, to an artificial T-cell receptor, T-body, monoclonal immunoreceptor, chimeric T-cell receptor, or chimeric immunoreceptor, and encompasses engineered receptors that implant artificial specificity onto specific immune effector cells. A CAR can be used to confer specificity of a monoclonal antibody onto T cells, thereby enabling the generation of a large number of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, the CAR is directed, for example, to the specificity of the cell toward a tumor-associated antigen. In some embodiments, the CAR comprises an intracellular activation domain (which causes the T cell to be activated upon coupling with a targeting moiety, such as a target tumor cell), a transmembrane domain, and an extracellular domain that may vary in length and includes a disease- or disorder-associated, for example, tumor-antigen binding region. In certain embodiments, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane domain and an endodomain. Specificity of other CAR designs may be derived from a receptor ligand (e.g., peptide) or a pattern-recognition receptor, such as decin. In certain cases, the spacing of the antigen-recognition domain may be modified to reduce activation-induced cell death. In certain cases, the CAR includes domains for additional co-stimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP 10 / 12 and / or OX40, ICOS, TLR (e.g., TLR2), etc. In some cases, molecules may be co-expressed with CARs, including co-stimulating molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally remove T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.Furthermore, those skilled in the art will understand that the co-stimulating domain does not need to be encoded solely by the full nucleotide sequence of the gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to induce a desired immune response.

[0054] As used herein, the term "anti-tumor effect" refers to a biological effect that may manifest as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the pathology of cancer. The "anti-tumor effect" may also manifest as the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors in the first place.

[0055] The term "auto-antigen" means any self-antigen that is mistakenly recognized as a foreign substance by the immune system according to the present invention. Auto-antigens include, but are not limited to, cell surface receptors, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins.

[0056] As used herein, the term “self” means any substance derived from an entity that is later reintroduced into the same entity.

[0057] As used in this specification, the term “homologous” refers to a substance derived from an animal that is later introduced into another animal of the same species.

[0058] The term "therapeutic effective dose" refers to an amount of a composition that elicits a biological or medical response in a tissue, system, or subject sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutic effective dose" includes an amount of a composition sufficient to prevent or alleviate to some extent the onset of one or more signs or symptoms of the disorder or disease being treated (e.g., a solid tumor) when administered. The therapeutic effective dose will vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0059] As used herein, the term “treat” a disease means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject.

[0060] "Concomitant" administration with one or more additional therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0061] As used herein, the term “pharmaceuticalally acceptable” refers to a substance including, but not limited to, a salt, carrier, or diluent that is relatively non-toxic and does not eliminate the biological activity or properties of the compound; that is, the substance may be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any component of the composition containing it.

[0062] "Encoding" refers to the unique characteristics of a specific sequence of nucleotides in polynucleotides, such as genes, cDNA, or mRNA, which act as a template for the synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological characteristics produced therefrom. Thus, a gene encodes a protein when the transcription and translation of the mRNA corresponding to that gene produce a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally provided in sequence lists, and the non-coding strand used as a template for the transcription of the gene or cDNA, may be referred to as encoding the protein or other product of that gene or cDNA.

[0063] "Isolated" means that it has been altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from their coexisting materials in their natural state are "isolated." The isolated nucleic acids or proteins may exist in a substantially purified form or in non-natural environments, such as host cells, for example.

[0064] Unless otherwise specified, “nucleotide sequences encoding amino acid sequences” include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0065] The terms “patient,” “subject,” “individual,” etc. are used interchangeably herein and refer to any animal that conforms to the method described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0066] As used herein with respect to antibodies, the term “specifically binds” means an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such species-cross-reactivity does not specifically change the classification of the antibody in itself. In another example, an antibody that specifically binds to an antigen may also bind to other allelic forms of the antigen. However, such cross-reactivity does not specifically change the classification of the antibody in itself. In some cases, the term “specifically binds” or “specifically binds” may be used in relation to the interaction between an antibody, protein, or peptide and a second chemical species, where the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, it may be used to mean that the antibody generally recognizes and binds to a specific protein structure rather than the protein itself. If the antibody is specific to epitope "A", the presence of a molecule containing epitope A (or free unlabeled A) in a reaction containing the antibody and labeled "A" will reduce the amount of labeled A bound to the antibody.

[0067] In some embodiments, the specific binding is at least about 1 x 10 -8 It can be characterized by an equilibrium dissociation constant less than or equal to M (e.g., a smaller K D (indicating a tighter bond). Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. Furthermore, a multi-specific antibody that binds to a first antigen and one or more additional antigens, or a bispecific antibody that binds to two different regions of an antigen, is nevertheless considered to be an "specifically binding" antibody as used herein.

[0068] A solid tumor is a tumor containing a mass of at least about 10 or at least about 100 tumor cells. A solid tumor can be a soft tissue tumor, a primary solid tumor, or a metastatic lesion.

[0069] Examples of solid tumors include sarcomas, adenocarcinomas, and carcinomas affecting various organ systems, such as the liver, lungs, breasts, lymphocytes, gastrointestinal tract (e.g., colon), urinary tract (e.g., kidneys, urothelial cells), prostate, and pharynx. Adenocarcinomas include most malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. In one embodiment, the cancer is melanoma, e.g., advanced melanoma. Metastatic lesions of the aforementioned cancer may 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, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid, cancer of the parathyroid, adrenal cancer, soft tissue sarcoma, cancer of the urethra, cancer of the penis, solid tumors of children, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal cancer, squamous cell carcinoma, environmentally induced cancer including asbestos-induced cancer, and combinations of said cancers. In a preferred embodiment, solid tumor cells express or overexpress TyrD or a fragment thereof. In some embodiments, solid tumor cells express or overexpress an HLA:peptide complex containing a TyrD fragment. In some embodiments, the TyrD fragment is TyrD 369 -377is. In some embodiments, the HLA is a Class I HLA such as HLA-A2. In some embodiments, the solid tumor cell is HLA-A2 / TyrD 369 -377 Expresses or over-expresses.

[0070] In some embodiments, solid tumor cells express or over-express Glypican 3 (GPC3). In some embodiments, solid tumor cells are US 7,919,086; WO 2014 / 180306; WO 2018 / 019772; WO 2016 / 049459; WO 2003 / 000883; WO 2006 / 046751; WO 2007 / 047291; WO 2016 / 086813; WO 2016 / 047722; WO 2016 / 036973; Reference [Cancer Res. 2008;68:9832-9838]; An epitope of GPC3 specifically bound by an anti-GPC3 antibody, a T cell receptor, or a chimeric antigen receptor as described in Proc Natl Acad Sci US A. 2013 Mar 19;110(12):E1083-91 is expressed or overexpressed, each of which is incorporated by reference in whole and for all purposes, and in particular by reference to a binding domain, an antibody, an antibody fragment, a complementarity determining region, a polypeptide containing said complementarity determining region, a nucleic acid encoding said complementarity determining region, and epitope specificity and an assay for determining epitope specificity as described herein. In some embodiments, solid tumor cells express or overexpress an epitope of glypican3 specifically bound by the anti-GPC3 antibody GC33. In some embodiments, solid tumors express or overexpress 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 GPC3298 -306 Expresses or overexpresses an HLA:peptide complex containing a peptide. Oncoimmunology See . 2012 Nov 1; 1(8): 1448-1450.

[0071] "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 sufficient cis-acting elements for expression; other elements for expression are a host cell or In vitro It can be supplied by an expression system. The expression cassette may be a component of a vector such as a cosmid, plasmid (e.g., contained in a naked or liposome), or virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). The expression cassette may be in a host cell such as a γδ T cell.

[0072] Scope: Throughout this disclosure, various aspects of the invention may be presented in the form of a range. It should be understood that descriptions in the form of a range are merely for convenience and brevity and should not be interpreted as strictly limiting the scope of the invention. Accordingly, descriptions of a range should be deemed to specifically disclose individual numerical values ​​within that range, as well as all possible sub-ranges. For example, a description of a range such as 1 to 6 should be deemed to specifically disclose individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the width of the range.

[0073] Chimeric antigen receptor constructs:

[0074] Aspects of the present invention include a nucleic acid encoding a CAR, and constructs and vectors containing such nucleic acid. In some cases, the nucleic acid is, for example, a heterogeneous component of an expression cassette. In some embodiments, the nucleic acid is, for example, a heterogeneous component of a retroviral vector. In some embodiments, the nucleic acid is, for example, a heterogeneous component of an αβ or γδ T cell, preferably a γδ T cell. In some embodiments, the nucleic acid is, for example, γ + T cells and / or δ + It is a component of the heterogeneity of T cells. In some embodiments, the nucleic acid is, for example, α - T cells and / or β - It is a component of the heterogeneity of T cells.

[0075] This specification describes nucleic acids encoding a CAR binding domain that specifically binds to tumor-associated antigens (TAAs) expressed on the surface of solid tumor cells. An exemplary TAA is tyrosinase (TyrD) or a peptide fragment thereof. In some cases, the TAA is glypican3 or a peptide fragment thereof. In some cases, the TAA is a peptide bound to an HLA molecule, such as a class I HLA molecule. A tyrosinase peptide bound to a class I HLA molecule (also referred to interchangeably herein as HLA-restricted tyrosinase epitope, HLA-restricted tyrosinase epitope, and MHC-restricted tyrosinase antigen) is derived from a tyrosinase enzyme (Gene Bank Accession No.: NP_000363.1), is typically 8-10 amino acids in length, and binds to the heavy chain α1-α2 groove through two or three anchor residues that interact with the corresponding binding pocket in the HLA molecule.

[0076] Tyrosinase is a membrane-associated N-linked glycoprotein and a key enzyme in melanin synthesis. It is expressed in all healthy melanocytes and in almost all melanoma tumor samples (H. Takeuchi, et al., 2003; S. Reinke, et al., 2005). Peptides derived from this enzyme exist on MHC class I molecules and are recognized by autolytic 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 Immunotherapy. 54:187-207, 2005]. Additional tumor tyrosinase HLA-restriction peptides derived from tumor-associated antigens (TAAs) can be found at Istituto Nazionale per lo Studio e la Cura dei Tumori on the website www.istitutotumori.mi.it.

[0077] Non-restrictive examples of MHC class I restricted tyrosinase antigenic peptides are provided in WO2008 / 120202, for example, Table 139 of WO2008 / 120202, which is incorporated herein by reference in its entirety. According to some embodiments of the present invention, the tyrosinase antigenic peptide is TyrD 369 -377 It is a peptide. HLA ( for exampleBinding domains that specifically bind to an epitope in TyrD, including but not limited to those that bind in a restrictive manner (e.g., Class I HLA), include but not limited to those described in WO 2016 / 199140; WO 2016 / 199141; US ​​9688739; and the co-pending application PCT / IB2017 / 053539, each of which is incorporated by reference for all purposes including but not limited to compositions and methods for identifying, preparing, and using a binding domain that specifically binds to TyrD or an epitope in TyrD in an HLA-restrictive or HLA-independent manner.

[0078] A GPC3 peptide that binds to a class I HLA molecule (also referred to interchangeably herein as HLA-restricted GPC3 epitope, HLA-restricted GPC3 epitope, and MHC-restricted GPC3 antigen) is derived from glypican3 protein (gene bank accession number: NM_001164617.2), is typically 8-10 amino acids long, and binds to the heavy chain α1-α2 groove through two or three anchor residues that interact with the corresponding binding pocket in the HLA molecule.

[0079] As used herein, a binding domain, CAR, or CAR T cell that specifically binds to TyrD and / or specifically binds to an epitope within TyrD comprises, but is not limited to, a binding domain, CAR, or CAR T cell that specifically binds to a TyrD peptide fragment. A binding domain, CAR, or CAR T cell that specifically binds to a TyrD peptide fragment may specifically bind to the TyrD peptide fragment referenced in an HLA-restricted manner. Similarly, as used herein, a cell expressing TyrD on the surface of a cell comprises a cell expressing or overexpressing a TyrD peptide fragment on the surface of a cell, as in a peptide:HLA complex.

[0080] As used herein, a binding domain, CAR, or CAR T cell that specifically binds to GPC3 and / or specifically binds to an epitope within GPC3 comprises, but is not limited to, a binding domain, CAR, or CAR T cell that specifically binds to a GPC3 peptide fragment. A binding domain, CAR, or CAR T cell that specifically binds to a GPC3 peptide fragment may specifically bind to the GPC3 peptide fragment referenced in an HLA-restricted manner. Similarly, as used herein, a cell expressing TyrD on the surface of a cell comprises a cell expressing or over-expressing a GPC3 peptide fragment on the surface of a cell, as in a peptide:HLA complex.

[0081] In some embodiments, the binding domain binds to an antigen expressed in a full-length functional polypeptide on the surface of a cell. In some embodiments, the binding domain binds to an antigen presented on an MHC:antigen complex. In some embodiments, the binding domain binds to an antigen in an HLA-restricted manner. Binding domains exhibiting specificity for the MHC:antigen complex are described, for example, in WO / 2016 / 199140 and WO / 2016 / 199141.

[0082] In some embodiments, the isolated nucleic acid encodes an anti-TyrD binding domain having CDRH1 containing TSGMGVS (SEQ No.: 33), CDRH2 containing HIYWDDDKRYNPSLKS (SEQ No.: 34), CDRH3 containing KDYGSSFYAMHY (SEQ No.: 35), CDRL1 containing KASQDIHNYIA (SEQ No.: 36), CDRL1 containing YTSTLQP (SEQ No.: 37), and / or CDRL2 containing LQYDNLWT (SEQ No.: 38).

[0083] In some embodiments, the isolated nucleic acid encodes an anti-GPC3 binding domain having CDRH1 containing DYEMH (SEQ No.: 39) (or GYTFTDYEMH (SEQ No.: 40)), CDRH2 containing ALDPKTGDTAYSQKFKG (SEQ No.: 41), CDRH3 containing FYSYTY (SEQ No.: 42), CDRL1 containing RSSQSLVHSNRNTYLH (SEQ No.: 43), CDRL2 containing KVSNRFS (SEQ No.: 44), and / or CDRL3 containing SQNTHVPPT (SEQ No.: 45).

[0084] The present disclosure also considers anti-TyrD or anti-GPC3 binding domains that compete for binding with the sequence provided herein. Those skilled in the art can determine, using known methods, whether the anti-TyrD binding domain binds to the same epitope as the reference antibody or binding domain, or competes for binding with it. For example, to determine whether a test antibody binds to the same epitope as the reference binding domain, the reference binding domain may be allowed to bind to TyrD under saturation conditions. Next, the ability of the test binding domain to bind to the TyrD molecule may be evaluated. If the test binding domain can bind to TyrD following saturation binding with the reference binding domain, it may be concluded that the test binding domain binds to an epitope different from that of the reference binding domain. On the other hand, if the test binding domain cannot bind to TyrD following saturation binding with the reference binding domain, the test binding domain may bind to the same epitope as that bound by the reference binding domain.

[0085] To determine whether a binding domain competes for binding with a reference binding domain, the binding methodology described above is performed in two directions. In the first direction, the reference binding domain binds to TyrD under saturation conditions, and then the binding of the test binding domain to the TyrD molecule is evaluated. In the first direction, the test binding domain binds to the TyrD molecule under saturation conditions, and then the binding of the reference binding domain to the TyrD molecule is evaluated. If, in both directions, only the first (saturated) binding domain can bind to the TyrD molecule, it is concluded that the test binding domain and the reference binding domain are competing for binding to TyrD. As is understood by those skilled in the art, a binding domain competing for binding with a reference binding domain does not necessarily have to bind to the same epitope as the reference binding domain, but can sterically block the binding of the reference binding domain by binding to overlapping or adjacent epitopes. The method described above can likewise be applied to the anti-TyrD binding domain to determine competition with the anti-TyrD binding domain and epitope binding.

[0086] Two binding domains bind to the same or overlapping epitopes, where each competitively inhibits (blocks) the binding of the other to the antigen. That is, 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%, when evaluated in a competitive binding assay. for example(Refer to Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, in an antigen that reduces or eliminates the binding of one binding domain, if essentially all amino acid mutations reduce or eliminate the binding of another, the two binding domains have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one binding domain reduce or eliminate the binding of another, the two binding domains have overlapping epitopes.

[0087] Additional routine experiments ( for example Next, peptide mutation and binding analysis can be performed to determine whether the observed lack of binding in the test binding domain is actually due to binding to the same epitope as the reference binding domain, or whether stereoblocking (or other phenomena) is responsible for 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.

[0088] The present disclosure provides antibodies and CARs having "substantial identity" or "substantial similarity" to the sequences provided herein in the CDR or framework region. When referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantively identical" indicates that when optimally aligned with another nucleic acid (or a complementary strand of another nucleic acid), there is nucleotide sequence identity in % of nucleotide bases, 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%, as measured by any well-known sequence identity algorithm such as FASTA, BLAST, or GAP as discussed below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule in a specific example.

[0089] As applied to polypeptides, the term “substantially similar” or “substantially similar” means that when optimally aligned by a program GAP or BESTFIT using default gap weights, for example, 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, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitution" is the substitution of an amino acid residue with another amino acid residue having a side chain (R group) that has similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitution will not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other due to conservative substitution, the conservative nature of the substitution can be modified by up-adjusting the percentage or degree of similarity. Means for this adjustment are well known to those skilled in the art. for example, refer to Pearson (1994) Methods Mol. Biol. 24: 307-331 incorporated herein by reference. Examples of a group of amino acids having side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; 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 having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., (1992) Science 256: 1443 45, incorporated herein by reference. A “moderately conservative” substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0090] Sequence identity and / or similarity for polypeptides is typically measured using sequencing software. Protein analysis software matches similar sequences using measures of 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, which, when used with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutates. for example, refer to GCG version 6.1. Polypeptide sequences can also be compared using default or recommended parameters; FASTA with the program in GCG version 6.1. FASTA ( for example FASTA2 and FASTA3) provide alignment and percentage sequence identity of the region that best overlaps between the query and search sequences (Pearson (2000) Same as above The sequences can also be compared using the Smith-Waterman homology search algorithm, which uses an affine gap search with a BLOSUM matrix of 62, a gap open penalty of 12 and a gap extension penalty of 2. Another preferred algorithm for comparing the sequences disclosed herein with a database containing a large number of sequences from other organisms is the computer program BLAST, in particular BLASTP or TBLASTN, which uses default parameters. for example See Altschul et al., (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0091] The present specification provides anti-TyrD or anti-GPC3 comprising variants of any HCVR, LCVR, and / or CDR amino acid sequence disclosed herein having one or more substitutions (e.g., conservative substitutions). For example, the present disclosure comprises, for example, comprising an anti-TyrD CAR having an HCVR, LCVR, and / or CDR amino acid sequence having 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 compared to any of the HCVR, LCVR, and / or CDR 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 substitution (e.g., conservative amino acid substitution) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0092] Similarly, the present disclosure is, for example, comprising an anti-GPC3 CAR having an HCVR, LCVR, and / or CDR amino acid sequence having 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 compared to any of the HCVR, LCVR, and / or CDR 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 substitution (e.g., conservative amino acid substitution) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0093] The exemplary binding domains described herein typically comprise a heavy chain region followed by a light chain region (VH-VL) in sequence from the amino to the carboxyl terminus. Where a 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 an alternative embodiment in which the order of the VH and VL regions is reversed, for example, in a CAR comprising a scFV or scFv binding domain. Accordingly, the description of the VH-VL order also describes an alternative VL-VH order, for example, in a CAR comprising a scFV or scFv binding domain. Furthermore, the description of the VL-VH order also describes an alternative VH-VL order, for example, in a CAR comprising a scFV or scFv binding domain.

[0094] Generally, a CAR encoding a nucleic acid described herein comprises an extracellular linker portion encoding a peptide linker that connects a binding domain to a transmembrane domain. An exemplary linker portion is a linker portion encoding a CD8α hinge domain, for example , including but not limited to SEQ No. 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ No. 2 (TTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). Typically, a region encoding a peptide linker ( for example , CD8α hinge domain) is the 3' of the region encoding the coupling domain and the 5' of the region encoding the transmembrane domain.

[0095] The CAR encoding the nucleic acid described herein includes a transmembrane domain. The transmembrane domain includes an extracellular antigen binding domain, for example , and the hinge can be connected to one or more intracellular signaling components. For example, the transmembrane domain is an antigen-binding domain, for example , and the hinge can be connected to the CD3ζ signaling domain and optionally to one or two co-stimulating endodomains. An exemplary transmembrane domain is, without limitation, the CD8α transmembrane domain, for example Sequence No. 3 (IWAPLAGTCGVLLLSLVITLYC) contains. Typically, a transmembrane domain ( for example The region encoding the CD8α transmembrane domain) is a peptide linker ( for example It is the 3' of the region encoding the CD8α hinge domain and the 5' of the region encoding one or more cytoplasmic domains.

[0096] In some embodiments, the isolated nucleic acid encodes a cytoplasmic region containing one or more cytoplasmic domains. The region encoding the cytoplasmic region is typically the 3' of the region encoding the transmembrane domain. The cytoplasmic domain is typically associated with γδ T cell proliferation, cytotoxic activity, and / or pro-inflammatory cytokine expression ( for example It is a signaling domain that provides an activation signal for , TNF-α or IFNγ). An exemplary cytoplasmic domain is a CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is SEQ No. 4 (RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR) or comprises the same. In some embodiments, the CD3ζ signaling domain is SEQ No. 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the cytoplasmic region is a plurality of ( for example , 2, 3, 4, 5, or 6) signaling domains, e.g., multiple ( for example , 2, 3, 4, 5 or 6) containing a CD3ζ signaling domain, and for example Each is independently selected from SEQ ID NOs: 4 and 5. In some embodiments, the cytoplasmic region is a plurality of ( for example , 2, 3, 4, 5, or 6) contains a non-CD3ζ signaling domain and a CD3ζ signaling domain. In some embodiments, the cytoplasmic region contains a non-CD3ζ signaling domain and a plurality of ( for example , 2, 3, 4, 5, or 6) contains a CD3ζ signaling domain. Additional or alternative signaling domains are included without limitation.

[0097] The cytoplasmic region may contain one or more co-stimulating endodomains. The region encoding one or more co-stimulating endodomains may be the 5' or 3' of the region encoding the signaling domain. In some embodiments, the region encoding one or more co-stimulating endodomains is the 5' of the region encoding the signaling domain. In some embodiments, the region encoding one or more co-stimulating endodomains is the 5' of the signaling domain, and an additional region encoding one or more co-stimulating endodomains is the 3' of the signaling domain. Exemplary co-stimulating endodomains include, without limitation, CD28; CD137 (4-1BB); CD278 (ICOS); CD27; CD134 (OX40); and TLR2 co-stimulating endodomains, and combinations thereof.

[0098] In some embodiments, additional signaling schemes may be included to increase the proliferation, persistence, and / or cytotoxic activity of γδ-T cells described herein. For example, in some embodiments, the CAR construct may encode a soluble common gamma-chain cytokine at the 3' end of the isolated nucleic acid. The common gamma-chain cytokine encoding region may be linked to the 5' portion of the CAR construct via a T2A linker encoding region so that the common gamma-chain cytokine is cleaved from the CAR polypeptide and secreted by the cell.

[0099] In some embodiments, the composition encodes at least one 4-1BB co-stimulating endodomain, and optionally a second co-stimulating endodomain selected from 4-1BB, ICOS, CD28, and CD27 co-stimulating endodomains. In some embodiments, the composition encodes at least two 4-1BB co-stimulating endodomains, or two 4-1BB co-stimulating endodomains, in combination with one, two, three, or four or more co-stimulating endodomains selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB co-stimulating endodomain comprises SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL).

[0100] In some embodiments, the composition encodes one CD27 co-stimulation endodomain and optionally a second co-stimulation endodomain selected from 4-1BB, ICOS, CD28, and CD27 co-stimulation endodomains. In some embodiments, the composition encodes a CD27 co-stimulation endodomain and a 4-1BB co-stimulation endodomain. In some embodiments, the composition encodes two CD27 co-stimulation endodomains. In some embodiments, the CD27 co-stimulation endodomain comprises SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[0101] In some embodiments, the composition encodes a secretory signal, e.g., SEQ No. 12 (MALPVTALLLPLALLLHAARP), operably linked to promote the secretion of a C-terminal polypeptide, such as a cytokine, that supports the activation, cytotoxicity, and / or persistence of T cells (e.g., CAR-T cells). In some embodiments, the secretory signal is the secretory signal of SEQ No. 26 (MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEA). In some embodiments, the composition encodes a secretion signal, e.g., SEQ: 12, operably coupled to promote the secretion of IL-15 or its active fragment, e.g., SEQ: 14 (NWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Other IL-15 sequences comprising a codon-optimized nucleic acid sequence encoding sIL15 are disclosed in WO 2007 / 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.

[0102] In some embodiments, the construct encodes one or more multi-cistron linker regions between a secretion signal operably linked to, for example, a signaling domain and / or a co-stimulating endodomain to promote the secretion of a cytokine. A multi-cistron linker region is a region of a polypeptide sequence or RNA sequence that facilitates the production of multiple separated polypeptides from a single transcription product. In some embodiments, the multi-cistron linker region encodes a cleavage sequence. Suitable cleavage sequences include self-cleavage sequences such as P2A, F2A, E2A, or T2A cleavage sequences and / or sequences cleaved by endogenous proteases such as purines.

[0103] In some embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a purine cleavage sequence. In some embodiments, the cleavage sequence is a P2A and purine cleavage sequence. In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ No. 15 (SGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is the purine cleavage sequence of SEQ No. 16 (RAKR). In some embodiments, the cleavage sequence is the P2A+purine cleavage sequence of SEQ No. 17 (RAKRSGSGATNFSLLKQAGDVEENP GP). In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ No. 25 (GSGATNFSLLKQAGDVEENPGP).

[0104] In some embodiments, the cleavage sequence is or includes the P2A cleavage sequence of SEQ No. 27 (ATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is or includes the F2A cleavage sequence of SEQ No. 28 (VKQTLNNFDLLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or includes the E2A cleavage sequence of SEQ No. 29 (QCTNYALLKLAGDVESNPGP). In some embodiments, the cleavage sequence is or includes the T2A cleavage sequence of SEQ No. 30 (EGRSLLTCGDVEENPGP). In certain embodiments, multiple self-cleavage sequences may encode a carboxyl terminus for a signaling and / or co-stimulating domain and an amino terminus for an encoded secreted cytokine (e.g., a common gamma-chain cytokine such as IL-15), and preferably, the multiple self-cleavage sequences are independently selected from the group consisting of P2A cleavage sequences, T2A cleavage sequences, E2A cleavage sequences, and F2A cleavage sequences. In certain embodiments, one or more self-cleavage sequences and one or more sequences cleaved by an endogenous protease are encoded in the compositions described herein. In certain embodiments, an endogenous protease recognition site encodes an amino terminus for a self-cleavage sequence.

[0105] In some embodiments, the multi-cistron linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by sequence number: 31 ().

[0106] Another exemplary internal ribosome entry site is sequence number: 32 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCGTCATGTGTGC AACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGCAGGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGACTGGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC) is encoded by

[0107] Additional suitable internal ribosomal 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 on iresite.org, those described in WO 2018 / 215787, the sequence described in Gene Bank accession number KP019382.1, and the IRES element described in Gene Bank accession number LT727339.1, the contents of which are incorporated in whole and for all purposes, in particular for reference to internal ribosomal entry sites and their use as described herein.

[0108] Additional multi-cystron linker regions including cutting self-cutting and IRES elements are disclosed in US 2018 / 0360992 and US 8,865,467.

[0109] In some embodiments, the isolated nucleic acid is SEQ ID NO: 8 (MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCKASQDIHNYIAWYQQKPGKAPKLLIHYTSTLQPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLWTFGQGT KVEIKRGGGSGGGGSGGGGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARKDYG SSFYAMHYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), hD11 anti-TyrD binding domain (anti-TyrD 369 - 377 It encodes an hD11-CD8-BBz polypeptide containing a CD8α hinge and transmembrane region, a 4-1BB co-stimulating endodomain, and a CD3ζ signaling domain.

[0110] In some embodiments, anti-TyrD 369 -377

[0111] In some embodiments, the isolated nucleic acid comprises a codon-optimized sequence encoding a CD8α hinge region. An exemplary codon-optimized CD8α hinge region nucleic acid sequence includes, but is not limited to, Sequence No. 10 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCACA GCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8α hinge region is encoded by the following sequence Sequence No. 11 (ACCACGACGCCAGCG CCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT).

[0112] In some embodiments, the isolated nucleic acid is SEQ No.: 18 (*), anti-TyrD hD11 (anti-TyrD 369 -377 The hD11-CD8-BBz-sIL15 polypeptide containing a binding domain, CD8α hinge and transmembrane region, 4-1BB co-stimulating endodomain, CD3ζ signaling domain, purine-P2A cleavage sequence and IL-15 domain encodes a secretory signal operably linked to the IL-15 domain.

[0113]

[0114] In some embodiments, the isolated nucleic acid is SEQ ID NO: 20 (MSVPTQVLGLLLLWLTDARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYS YTYWGQGTLVTVSSGGGGSGGGGSGGGGDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCS QNTHVPPTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*), a polypeptide containing a GC33 anti-GPC3 binding domain, a CD8α hinge and transmembrane region, a 4-1BB co-stimulating endodomain, and a CD3ζ signaling domain.

[0115]

[0116] In some embodiments, the isolated nucleic acid encodes a polypeptide containing the sequence number: 22 (*), GC33 anti-GPC3 binding domain, CD8α hinge and transmembrane region, 4-1BB co-stimulating endodomain, CD3ζ signaling domain, purine and P2A cleavage region, and a secretory signal operably linked to an IL-15 domain.

[0117]

[0118]

[0119] 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 (e.g., a gamma retrovirus vector), or a lentivirus vector. In some embodiments, the isolated nucleic acid contains a binding domain, a transmembrane domain, and one or more signaling and / or co-stimulating endodomains, or for example , and its adjacent portion is incorporated into the genome of a host cell, such as a host γδ T cell. In an exemplary embodiment, the isolated nucleic acid is a retroviral vector.

[0120] γδ T cells:

[0121] An embodiment of the present invention comprises γδ T cells that functionally express the isolated nucleic acid described herein and thereby express CAR on the surface of the γδ T cells.

[0122] An aspect of the present invention relates to solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAA). In vitro or In vivo γδ T cells having cytotoxic activity may be included additionally or alternatively. In some cases, cytotoxic activity is innate. In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely attributed to the presence of a CAR construct having a binding domain that specifically binds to TAAs expressed on the surface of solid tumor cells. In some cases, the solid tumor cell death activity of said γδ T cells is at the innate level of control γδ T cells. In vitro and / or In vivo solid tumorsIt indicates that it is greater than apoptotic activity. In some cases, control γδ T cells do not contain a CAR construct. In some cases, 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-stimulating endodomain described herein.

[0123] In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely due to TyrD or epitopes within TyrD, e.g., TyrD 369 -377 It is attributed to the presence of CAR constructs having a binding domain that specifically binds to. In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely due to HLA restriction ( for example , class I HLA-restricted) in the manner of TyrD or epitopes within TyrD, e.g., TyrD 369 -377 It is attributed to the presence of CAR constructs having a binding domain that specifically binds to HLA-A2 / TyrD. In some cases, cytotoxicity is at least partially, significantly (> about 25%), or entirely, HLA-A2 / TyrD 369 -377 This is due to the presence of a CAR construct having a binding domain that specifically binds to it. In some cases, γδ T cells functionally express a CAR encoded by the isolated nucleic acid described herein that specifically binds to TyrD or a peptide fragment thereof.

[0124] In some embodiments, the γδ T cells described herein are HLA-restricted ( for example It may exhibit cytotoxicity restricted by HLA class I. In other embodiments, most (> 50%), substantially all (> 90%), or all cytotoxic activity is restricted by HLA ( for example, HLA class I restriction) is not observed. HLA-limited cytotoxic activity is HLA ( for example , HLA class I) (null) tumor cell lines In vitro Cytotoxicity vs. HLA+ ( for example , HLA Class I + ) regarding tumor cell lines In vitro Cytotoxicity can be evaluated by comparing. In some embodiments, HLA-limited cytotoxic activity is provided at least partially, significantly (> 25%), or wholly by the use of a T cell receptor-like binding domain. A binding domain such as a T cell receptor is a binding domain that specifically recognizes an antigen when presented on the surface of a cell as a complex with an MHC molecule. The T cell receptor-like binding domain is, for example It is additionally listed in WO 2016 / 199141.

[0125] The γδ T cells described herein may exhibit robust and / or sustained solid tumor cell apoptotic activity. In some cases, the solid tumor cell apoptotic activity may persist for at least about 6 to 120 days or at least about 6 to 180 days from the initial contact with the solid tumor cell. In some cases, the solid tumor cell apoptotic activity of the γδ T cells described herein or their progeny may persist for at least about 6 to 120 days or at least about 6 to 180 days from the initial contact with the solid tumor cell or from the administration of the γδ T cells described herein. This sustained solid tumor cell apoptotic activity In vitro , In vivo or In vitro and In vivo It can appear in both.

[0126] Aspects of the present invention may comprise γδ T cells that proliferate in response to contact with cells exhibiting cell surface expression or overexpression of tumor-associated antigens (TAAs), either additionally or alternatively. Cells exhibiting cell surface expression of tumor-associated antigens (TAAs) may be normal cells, such as normal endothelial cells. Cells exhibiting cell surface expression or overexpression of tumor-associated antigens (TAAs) may be solid tumor cells. In some cases, proliferation is an innate activity. In some cases, proliferation is at least partially, significantly (> about 20% or > about 25%), or entirely attributed to the presence of a CAR construct having a binding domain that specifically binds to TAAs expressed on the surface of the cell. In some cases, γδ T cells exhibit a greater level compared to control γδ T cells. In vitro and / or In vivo It exhibits proliferation. In some cases, control γδ T cells do not contain a CAR construct. In some cases, 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-stimulating endodomain described herein.

[0127] In some cases, proliferation is at least partially, significantly (> about 20 or > about 25%), or entirely 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 proliferate in response to contact with cells exhibiting cell surface expression of TyrD functionally express a TyrD-specific CAR encoded by the isolated nucleic acid described herein.

[0128] The γδ T cells described herein may exhibit robust and / or sustained proliferation in a host organism containing cells that exhibit cell surface expression or overexpression of tumor-associated antigens (TAA). In some cases, proliferation may persist for at least about 6 to 120 days, or at least about 6 to 180 days, from a first contact with cells that exhibit cell surface expression or overexpression of tumor-associated antigens (TAA) or from the date of administration of γδ T cells to the host organism. In some cases, proliferation of the γδ T cells described herein or their progeny in a host organism containing cells that exhibit cell surface expression or overexpression of tumor-associated antigens (TAA) may persist for at least about 6 to 120 days, or at least about 6 to 180 days, from a first contact with the cells or from the date of first administration of γδ T cells to the host organism. In some cases, proliferation in a host organism is attributed at least partially, significantly (> about 20% or > about 25%), or entirely 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 proliferating in a host organism, including cells exhibiting cell surface expression of TyrD, functionally express a TyrD-specific CAR encoded by the isolated nucleic acid described herein.

[0129] 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 a cell expressing or overexpressing TyrD or a peptide fragment thereof on the surface of the cell. In some embodiments, the γδ T cells described herein or their progeny express or continuously express pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with a host organism, for example, a cell expressing or overexpressing TyrD or a peptide fragment thereof on the surface of the cell, for example.

[0130] In some embodiments, γδ T cells, or pharmaceutical compositions containing γδ T cells, do not essentially exhibit or do not exhibit a graft-versus-host disease when introduced into a homologous host. In some embodiments, γδ T cells, or pharmaceutical compositions containing γδ T cells, exhibit a clinically acceptable level of graft-versus-host disease when introduced into a homologous host. In some embodiments, a clinically acceptable level is a amount of graft-versus-host disease that does not require discontinuation of γδ T cell therapy to achieve therapeutically effective treatment. In some embodiments, a clinically acceptable level of graft-versus-host disease (GvHD) is an acute reaction less severe than Grade C according to the applicable IBMTR grading scale. The severity of acute graft-versus-host disease is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. An overall grade is calculated by combining the stages of individual organ involvement, which has prognostic significance. Grade I(A) GvHD is characterized as mild, Grade II(B) GvHD as moderate, Grade III(C) as severe, and Grade IV(D) as life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997; 97:855):

[0131] ● Grade A - Stage 1 cutaneous involvement alone without liver or gastrointestinal involvement (maculopapular rash covering more than 25 percent of the body)

[0132] ● Grade B - Stage 2 skin involvement; Stage 1 to 2 intestinal or liver involvement

[0133] ● Grade C - Stage 3 involvement of any organ system (generalized erythrocytoma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 1500 to 2000 mL / day)

[0134] ● Grade D - Stage 4 involvement of any organ system (generalized erythrocytoma with blister formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day or pain or bowel obstruction).

[0135] Also, Schoemans et al. Bone Marrow Transplantation Refer to pages 1401-1415 of volume 53 (2018), for example, Tables 1 and 2 which disclose criteria for evaluating and grading acute GvHD.

[0136] In some embodiments, γδ T cells or pharmaceutical compositions containing γδ T cells exhibit a reduced or substantially reduced graft-versus-host response when introduced into an allogeneic host compared to the graft-versus-host response exhibited by control αβ T cells or a control pharmaceutical composition containing control αβ T cells administered to an allogeneic host. In some cases, the control αβ T cells are allogeneic non-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.

[0137] The γδ T cells described herein may be δ1, δ2, δ3, or δ4 γδ T cells, or a combination thereof. In some cases, γδ T cells are mostly (>50%), substantially (>90%), essentially all, or completely δ2 - They are γδ T cells. In some cases, γδ T cells are mostly (>50%), substantially (>90%), essentially all, or completely δ1 γδ T cells.

[0138] γδ T cells can be obtained from allogeneic or autodonors. γδ T cells can be partially or wholly purified, or unpurified. In vitro It can be expanded. In vitroMethods and compositions for expansion include, without limitation, those described in WO 2017 / 197347. Expansion may be performed before or after, or before and after, the CAR construct is introduced into the γδ T cell(s).

[0139] The γδ T cells described in this specification are stored for use in adoptive cell delivery, for example It can be frozen.

[0140] Methods to inhibit or kill tumor cells

[0141] One or multiple non-engineered, γδ T-cell populations, engineered γδ T-cell populations, and / or mixtures thereof having cytotoxic activity against solid tumor cells may be administered to a subject in any order or concurrently. When administered concurrently, the multiple non-engineered, γδ T-cell populations, engineered γδ T-cell populations, and / or mixtures thereof of the present invention may be provided in a single integrated form, such as an intravenous injection, or in multiple forms, such as multiple intravenous infusions, sc, injections, or pills. The non-engineered, γδ T-cell populations, engineered γδ T-cell populations, and / or mixtures thereof of the present invention may be packaged together or separately in a single package or multiple packages. One or all of the non-engineered, γδ T-cell populations, engineered γδ T-cell populations, and / or mixtures thereof of the present invention may be provided in multiple doses. If not done simultaneously, the time between multiple doses may vary considerably, ranging from about 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or up to about 1 year. In some cases, the non-engineered, enriched γδ T-cell population, engineered, enriched γδ T-cell population, and / or mixtures thereof of the present invention, after administration to the subject, in the subject's body In vivoThey may proliferate. One or more non-engineered γδ T-cell populations, one or more engineered γδ T-cell populations, and / or mixtures thereof may be frozen to provide cells for multiple treatments with the same cell preparation. One or more non-engineered γδ T-cell populations, one or more engineered γδ T-cell populations, and / or mixtures thereof of the present disclosure, and pharmaceutical compositions containing them may be packaged as a kit. The kit may include instructions (e.g., written instructions) for the use of the non-engineered γδ T-cell populations, engineered γδ T-cell populations, and / or mixtures thereof, and compositions containing them.

[0142] In some cases, a method for treating a solid tumor comprises administering a therapeutically effective amount of a non-engineered γδ T-cell population, an engineered γδ T-cell population, and / or a mixture thereof to a subject, said administration treating the solid tumor. In some embodiments, a therapeutically effective amount of a non-engineered γδ T-cell population, an engineered γδ T-cell population, and / or a 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, δ hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, δ 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, a therapeutically effective amount of a non-engineered γδ T-cell population, an engineered γδ T-cell population and / or a mixture thereof is administered for at least one week. In some embodiments, a therapeutically effective amount of a non-engineered γδ T-cell population, an engineered γδ T-cell population and / or a mixture thereof is administered for at least two weeks.

[0143] The non-engineered γδ T-cell populations, engineered γδ T-cell populations, and / or mixtures thereof described herein may be administered before, during, or after the onset of a disease or pathological condition, and the timing of administration of pharmaceutical compositions containing γδ T-cell populations may vary. For example, γδ T-cell populations may be used as a prophylactic agent and may be administered continuously to subjects predisposed to a pathological condition or disease to reduce the likelihood of the disease or pathological condition developing. Initial administration may be made via any substantially any route, such as by any route described herein, using any formulation described herein. In some examples, the administration of the γδ T-cell populations of this disclosure is intravenous. One or multiple doses of a γδ T-cell population may be administered as soon as feasible after the onset of a solid tumor and for the time required for the treatment of the immune disease, e.g., 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, or about 1 month to about 3 months. In some embodiments, one or multiple doses of a γδ T-cell population may be administered years after the onset of cancer and before or after other treatments.

[0144] In some embodiments, a γδ T-cell population is administered simultaneously or sequentially with one or more methods that elevate common gamma chain cytokine(s). As used herein, "one or more methods for raising common gamma chain cytokine(s): refer to a method or combination of methods for altering the physiological state of a subject such that the level of at least one common gamma chain cytokine is raised in the subject." In some embodiments, the method raises the level of one or more common gamma chain cytokine(s) selected from the group consisting of IL-2, IL-7, and IL-15, preferably wherein the method raises the level of IL-15 in the subject. In some embodiments, the method comprises lymphocyte removal. In some embodiments, the method comprises administering one or more common gamma chain cytokine(s) to the subject. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, is administered. In some embodiments, the method comprises secreting the common gamma chain cytokine(s) from the administered, for example, γδ T cells. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, is secreted.

[0145] In some embodiments, administering one or more methods for increasing common gamma chain cytokine(s) involves lymphocyte removal prior to introducing γδ T cell(s). In some embodiments, administering one or more methods for increasing common gamma chain cytokine(s) involves administering an amount of common gamma chain cytokine(s) effective for increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδ T cell(s) simultaneously with or sequentially with the introduction of γδ T cell(s), preferably wherein the method comprises administering IL-2 or one or more mimics thereof, and more preferably wherein the method comprises administering IL-15 or one or more mimics thereof. The amount of common gamma chain cytokine(s) administered may be an amount effective for increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδ T cell(s) before and / or after the introduction of γδ T cell(s). Exemplary amounts of IL-15 include between 0.01 and 10 μg / kg / dose every 24 hours without limitation. Exemplary amounts of IL-2 include approximately 3 x 10⁶ every 8 to 48 hours without limitation. 6 Up to about 22x10 6 It includes units. For example, the dosing regimen for IL2 in RCC is 600,000 international units / kg (0.037 mg / kg) IV q8hr infused over 15 minutes for up to 14 doses.

[0146] In some embodiments, administering one or more methods for elevating common gamma chain cytokine(s) includes administering an amount of common gamma chain cytokine(s) effective for lymphocyte removal and increasing the proliferation, cytotoxic activity, persistence, or combination thereof of the introduced γδ T cell(s) before administering the γδ T cell(s), either simultaneously or sequentially with the introduction of the γδ T cell(s).

[0147] Examples

[0148] Examples 1

[0149] 1 x 10⁶ in modified culture medium pre-coated with anti-Vδ1 antibody D1-08 or D1-35 for 5 days in the presence of IL-2 (100 U / mL) in a 24-well plate (Costar) 6 Human PBMCs were activated at 1 / mL. On day 5, cell cultures were transfected with a γ-retroviral construct encoding the anti-TyrD chimeric antigen receptor (SEQ No. 8) in the presence of retronectin. On day 6, cells were returned to modified cell culture medium and further expanded with supplementation and IL-2 replacement as needed. On days 17, 18, or 19, cells were harvested, and remaining αβ T cells were depleted using the AutoMACS® kit (Miltenyi Biotec). The purity and transfection efficiency of the γδ cell population were evaluated by FACS. Simultaneously, untransfected cell cultures were expanded in the same manner without the addition of retroviral supernatant. As shown in Figure 2, untransfected expanded Vδ1 cells expressed tyrosinase and Tyr 369 -377 It induced some degree of cytotoxicity in 526 and WM266.1-Luc melanoma cell lines known to present the peptide. This cytotoxicity was enhanced by the introduction of anti-TyrD CAR. Cytotoxicity was determined by total luminescence measurements in 96-well plates by adding the luminescent substrate D-luciferin (Perkin Elmer) after 18 hours of co-incubation at the indicated E / T ratio.

[0150] Examples 2

[0151] WM266.4-Luc cells (4x10 per animal) 6) was implanted subcutaneously into NSG mice (Jackson Labs). The tumor was 100-200 mm 3 When it reaches the size, the animal is 6x10 6 The animals were treated with anti-TyrD CAR+ Vδ1 cells. Throughout the study, the animals were administered IL-2 (60,000 U / dose) three times a week in combination. The results are illustrated in Figure 3. As shown in Figure 3, animals administered with anti-TyrD CAR+ Vδ1 cells demonstrated potent control of tumor burden.

[0152] Examples 3

[0153] The Tyr CAR construct was introduced into Vδ1 T cells as described above, and the cells were expanded and tested in a cytotoxicity assay against WM266.4-Luc cells. A non-TyrD targeting CAR construct was used as a control. The results are illustrated in Figure 5 and show increased cytotoxicity conferred by the anti-TyrD CAR construct.

[0154] Examples 4

[0155] 1 x 10 in the growth medium 6Human PBMCs at 1 / mL were activated in 24-well plates (Costar) pre-coated with anti-Vδ1 antibodies D1-08 or D1-35 in the presence of IL-2 (100 U / mL) for 5 days. On day 5, cell cultures were transfected with γ-retroviral constructs encoding the anti-GPC3 chimeric antigen receptor (SEQ No. 20 (GC33 CAR) or SEQ No. 22 (GC33 CAR + sIL15 and GC33 CAR + CO sIL15)) in the presence of retronectin. GC33 CAR is encoded by the nucleic acid sequence of SEQ No. 21; GC33 CAR + sIL15 is encoded by the nucleic acid sequence of SEQ No. 23; GC33 CAR + CO sIL15 contains a codon-optimized sIL15 encoding region and is encoded by the nucleic acid sequence of SEQ No. 24. On day 6, the cells were returned to growth medium and further expanded with supply and IL-2 replacement as needed. On days 17, 18, or 19, the cells were harvested, and the remaining αβ T cells were depleted using the AutoMACS® kit (Miltenyi Biotec). The purity and transduction efficiency of the γδ cell population were evaluated by FACS (Fig. 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 the manufacturer-recommended 1:500 dilution.

[0156] In parallel, untransfected cell cultures were expanded in the same manner without the addition of retroviral supernatant. The expanded cells were tested in an in vitro cytotoxicity assay against GPC3-positive cells (HepG2, Hep3B, PLC / PRF / 5). As shown in Figure 7, untransfected expanded Vδ1 cells induce some degree of cytotoxicity against liver cancer cell lines known to express GPC3. This cytotoxicity is enhanced by the introduction of GPC3 CARs with or without the sIL15 cytokine engineered to be co-expressed. Cytotoxicity was determined by total luminescence measurements in 96-well plates by adding the luminescence substrate D-luciferin (Perkin Elmer) after 18 hours of co-incubation at the indicated E / T ratio.

[0157] * * *

[0158] The foregoing merely illustrates the principles of the invention. Those skilled in the art will understand that various arrangements embodying the principles of the invention and included within the spirit and scope thereof may be devised, even if not explicitly described or illustrated in this specification. Furthermore, all embodiments and conditional language cited in this specification are intended primarily to help the reader understand the principles of the invention and the concepts to which the inventors contributed to developing the technology, and should be interpreted as not being limited to such specifically cited embodiments and conditions. Moreover, all statements in this specification referring to specific embodiments as well as the principles and aspects of the invention are intended to encompass both their structural and functional equivalents. Additionally, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, that is, all developed elements that perform the same function regardless of structure. Accordingly, the scope of the invention is not intended to be limited to the exemplary aspects illustrated and described in this specification. Rather, the scope and spirit of the invention are embodied by the appended claims.

Claims

Claim 1 A δ1 γδ T cell comprising: a. a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) a binding domain that specifically binds to a tumor-associated antigen (TAA) expressed on the surface of a solid tumor cell, (ii) a CD8α hinge domain, (iii) a CD8α transmembrane domain, (iv) a costimulatory signaling domain selected from a 4-1BB costimulatory signaling domain and a CD27 costimulatory signaling domain, and (v) a CD3ζ signaling domain; or b. a polypeptide comprising a CAR having an amino acid sequence encoded by the nucleic acid of a, wherein the δ1 γδ T cell functionally expresses the binding domain of the nucleic acid-encoded CAR or polypeptide on the surface of the δ1 γδ T cell. Claim 2 In claim 1, the above (i)-(v) are δ1 γδ T cells in the 5' to 3' order. Claim 3 In claim 1, the TAA is a δ1 γδ T cell comprising an adjacent region of TyrD. Claim 4 A δ1 γδ T cell according to paragraph 3, wherein the adjacent region of the TyrD comprises at least 4 or at least about 4 adjacent amino acids of the TyrD and 12 or fewer or about 12 or fewer, or 7, 8 or 9 adjacent amino acids of the TyrD. Claim 5 In paragraph 4, the adjacent area of ​​the above TyrD is the TyrD 369-377 Phosphorus δ1 γδ T cell. Claim 6 In claim 1, the antigen is a protein-peptide complex, the protein is an MHC protein, the binding domain binds to the complex in an HLA restriction manner, and the binding domain that specifically binds to the TAA peptide MHC complex is HLA-A2 / TyrD 369-377 δ1 γδ T cells that specifically bind to it. Claim 7 In claim 1, the binding domain specifically binds to an epitope bound by an antibody comprising the following, or competes with an antibody comprising the following: a. CDRH1 comprising TSGMGVS (SEQ No.: 33), b. CDRH2 comprising HIYWDDDKRYNPSLKS (SEQ No.: 34), c. CDRH3 comprising KDYGSSFYAMHY (SEQ No.: 35), d. CDRL1 comprising KASQDIHNYIA (SEQ No.: 36), e. CDRL2 comprising YTSTLQP (SEQ No.: 37), and f. CDRL3 comprising LQYDNLWT (SEQ No.: 38). Claim 8 In claim 1, the binding domain specifically binds to an epitope within GPC3 expressed on the surface of a solid tumor cell, in a δ1 γδ T cell. Claim 9 In claim 8, the binding domain is a δ1 γδ T cell characterized by one or more of the following: i) comprising one or more of the following complementarity determining regions (CDRs); ii) binding to the same GPC3 epitope as an antibody comprising one or more of the following CDRs; and iii) competing for binding to the GPC3 epitope with an antibody comprising one or more of the following CDRs: a. CDRH1 comprising the sequence of DYEMH (SEQ No.: 39) (or GYTFTDYEMH (SEQ No.: 40)); b. CDRH2 comprising the sequence of ALDPKTGDTAYSQKFKG (SEQ No.: 41); c. CDRH3 comprising the sequence of FYSYTY (SEQ No.: 42); d. CDRL1 comprising the sequence of RSSQSLVHSNRNTYLH (SEQ No.: 43); e. CDRL2 containing the sequence of KVSNRFS (Sequence No.: 44), and f. CDRL3 containing the sequence of SQNTHVPPT (Sequence No.: 45). Claim 10 In claim 1, the CAR comprises a δ1 γδ T cell comprising one or more of the following: a. a CD8α hinge domain comprising SEQ ID No. 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID No. 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY), b. a CD8α transmembrane domain comprising SEQ ID No. 3 (IWAPLAGTCGVLLLSLVITLYC), and c. CD3ζ signaling domains including the following: (i) Sequence No.: 4(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or (ii) Sequence No.: 5(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). Claim 11 In claim 10, the CAR comprises a. a 4-1BB co-stimulatory signaling region containing SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), or b. a CD27 co-stimulatory signaling region containing SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or the nucleic acid is a δ1 γδ T cell encoding a 4-1BB co-stimulatory signaling region containing SEQ ID NO: 6 and a CD27 co-stimulatory signaling region containing SEQ ID NO:

7. Claim 12 In claim 1, the nucleic acid further encodes the following: a. a secreted cytokine, b. a secreted common gamma-chain interleukin, c. a secreted IL-15 which may include the sequence of SEQ ID NO: 14, or may include the sequence of SEQ ID NO: 14 operably linked to the secretion signal sequence of SEQ ID NO: 12, or may include the sequence of SEQ ID NO: 14 operably linked to the secretion signal sequence of SEQ ID NO: 26, or d. a secreted common gamma-chain interleukin which may be IL-15, and a multi-cistron linker region that is an amino terminus for the interleukin or interleukin secretion signal which may include any one of the sequences of SEQ ID NO: 15-17, 25, or 27-30 or a combination thereof, or a multi-cistron linker region that may encode an internal ribosome entry site, e.g., SEQ ID NO: 31 or 32. Claim 13 In claim 12, a δ1 γδ T cell characterized by one or more of the following: a. the secretion signal comprises the sequence of SEQ No. 12 or SEQ No. 26; b. the sIL15 domain comprises the sequence of SEQ No. 14; c. the P2A cleavage sequence comprises the sequence of SEQ No. 15 or SEQ No. 25; d. the purine cleavage sequence comprises the sequence of SEQ No. 16; and e. the CAR comprises the sequences of SEQ No. 17, SEQ No. 12, and SEQ No. 14 in the amino-to-carboxyl order. Claim 14 In claim 1, a. the binding domain is HLA-A2 / TyrD 369-377 a. A δ1 γδ T cell in which the binding domain specifically binds to GPC3, and the nucleic acid encodes SEQ No. 8 or SEQ No. 18, or b. the binding domain specifically binds to GPC3, and the nucleic acid encodes SEQ No. 20 or 22. Claim 15 In claim 14, the nucleic acid is a δ1 γδ T cell comprising the sequence of SEQ ID NO: 9, SEQ ID NO: 19, SEQ ID NO: 21, 23, or 24. Claim 16 A cell population comprising a plurality of δ1 γδ T cells according to claim 1, wherein the cell population is characterized by one or both of the following: a. said plurality is at least about 10 8 10 δ1 γδ T cells, or about 10 8 10 to 10 δ1 γδ T cells 11 a. comprising δ1 γδ T cells, and b. a composition comprising at least 60%, 80%, or about 60% or 80% to about 90% or 95% δ1 γδ T cells. Claim 17 A method for producing the δ1 γδ T cells of claim 1 or the cell population of claim 16, comprising transfecting the δ1 γδ T cell(s) with the nucleic acid defined in claim 1, and characterized by one or both of the following: a. the method comprises retroviral transduction or gamma retroviral transduction; and b. the method comprises in vitro expansion of the δ1 γδ T cell(s), wherein the in vitro Expansion is performed before or after nucleic acid transfection, or before and after transfection. Claim 18 A pharmaceutical composition for treating cancer comprising a pharmaceutically acceptable excipient and the δ1 γδ T cells of claim 1 or the cell population of claim 18. Claim 19 A drug for killing solid tumor cells, comprising a pharmaceutical composition comprising an effective amount of tumor cell killing δ1 γδ T cells of claim 1, a cell population of claim 16, or a pharmaceutically acceptable excipient and said δ1 γδ T cells or said cell population, wherein solid tumor cells are in contact with said δ1 γδ T cells, said cell population, or said pharmaceutical composition, and the drug is capable of treating cancer in a subject requiring cancer treatment. Claim 20 In claim 19, the agent is one in which a therapeutically effective amount of the δ1 γδ T cell(s) or the pharmaceutical composition is introduced into a host organism comprising solid tumor cells, and which can reduce the in vivo tumor burden in the host organism compared to a control organism, increase the average survival time of the host organism compared to a control organism, or reduce the in vivo tumor burden in the host organism and increase the average survival time of the host organism compared to a control organism, wherein the control organism is not treated with the δ1 γδ T cell(s) or the pharmaceutical composition. Claim 21 In claim 20, a therapeutically effective amount of the δ1 γδ T cell(s) or the pharmaceutical composition is introduced into a host organism comprising solid tumor cells, and one or more agonists for elevating common gamma chain cytokine(s) are administered simultaneously or sequentially, and the agent is characterized by one or both of the following: a. The administration of one or more agents for elevating common gamma-chain cytokine(s) comprises administering an amount of common gamma-chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced δ1 γδ T cell(s) simultaneously or sequentially with the introduction of δ1 γδ T cell(s), wherein IL-2 may be administered or IL-15 may be administered, and the one or more agents for elevating common gamma-chain cytokine(s) may comprise an amount of common gamma-chain cytokine(s) effective to increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced δ1 γδ T cell(s) before, after, or both before and after the introduction of δ1 γδ T cell(s), and b. One or more agonists for increasing common gamma chain cytokine(s) deplete lymphocytes before introducing δ1 γδ T cell(s), or one or more agonists for increasing common gamma chain cytokine(s) induce secretion of one or more common gamma chain cytokine(s) from introduced δ1 γδ T cell(s). Claim 22 A method for preparing a drug for treating solid tumor cell cancer in a subject requiring treatment for solid tumor cell cancer, characterized by using a pharmaceutical composition comprising an effective amount of tumor cell apoptosis δ1 γδ T cells of claim 1, the cell population of claim 16, or a pharmaceutically acceptable excipient and said δ1 γδ T cells or said cell population for solid tumor cell apoptosis. Claim 23 A pharmaceutical composition for treating cancer in a subject requiring cancer treatment, comprising a therapeutically effective amount of δ1 γδ T cells, wherein the cancer comprises solid tumor cells exhibiting cell surface expression of TyrD or GPC3, and characterized by one or both of the following: i) one or more agonists for elevating common gamma chain cytokine(s) are administered concurrently or sequentially with the administration of δ1 γδ T cells, and ii) multiple administrations of δ1 γδ T cells are performed, wherein the interval between multiple administrations is at least about 1 week, at least about 2, 3, 4, 5, 6, 7, 8, or 12 weeks, or no more than once every 6 or 12 months. 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