Compositions and methods relating to genetically modified and ungenetically modified γδ-T cells for the treatment of solid tumors

Abstract

To provide compositions and methods to improve the specificity or selectivity of cells.SOLUTION: Aspects of the present invention include compositions and methods for treatment of 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 can comprise an anti-TryD binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a CD3ζ signalling domain, a combination thereof, or all thereof. The CAR construct can comprise an anti-GPC3 binding domain, a CD8α hinge and transmembrane domain, a costimulatory domain, a CD3ζ signalling domain, a combination thereof, or all thereof.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 739826, filed 1 October 2018, the contents of which are incorporated herein by reference for all purposes.

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

[0003] Adoptive immunotherapy has been continuously revisited for over 30 years, from early approaches focused on basic lymphokine activation and / or tumor infiltration to more recent strategies involving the genetic engineering of these immune cells to express genetically modified antigen receptors such as chimeric antigen receptors (CARs). While some hints and indications of the curative potential of these approaches have been observed, much remains to be done. In particular, the success of tumor eradication with CAR-T lymphocytes depends on the persistence and effector function of CAR-T cells, and an excess of either can induce a graft-versus-host effect in the patient. Furthermore, there is the problem of a lack of available positive stimuli and the presence of an inhibitory environment, especially in solid tissues. Therefore, in this art, countless co-stimulation strategies have been tested with both T cells and NK cells, particularly αβT cells, with the aim of balancing efficacy and safety. Notably, given the current lack of understanding regarding the co-stimulation requirements of γδT cells compared to αβT cells, any practical translation of these various approaches to γδT cells is uncertain at best. For example, see Ribot et al., "Searching for “signal 2”: costimulation requirements of γδ T cells," Cell.Mol.Life Sci.(2011)68:2345-2355.

[0004] Therefore, improved strategies are still needed to improve cell specificity or selectivity, for example by reducing or avoiding the graft-versus-host (GVH) effect; to improve cell safety, for example by avoiding the suppression of effector function; to improve efficacy against solid tumor cells; and to improve cell activity and / or survival upon administration to a target. We provide methods, cells, compositions, kits, and systems that meet such needs. [Overview of the Initiative]

[0005] Aspects of the present invention include an isolated nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises a binding domain that specifically binds to a 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 comprises an isolated nucleic acid sequence that binds to the complex, HLA-restricted, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling region selected from the 4-1BB costimulatory signaling region and the CD27 costimulatory signaling region, and a CD3ζ signaling domain. Aspects of the present invention further include unmodified γδT cells as described herein, and modified γδT cells comprising nucleic acids encoding the CAR construct as described herein, wherein the γδT cells functionally express the nucleic acid encoding the CAR on the surface of the γδT cells.

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

[0007] 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 solid tumor cells and optionally the binding domain therein binds to the complex in an HLA-restricted manner; (b) a hinge domain such as a CD8α hinge domain; (c) a transmembrane domain such as a CD8α transmembrane domain; (d) a co-stimulatory signaling region or a combination of co-stimulatory signaling regions, wherein the co-stimulatory signaling region(s) is optionally selected from the 4-1BB (CD137) co-stimulatory signaling region and the CD27 co-stimulatory signaling region; and (e) a signaling domain such as a CD3ζ signaling domain. In some embodiments, the aforementioned elements (a) to (e) are encoded in 5' to 3' order on the sense strand of the isolated nucleic acid.

[0008] In some embodiments, TAA includes the region adjacent to TyrD. In some embodiments, the region adjacent to TyrD includes at least 4, or at least about 4 and 12 or less, or about 12 or less, 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 region adjacent to TyrD is TyrD 369-377 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 it.

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

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

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

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

[0013] In some embodiments, the CAR comprises a 4-1BB costimulatory signaling region comprising SEQ ID NO: 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), or a CD27 costimulatory signaling region comprising SEQ ID NO: 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP), or a nucleic acid sequence encoding a 4-1BB costimulatory signaling region comprising SEQ ID NO: 6 and a CD27 costimulatory signaling region comprising SEQ ID NO: 7.

[0014] In any one of the foregoing or in some embodiments as described herein, the isolated nucleic acid encodes a secreted cytokine, or a secreted common gamma chain interleukin, or a secreted common gamma chain interleukin such as IL-15, and preferably, this secreted common gamma chain interleukin such as IL-15 comprises an interleukin polypeptide sequence operably linked to a secretion signal sequence (e.g., the secretion signal of SEQ ID NO: 12 or 26). In some embodiments, the isolated nucleic acid encodes secreted IL-15, and preferably, wherein IL-15 comprises the sequence of SEQ ID NO: 14, more preferably, wherein IL-15 comprises 14 sequences operably linked to the secretion signal sequence of SEQ ID NO: 12, or wherein IL-15 comprises the sequence of SEQ ID NO: 14 operably linked to the secretion signal sequence of SEQ ID NO: 26. In some cases, the secreted cytokine, common gamma chain interleukin and / or IL-15 is encoded at the carboxy terminus with a binding region, hinge and transmembrane domain, signaling domain and / or costimulatory endodomain. In some cases, the secreted cytokine, common gamma chain interleukin and / or IL-15 is encoded 3' of the sense strand of a binding region, hinge and transmembrane domain, signaling domain and / or costimulatory endodomain.

[0015] In some embodiments, the nucleic acid encodes a multicistronic linker region configured to facilitate the translation of IL-15 as CAR and secreted cytokines, common gamma-chain cytokines, or separate polypeptides. In some embodiments, the multicistronic linker region encodes an autocleavage sequence and / or a cleavage polypeptide sequence. In some cases, the autocleavage sequence is a P2A, F2A, T2A, or E2A autocleavage sequence. In some cases, the cleavage sequence is a furin cleavage sequence. In some cases, the cleavage sequence (e.g., a furin cleavage sequence) is the amino terminus of the autocleavage sequence. In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. In some embodiments, the nucleic acid encodes the amino terminus of the multicistronic linker region to an interleukin or cytokine or an interleukin or cytokine secretion signal, preferably therein, this multicistronic linker region includes any one of the sequences of SEQ ID NOs: 15-17, 25, or 27-30 or a combination thereof, or encodes an internal ribosome entry site of, for example, SEQ ID NOs: 31 or 32.

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

[0017] In some embodiments, the binding domain is HLA-A2 / TyrD 369-377The nucleic acid specifically binds to and encodes sequence number 8 or sequence number 18. In some embodiments, the binding domain specifically binds to GPC3, and the nucleic acid encodes sequence number 20 or 22. In some embodiments, the nucleic acid includes the sequence of sequence number 9, sequence number 19, sequence number 21, 23, or 24.

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

[0019] In another embodiment, the present invention provides γδ, T cells comprising, for example, the polypeptide described herein or a CAR-encoding nucleic acid as described herein, wherein the cells functionally express the CAR-encoding nucleic acid on the polypeptide-binding domain or on the cell surface. In some embodiments, the cells exhibit in vitro and / or in vivo cytotoxic activity against solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAAs). In some embodiments, the solid tumor cytotoxic activity of these cells is greater than the innate level of in vitro and / or in vivo solid tumor cytotoxic activity in control cells without a CAR construct. In some embodiments, the cells are HLA class I + The solid tumor cytotoxic activity is increased against solid tumor cells. In some embodiments, the solid tumor cytotoxic activity or increased solid tumor cytotoxic activity persists for about, at least, or at least about 6 to 180 days after initial contact with solid tumor cells.

[0020] In some embodiments, cells proliferate in response to contact with solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAAs). In some embodiments, cells proliferate in response to contact with solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAAs) compared with control cells that do not functionally express CAR-encoding nucleic acids on their cell surface. In some embodiments, cells proliferate in a host organism containing solid tumor cells exhibiting tumor-associated antigens (TAAs). In some embodiments, the cell proliferation or increased cell proliferation of cells lasts for about, at least, or at least about 6 to 180 days after initial contact with solid tumor cells. In some embodiments, cells express one or more pro-inflammatory cytokines, optionally, therein, one or more pro-inflammatory cytokines, preferably in greater amounts than control cells that do not functionally express CAR-encoding nucleic acids on their cell surface, after contact with solid tumor cells.

[0021] In some embodiments, compared to the graft-versus-host response shown by αβT cells administered to an allogeneic host, when introduced to an allogeneic host, the graft-versus-host response shown by the cells is reduced, substantially reduced, substantially absent, or absent. In some embodiments, compared to the graft-versus-host response shown by αβT cells administered to an allogeneic host, when introduced to an allogeneic host, the graft-versus-host response shown by, for example, γδT cells is reduced, substantially reduced, substantially absent, or absent. In some embodiments, the T cells are γT cells. In some embodiments, the T cells are δT cells. In some embodiments, the T cells are γδT cells. In some embodiments, the T cells are δ1, δ2, δ3, or δ4T cells, preferably δ2. - δT cells, more preferably δ1δT cells. In some embodiments, the T cells are δ1, δ2, δ3 or δ4γδT cells, preferably δ2 - γδT cells, more preferably δ1γδT cells.

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

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

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

[0025] In another embodiment, the present invention provides a method for killing solid tumor cells, the method comprising contacting solid tumor cells with an amount effective for killing tumor cells of the cells or a plurality of cells or a pharmaceutical composition described herein, or cells or a plurality of cells or a pharmaceutical composition as described herein. In some cases, the cells or a plurality of cells are, for example, γδ, T cells(s).

[0026] In some embodiments, the method involves introducing a therapeutically effective amount of cells or a pharmaceutical composition, such as γδ, T cells, or more, into a host organism containing solid tumor cells. In some embodiments, the method involves introducing a therapeutically effective amount of cells or a pharmaceutical composition, such as γδ, T cells, or more, into a host organism containing solid tumor cells, and simultaneously or subsequently administering one or more methods that increase common gamma chain cytokines.

[0027] In some embodiments, one or more methods of administration for increasing common gamma chain cytokines (or more) involve administering an effective amount of common gamma chain cytokines (or more) concurrently with the introduction of cells (or more) or sequentially, thereby increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced cells (or more), preferably comprising the administration of IL-2, and more preferably comprising the administration of IL-15. In some embodiments, one or more methods of increasing common gamma chain cytokines (or more) involve administering an effective amount of common gamma chain cytokines (or more) before and / or after the introduction of cells (or more) thereby increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced cells (or more).

[0028] In some embodiments, one or more methods for increasing common gamma chain cytokines include lymphocyte depletion before introducing γδT cells. In some embodiments, one or more methods for increasing common gamma chain cytokines include the secretion of one or more common gamma chain cytokines from the introduced cells. In some embodiments, the method reduces the in vivo tumor mass of a host organism and / or increases the mean survival time of the host organism compared to a control organism, where the control organism is not treated with cells or pharmaceutical compositions. In some embodiments, the method is a method for treating cancer of a target requiring treatment.

[0029] In another embodiment, the present invention provides the use of any one of the following cells or cells as described herein (e.g., γδT cells), a plurality of such cells, or a pharmaceutical composition containing such cells in the preparation of a drug for treating solid tumor cell cancer of a subject requiring treatment, in an amount effective for killing tumor cells. In another embodiment, the present invention provides a method for treating cancer of a subject requiring treatment, 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.

[0030] In some embodiments, the method includes administering one or more methods that increase common gamma chain cytokines concurrently with or consecutively with the administration of cells. In some embodiments, the method includes performing multiple administrations of cells, where the interval between these multiple administrations is at least about one week, preferably at least about two, three, four, five, six, seven, eight, or twelve weeks, and / or once every six or twelve months or less.

[0031] In another embodiment, the present invention provides a pharmaceutical composition for use with either the method described above or the method described herein.

[0032] Reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each publication, patent, or patent application has been specifically and individually cited for incorporation by reference. [Brief explanation of the drawing]

[0033] [Figure 1]A schematic diagram of an embodiment of a chimeric antigen receptor (CAR) containing one costimulatory signaling endodomain (left) or two costimulatory signaling endodomains (right) is shown. The costimulatory signaling endodomain as used herein is also called a costimulatory endodomain. Exemplary costimulatory signaling endodomains useful in exemplary CARs include, but are not limited to, CD28, CD137 (4-1BB), CD278 (ICOS), CD27, CD134 (OX40), TLR2, and combinations thereof. [Figure 2] This document demonstrates the in vitro cytotoxicity of the genetically modified and unmodified γδT cells described herein against the 526 and WM266.1-Luc melanoma cell lines. [Figure 3] This document demonstrates the in vivo therapeutic efficacy of the γδT cells described herein in a subcutaneous WM266.4 cell NOD scid gamma (NSG) mouse model. [Figure 4] For example, this document shows the manufacturing process for producing genetically modified and unmodified γδCAR-T cells to treat solid tumors. [Figure 5] This demonstrates the cytotoxic activity of Vδ1 T cells transduced with a control CAR construct or a construct targeting a tyrosinase polypeptide. [Figure 6] This study demonstrates the transduction efficiency of Vδ1 cells with anti-glypican 3 (GPC3) such as soluble IL-15 (sIL15) (SEQ ID NO: 14) and codon-optimized (WO2007 / 037780A2) sIL15. [Figure 7] The anti-GPC3CAR construct exhibits cytotoxic activity in either transduced or untransduced Vδ1 T cells against a panel of liver cancer cell lines with varying levels of GPC3 expression. [Modes for carrying out the invention]

[0034] Definition: For the purpose of interpreting this specification, the following definitions shall apply, where appropriate, including the plural form of a singular term and vice versa. In the event of any conflict between any definition described herein and any document incorporated herein by reference, the definition described below shall prevail. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0035] As used herein, the term "about" is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from a given value, where such variation is appropriate for carrying out the disclosed method.

[0036] As used herein, the term “γδT cells (gamma delta T cells)” refers to a subset of T cells that express a different T cell receptor (TCR), namely γδTCR, on their surface, consisting of one γ chain and one δ chain. The term “γδT cells” includes all subsets of γδT cells, including but not limited to Vδ1, Vδ2, and Vδ3γδT cells, as well as naive, effector memory, central memory, and terminally differentiated γδT cells. As a further example, the term “γδT cells” includes Vδ4, Vδ5, Vδ7, and Vδ8γδT cells, as well as Vγ2, Vγ3, Vγ5, Vγ8, Vγ9, Vγ10, and Vγ11γδT cells. In some embodiments, γδT cells are Vδ1 - , Vδ2 - or Vδ1 - and Vδ2 -Compositions and methods for producing and using genetically modified and non-genetically modified γδT cells and / or their subtypes include, but are not limited to, those described in US2016 / 0175358, WO2017 / 197347, US9499788, US2018 / 0169147, US9907820, US2018 / 0125889 and US2017 / 0196910. The contents of each of these are incorporated by reference for any purpose, including these compositions and methods for producing and using genetically modified and non-genetically modified γδT cells and / or their subtypes. This application further considers T cells, or other genetically modified leukocytes or lymphocytes, expressing one γ chain or one δ chain in combination with a second polypeptide to optionally form a functional TCR. Genetically modified leukocytes or lymphocytes expressing one γ chain or one δ chain may be used in this method or may be present in the compositions described herein.

[0037] As used herein, the terms “T lymphocyte” or “T cell” refer to immune cells that express or have expressed CD3 (CD3+) and the T cell receptor (TCR+). T cells play a central role in cellular immunity. T cells that “express” CD3 and TCR are genetically engineered to eliminate the expression of CD3 and / or TCR on the cell surface.

[0038] As used herein, the terms "TCR" or "T cell receptor" refer to dimeric xenocellular surface signaling proteins that form alpha-beta or gamma-delta receptors or combinations thereof. αβTCRs recognize antigens presented by MHC molecules, while γδTCRs can recognize antigens independently of MHC presentation.

[0039] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encode antigen-presenting proteins on the cell surface. In humans, these genes are called human leukocyte antigen (HLA) genes. In this specification, the abbreviations MHC and HLA are used interchangeably.

[0040] As used herein, "activation" refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" specifically refers to T cells undergoing cell division.

[0041] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be untreated immunoglobulins of natural or recombinant origin, or they may be the immunoreactive portion of untreated immunoglobulins. Typically, antibodies are tetramers of immunoglobulin molecules. The antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0042] The term "antibody fragment" refers to a portion of an untreated antibody, specifically the antigen-determining variable region of an untreated antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0043] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation.

[0044] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformation. κ and λ light chains refer to the two main antibody light chain isotypes.

[0045] As used herein, the term "synthetic antibody" means an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage as described herein. Furthermore, this term should be interpreted as an antibody produced by synthesizing an antibody-encoding DNA molecule, the DNA molecule expressing an antibody protein or an amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is available and obtained using synthetic DNA or amino acid sequence techniques well known in the art.

[0046] As used herein, the terms “antigen” or “Ag” are defined as molecules that induce an immune response. This immune response may include either antibody production or activation of specific immune-qualified cells, or both. Those skilled in the art will understand that any macromolecule, including proteins or peptides, can serve as an antigen. Furthermore, antigens may be recombinant or derived from genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that induces an immune response will encode the term “antigen” as used herein. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by the full-length 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 one or more genes, and that these nucleotide sequences may be arranged in various combinations to induce a desired immune response. Furthermore, those skilled in the art will understand that antigens do not need to be encoded by a “gene” at all. It is readily apparent that antigens may be produced, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0047] The term "epitope" includes any protein, lipid, or carbohydrate determinant capable of specifically binding to immunoglobulins or T cell receptors. Epitope determinants typically consist of active surfaces of molecules such as amino acids, lipids, or sugar side chains, and usually possess specific three-dimensional structural properties as well as specific charge properties. Equilibrium dissociation constant (K) D ) is 10 -6 ~10 -12 It is said that when antibodies are within the M range, they specifically bind to the antigen.

[0048] As used herein, the term “chimeric antigen receptor (CAR)” may refer to, for example, an artificial T cell receptor, T-body, single-chain immune receptor, chimeric T cell receptor, or chimeric immune receptor, and may also include genetically modified receptors that impart artificial specificity to specific immune effector cells. CARs may be used to confer the specificity of a monoclonal antibody to T cells, thereby enabling the production of a large number of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, CARs direct, for example, the specificity of cells to tumor-associated antigens. In some embodiments, a CAR includes an intracellular activation domain (which enables the activation of T cells upon binding of the target portion to a target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain whose length may vary, and further includes a disease- or disease-related, for example, tumor-antigen binding region. In certain embodiments, a CAR includes a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody, fused to the CD3-zeta transmembrane domain and endodomain. Other CAR design specificities may derive from receptor ligands (such as peptides) or pattern recognition receptors such as Dectin. In certain cases, altering the spacing of antigen recognition domains can reduce activation-induced cell death. In certain cases, CARs include domains for adding co-stimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 / 12 and / or OX40, ICOS, TLR (e.g., TLR2). In some cases, molecules may be co-expressed with CARs such as co-stimulatory molecules, reporter genes for imaging (e.g., positron emission tomography), gene products that conditionally excise T cells upon prodrug addition, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. Furthermore, those skilled in the art will understand that the co-stimulatory domain does not need to be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention, without limitation, involves the use of partial nucleotide sequences of one or more genes, and that these nucleotide sequences are arranged in various combinations to induce a desired immune response.

[0049] As used herein, the term "antitumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer. Furthermore, the "antitumor effect" may be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent tumor development in the first place.

[0050] In this invention, the term "autoantigen" means any autoantigen that is mistakenly recognized as a foreign substance by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphorylated proteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins including cell surface receptors.

[0051] As used herein, the term “self-derived” is intended to refer to any material originating from the same individual that will be later reintroduced.

[0052] As used herein, the term "homogeneous" is intended to refer to any animal-derived material that is later introduced into another animal of the same species.

[0053] The term "therapeutically effective dose" refers to the amount of a composition that elicits a biological or medical response to a tissue, system, or subject, as required by researchers, veterinarians, physicians, or other clinicians. The term "therapeutically effective dose" includes an amount of a composition sufficient, when administered, to prevent the onset of a sign or symptom of a disease or illness being treated (e.g., a solid tumor) or to alleviate, to some extent, one or more of its signs or symptoms. The therapeutically effective dose varies depending on the composition, the disease and its severity, and the age, weight, and other factors of the subject being treated.

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

[0055] Administration "in combination with" one or more therapeutic drugs includes simultaneous (combined) administration and administration in any sequence.

[0056] As used herein, the term "pharmaceutically acceptable" refers to, but is not limited to, materials such as salts, carriers, or diluents that do not inhibit the biological activity or properties of a compound and are relatively non-toxic. In other words, such materials can be administered to an individual without causing undesirable biological effects or acting in a harmful manner with any of the components of a composition containing them.

[0057] "Code" refers to the inherent properties of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, and the biological properties derived therefrom, which serve as a template for synthesizing other polymers and macromolecules by biological methods having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. It is possible to say that both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, code for the protein or other product of that gene or cDNA.

[0058] "Isolated" means altered or removed from its natural state. For example, nucleic acids or peptides that naturally exist in a living animal are not "isolated," but the same nucleic acids or peptides that have been partially or completely separated from their naturally occurring coexisting substances are "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0059] 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 contain introns.

[0060] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal that is compliant with the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0061] With respect to antibodies, the term "specifically binding" as used herein means an antibody that recognizes a particular antigen but does not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen obtained from one species may also bind to that antigen obtained from one or more species. However, such interspecies reactivity does not in itself change the classification of the antibody according to its properties. In another example, an antibody that specifically binds to an antigen may bind to various alleles of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody according to its properties. In some cases, the terms "specific binding" or "specifically binding" may be used in relation to the interaction between an antibody, protein, or peptide and a second chemical species, where this interaction depends on the presence of a specific structure of the chemical species (e.g., an antigenic determinant or epitope). For example, an antibody usually means that it recognizes and binds to a specific protein structure rather than to the protein itself. If the antibody is specific to epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0062] 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 (for example, K D(The smaller the value, the stronger the binding.) Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis and surface plasmon resonance. Furthermore, multispecific antibodies that bind to a first antigen and one or more additional antigens, or bispecific antibodies that bind to two different regions of an antigen, are nevertheless considered “specifically binding” antibodies as used herein.

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

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

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

[0066] An "expression cassette" refers to a nucleic acid containing an expression regulatory sequence operably linked to a nucleic acid encoding the transcript or polypeptide to be expressed. An expression cassette contains sufficient cis-acting elements for expression, with other elements for expression being supplied by the host cell or in an in vitro expression system. Expression cassettes can be components of vectors such as cosmids, plasmids (e.g., naked or liposome-containing), or viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses). Expression cassettes may be present in host cells, such as γδT cells.

[0067] Scope: Throughout this disclosure, various aspects of the invention may be presented in range form. It should be understood that descriptions in range form are merely for convenience and conciseness and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible subranges, not just the individual numbers within that range. For example, a range description such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and individual numbers within ranges such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0068] Chimeric antigen receptor construct: Embodiments of the present invention include nucleic acids encoding CARs, as well as constructs and vectors containing such nucleic acids. In some cases, the nucleic acid is, for example, a component of a heterologous expression cassette. In some embodiments, the nucleic acid is, for example, a component of a heterologous retroviral vector. In some embodiments, the nucleic acid is, for example, a component of a heterologous αβ or γδ T cell, preferably a γδ T cell. In some embodiments, the nucleic acid is, for example, a heterologous γ + T cells and / or δ +It is a component of T cells. In some embodiments, nucleic acids are, for example, heterogeneous, α - T cells and / or β - It is a component of T cells.

[0069] Described herein are nucleic acids encoding CAR-binding domains that specifically bind to tumor-associated antigens (TAAs) expressed on the surface of solid tumor cells. Exemplary TAAs are tyrosinase (TyrD) or its peptide fragment. In some cases, TAAs are glypican 3 or its peptide fragment. In some cases, TAAs are peptides bound to HLA molecules, such as class I HLA molecules. Tyrosinase peptides that bind to class I HLA molecules (also interchangeably referred herein as HLA-restricted tyrosinase epitopes, HLA-restricted tyrosinase epitopes, and MHC-restricted tyrosinase antigens) are derived from the tyrosinase enzyme (Genebank Accession No: NP_000363.1), are typically 8–10 amino acid long, and bind to the α1–α2 groove of the heavy chain via 2 or 3 anchor residues that interact with the corresponding binding pocket in the HLA molecule.

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

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

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

[0073] As used herein, binding domains, CARs, or CAR T cells that specifically bind to TyrD and / or to epitopes within TyrD include, but are not limited to, binding domains, CARs, or CAR T cells that specifically bind to TyrD peptide fragments. Binding domains, CARs, or CAR T cells that specifically bind to TyrD peptide fragments are capable of specifically binding to a reference TyrD peptide fragment in an HLA-restricted manner. Similarly, as used herein, cells expressing TyrD on their surface include cells that express or overexpress TyrD peptide fragments on their surface, such as peptide:HLA complexes.

[0074] As used herein, binding domains, CARs, or CAR T cells that specifically bind to and / or to epitopes within GPC3 include, but are not limited to, binding domains, CARs, or CAR T cells that specifically bind to GPC3 peptide fragments. Binding domains, CARs, or CAR T cells that specifically bind to GPC3 peptide fragments are capable of specifically binding to a reference GPC3 peptide fragment in an HLA-restricted manner. Similarly, as used herein, cells expressing TyrD on their surface include cells expressing or overexpressing GPC3 peptide fragments on their surface, such as peptide:HLA complexes.

[0075] In some embodiments, the binding domain binds to the antigen so that it is expressed as a full-length functional polypeptide on the cell surface. In some embodiments, the binding domain binds to the antigen so that it is presented in the MHC:antigen complex. In some embodiments, the binding domain binds to the antigen with HLA restriction. Binding domains exhibiting specificity for the MHC:antigen complex are described, for example, in WO / 2016 / 199140 and WO / 2016 / 199141.

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

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

[0078] This disclosure also considers anti-TyrD binding domains or anti-GPC3 binding domains that compete for binding to the sequences provided herein. By known methods, it is possible to determine whether an anti-TyrD binding domain binds to the same epitope as the reference antibody or binding domain, or whether it competes for binding to that reference antibody or binding domain. For example, to determine whether a test antibody binds to the same epitope as the reference binding domain, it is possible to bind the reference binding domain to TyrD under saturated conditions. Next, it is possible to evaluate the ability of the test binding domain to bind to the TyrD molecule. If the binding domain under test is able to bind to TyrD following saturated binding with the reference binding domain, it can be concluded that the test binding domain binds to a different epitope than the reference binding domain. On the other hand, if the test binding domain is not able to bind to TyrD following saturated binding with the reference binding domain, it is possible that the test binding domain can bind to the same epitope as the epitope bound by the reference binding domain.

[0079] If a binding domain competes with a reference binding domain for binding, the binding methodology described above is carried out in two directions. In the first direction, the reference binding domain is bound to TyrD under saturated conditions, and then the binding of the test binding domain to the TyrD molecule is evaluated. In the second direction, the test binding domain is bound to the TyrD molecule under saturated 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 is able to bind to the TyrD molecule, it is concluded that the test binding domain and the reference binding domain compete for binding to TyrD. As will be understood by those skilled in the art, a binding domain competing for binding to a reference binding domain is not necessarily able 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 an overlapping or adjacent epitope. The methods described above for determining competition with and epitope binding of an anti-TyrD binding domain can similarly be applied to an anti-TyrD binding domain.

[0080] If two binding domains each competitively inhibit (block) the binding of the other to the antigen, they bind to the same or overlapping epitopes. That is, a 1x, 5x, 10x, 20x, or 100x excess of one binding domain inhibits the binding of the other by at least 50%, e.g., 75%, 90%, or even 99%, as measured in competitive binding assays (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one binding domain also reduce or eliminate binding of the other, the two binding domains have the same epitope. If several amino acid mutations that reduce or eliminate binding of one binding domain also reduce or eliminate binding of the other, the two binding domains have overlapping epitopes.

[0081] Furthermore, routine experiments (e.g., peptide mutation and binding analysis) can be performed to determine whether the observed lack of binding of the test binding domain is actually due to binding to the same epitope as the reference binding domain, or whether steric hindrance (or other phenomena) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative binding assay available in the art.

[0082] This disclosure provides antibodies and CARs having “substantially identical” or “substantially similar” to sequences provided herein in the CDR or framework region. The terms “substantially identical” or “substantially identical,” when referring to a nucleic acid or a fragment thereof, mean that the nucleotide sequence identity is present in, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the nucleotide bases when optimally aligned with other nucleic acids (or complementary strands of other nucleic acids). A nucleic acid molecule that is substantially identical to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0083] When applied to polypeptides, the term “substantial similarity” or “substantial similar” means that 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 when optimally aligned using a program such as GAP or BESTFIT with default gap weights. In some embodiments, the positions of non-identical residues differ due to conserved amino acid substitutions. A “conservative amino acid substitution” is when an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by a conservative substitution, the percentage or degree of identity may be adjusted up to restore the conservative nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.Instead, a conservative permutation is any change having a positive value in the PAM250 log-likelihood matrix, disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A “moderately conservative” permutation is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0084] Polypeptide sequence identity and / or similarity are typically measured using sequence analysis software. Protein analysis software matches similar sequences, used to measure similarity assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can use default parameters, to measure sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from various species of organisms, or between wild-type proteins and their mutant proteins. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA with default or recommended parameters, i.e., the GCG Version 6.1 program. FASTA (FASTA2 and FASTA3, etc.) provides optimal overlap region alignment and sequence identity percentage between the query sequence and the search sequence (Pearson (2000) above). Sequences can be compared using the Smith-Waterman homology search algorithm with an affine gap search using a gap start penalty of 12, a gap expansion penalty of 2, and a BLOSUM matrix of 62. When comparing a database containing a large number of sequences obtained from various organisms with the sequences disclosed herein, other preferred algorithms are the computer programs BLAST, particularly BLASTP or TBLASTN, using default parameters. See, for example, Altschul et al. (1990) J.Mol.Biol.215:403-410 and (1997) Nucleic Acids Res.25:3389-3402. Each of these contents is incorporated by reference.

[0085] This specification provides anti-TyrD CARs or anti-GPC3 CARs comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more substitutions (such as conservative substitutions). For example, this disclosure includes anti-TyrD CARs having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, an anti-TyrD CAR may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (such as conserved amino acid substitutions) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0086] Similarly, this disclosure includes anti-GPC3CARs having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein. For example, anti-GPC3CAR may include 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions (such as conserved amino acid substitutions) compared to any of the HCVR, LCVR, and / or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3) amino acid sequences disclosed herein.

[0087] The exemplary binding domains described herein typically include a heavy chain region followed by a light chain region (VH-VL) in the order from the amino terminus to the carboxyl terminus. Where a specific order of the VH and VL regions in a binding domain is explicitly or implicitly described, this disclosure may also be understood to describe alternative embodiments in which the order of the VH and VL regions is reversed, for example, in a CAR containing an scFV or scFv binding domain. Thus, a description of the VH-VL order also describes an alternative VL-VH order in a CAR containing an scFV or scFv binding domain, for example. Furthermore, a description of the VL-VH order also describes an alternative VH-VL order in a CAR containing an scFV or scFv binding domain, for example.

[0088] Generally, the nucleic acids encoding CARs described herein include an extracellular linker region encoding a peptide linker that ligates the binding domain to the transmembrane domain. Exemplary linker regions include, but are not limited to, the linker region encoding the CD8α hinge domain, e.g., SEQ ID NO: 1 (PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY) or SEQ ID NO: 2 (TTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY). Typically, the region encoding the peptide linker (e.g., the CD8α hinge domain) is the 3' region of the binding domain and the 5' region of the transmembrane domain.

[0089] The nucleic acids encoding CARs described herein include a transmembrane domain. The transmembrane domain can ligate an extracellular antigen-binding domain, e.g., a hinge, to one or more intracellular signaling components. For example, the transmembrane domain can ligate an antigen-binding domain, e.g., a hinge, to a CD3ζ signaling domain and optionally one or two costimulatory end domains. Exemplary transmembrane domains include, but are not limited to, the CD8α transmembrane domain, e.g., SEQ ID NO: 3 (IWAPLAGTCGVLLLSLVITLYC). Typically, the region encoding the transmembrane domain (e.g., the CD8α transmembrane domain) is the 3' region encoding the peptide linker (e.g., the CD8α hinge domain) and the 5' region encoding one or more cytoplasmic domains.

[0090] 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 a signaling domain that provides activation signals for γδT cell proliferation, cytotoxic activity, and / or pro-inflammatory cytokine expression (e.g., TNF-α or IFNγ). A model cytoplasmic domain is the CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain is or includes SEQ ID NO: 4 (RVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the CD3ζ signaling domain is or includes SEQ ID NO: 5 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR). In some embodiments, the cytoplasmic region includes multiple (e.g., 2, 3, 4, 5, or 6) signaling domains, such as multiple (e.g., 2, 3, 4, 5, or 6) CD3ζ signaling domains, each independently selected from, for example, SEQ ID NOs: 4 and 5. In some embodiments, the cytoplasmic region includes multiple (e.g., 2, 3, 4, 5, or 6) non-CD3ζ signaling domains and CD3ζ signaling domains. In some embodiments, the cytoplasmic region includes non-CD3ζ signaling domains and multiple (e.g., 2, 3, 4, 5, or 6) CD3ζ signaling domains. This includes, but is not limited to, substitute or additional signaling domains.

[0091] The cytoplasmic region may contain one or more costimulatory end domains. The region encoding one or more costimulatory end domains may be the 5' or 3' of the region encoding the signaling domain. In some embodiments, the region encoding one or more costimulatory end domains may be the 5' of the region encoding the signaling domain. In some embodiments, the region encoding one or more costimulatory end domains is the 5' of the signaling domain, and the additional region encoding one or more costimulatory end domains is the 3' of the signaling domain. Exemplary costimulatory end domains include, but are not limited to, the CD28, CD137(4-1BB), CD278(ICOS), CD27, CD134(OX40), and TLR2 costimulatory end domains and combinations thereof.

[0092] In some embodiments, additional signaling modes may be included to enhance the proliferation, persistence, and / or cytotoxic activity of the 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 an isolated nucleic acid. The common gamma chain cytokine coding region can be ligated to the 5' portion of the CAR construct via a T2A linker coding region, thereby cleaving the common gamma chain cytokine from the CAR polypeptide and secreting it by the cell.

[0093] In some embodiments, the construct encodes at least one 4-1BB costimulatory end-domain and optionally a second costimulatory end-domain selected from the 4-1BB, ICOS, CD28, and CD27 costimulatory end-domains. In some embodiments, the construct encodes two 4-1BB costimulatory end-domains combined with at least two 4-1BB costimulatory end-domains or one, two, three, or four or more costimulatory end-domains selected from 4-1BB, ICOS, CD28, and CD27. In some embodiments, the 4-1BB costimulatory end-domain includes sequence number 6 (KRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL).

[0094] In some embodiments, the construct encodes one CD27 costimulatory end-domain and optionally a second costimulatory end-domain selected from 4-1BB, ICOS, CD28, and CD27 costimulatory end-domains. In some embodiments, the construct encodes the CD27 costimulatory end-domain and the 4-1BB costimulatory end-domain. In some embodiments, the construct encodes two CD27 costimulatory end-domains. In some embodiments, the CD27 costimulatory end-domain includes sequence number 7 (QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQED YRKPEPACSP).

[0095] In some embodiments, the construct encodes a secretory signal, e.g., SEQ ID 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 (such as CAR-T cells). In some embodiments, the secretory signal is the secretory signal SEQ ID NO: 26 (MRISKPHLRSISIQKYLCLLNSHFLTEAGIHVFILGCFSAGLPKTEA). In some embodiments, the construct encodes a secretory signal, such as SEQ ID NO: 12, operably linked to promote the secretion of a common gamma chain cytokine such as IL-15 or its active fragment, e.g., SEQ ID NO: 14 (NWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS). Other IL-15 sequences, including codon-optimized nucleic acid sequences encoding sIL15, are disclosed in WO2007 / 037780. Exemplary common gamma-chain cytokines include IL-2 and IL-15. In some embodiments, common gamma-chain cytokines are selected from IL-2, IL-7, and IL-15.

[0096] In some embodiments, the construct encodes one or more multicistronic linker regions between, for example, a signaling domain and / or a costimulatory end domain and a secretory signal operable to promote cytokine secretion. The multicistronic linker region is a region of polypeptide or RNA sequence that facilitates the generation of multiple distinct polypeptides from a single transcript. In some embodiments, the multicistronic linker region encodes a cleavage sequence. Preferred cleavage sequences include self-cleaving sequences such as P2A, F2A, E2A, or T2A cleavage sequences and / or sequences cleaved by endogenous proteases such as furin.

[0097] In some embodiments, the cleavage sequence is a P2A cleavage sequence. In some embodiments, the cleavage sequence is a furin cleavage sequence. In some embodiments, the cleavage sequence is a P2A and furin cleavage sequence. In some embodiments, the cleavage sequence is the P2A cleavage sequence of sequence number 15 (SGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is the furin cleavage sequence of sequence number 16 (RAKR). In some embodiments, the cleavage sequence is the P2A + furin cleavage sequence of sequence number 17 (RAKRSGSGATNFSLLKQAGDVEENPGP). In some embodiments, the cleavage sequence is the P2A cleavage sequence of sequence number 25 (GSGATNFSLLKQAGDVEENPGP).

[0098] In some embodiments, the cleavage sequence is the P2A cleavage sequence of SEQ ID NO: 27 (ATNFSLLKQAGDVEENPGP) or includes the P2A cleavage sequence thereof. In some embodiments, the cleavage sequence is the F2A cleavage sequence of SEQ ID NO: 28 (VKQTLNNFDLLKLAGDVESNPGP) or includes the F2A cleavage sequence thereof. In some embodiments, the cleavage sequence is the E2A cleavage sequence of SEQ ID NO: 29 (QCTNYALLKLAGDVESNPGP) or includes the E2A cleavage sequence thereof. In some embodiments, the cleavage sequence is the T2A cleavage sequence of SEQ ID NO: 30 (EGRSLLTCGDVEENPGP) or includes the T2A cleavage sequence thereof. In some embodiments, the multiple self-cleavage sequences may encode the carboxyl terminus of a signaling and / or costimulatory domain, as well as the amino terminus of an encoded secretory cytokine (e.g., a common gamma chain cytokine such as IL-15), where the multiple self-cleavage sequences are preferably independently selected from the group consisting of P2A cleavage sequences, T2A cleavage sequences, E2A cleavage sequences, and F2A cleavage sequences. In one embodiment, one or more self-cleaving sequences and one or more sequences cleaved by an endogenous protease are encoded in the construct described herein. In a particular embodiment, the endogenous protease recognition site is encoded at the amino terminus of the self-cleaving sequence.

[0099] In some embodiments, the multicistronic linker region encodes an internal ribosome entry site. A typical internal ribosome entry site is encoded by Sequence ID No. 31().

[0100] Another exemplary internal ribosome entry site is encoded by Sequence ID 32 (AGCAGGTTTCCCCAACTGACACAAAACGTGCAACTTGAAACTCCGCCTGGTCTTTCCAGGTCTAGAGGGGTAACACTTTGTACTGCGTTTGGCTCCACGCTCGATCCACTGGCGAGTGTTAGTAACAGCACTGTTGCTTCGTAGCGGAGCATGACGGCCGTGGGAACTCCTCCTTGGTAACAAGGACCCACGGGGCCAAAAGCCACGCCCACACGGGCCCGTCATGTGTGCAACCCCAGCACGGCGACTTTACTGCGAAACCCACTTTAAAGTGACATTGAAACTGGTACCCACACACTGGTGACAGGCTAAGGATGCCCTTCAGGTACCCCGAGGTAACACGCGACACTCGGGATCTGAGAAGGGGACTGGGGCTTCTATAAAAGCGCTCGGTTTAAAAAGCTTCTATGCCTGAATAGGTGACCGGAGGTCGGCACCTTTCCTTTGCAATTACTGACCAC).

[0101] Further preferred internal ribosome entry sites include, but are not limited to, those described in Nucleic Acids Res. 2010 Jan;38 (Database issue):D131-6.doi:10.1093 / nar / gkp981 Epub 2009 Nov 16, those described in iresite.org, those described in WO2018 / 215787, the sequences described in GenBank accession No. KP019382.1, and the IRES elements described in GenBank accession No. LT727339.1. For all purposes, the entirety of these contents, in particular, of the internal ribosome entry sites and their uses described herein are incorporated herein by reference.

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

[0103] In some embodiments, the isolated nucleic acid is sequence number 8 (MSVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCKASQDIHNYIAWYQQKPGKAPKLLIHYTSTLQPGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDNLWTFGQGTKVEIKRGGGGSGGGGSGGGGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMGVSWIRQPPGKALEWLAHIYWDDDKRYNPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARK DYGSSFYAMHYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR hD11 anti-TyrD binding domain (anti-TyrD 369-377 It encodes the hD11-CD8-BBz polypeptide, which includes the CD8α hinge and transmembrane region, the 4-1BB costimulatory endodomain, and the CD3ζ signaling domain.

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

[0105] In some embodiments, the isolated nucleic acid contains a codon-optimized sequence encoding the CD8α hinge region. Exemplary codon-optimized CD8α hinge region nucleic acid sequences include, but are not limited to, SEQ ID NO: 10 (ACCACCACCCCTGCACCAAGGCCCCCGACTCCCGCGCCCACCATCGCGTCACA GCCTCTTAGCCTGCGACCGGAAGCATGCAGACCAGCTGCCGGGGGGGCCGTGCATACGAGAGGTTTGGACTTCGCCTGCGAT). In some embodiments, the CD8α hinge region is encoded by the following SEQ ID NO: 11 (ACCACGACGCCAGCG CCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT).

[0106] In some embodiments, the isolated nucleic acid is sequence number 18(*), anti-TyrD hD11(anti-TyrD 369-377 It encodes the hD11-CD8-BBz-sIL15 polypeptide, which includes a binding domain, CD8α hinge and transmembrane region, 4-1BB costimulatory endodomain, CD3ζ signaling domain, furin-P2A cleavage sequence, and a secretory signal operably linked to the IL-15 domain.

[0107]

[0108] In some embodiments, the isolated nucleic acid is sequence number 20 (MSVPTQVLGLLLLWLTDARCQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKTTTPAPRP It encodes a polypeptide comprising a GC33 anti-GPC3 binding domain, a CD8α hinge and transmembrane region, a 4-1BB costimulator endodomain and a CD3ζ signaling domain (*), a GC33 anti-GPC3 binding domain, a CD8α hinge and transmembrane region, a 4-1BB costimulator endodomain and a CD3ζ signaling domain.

[0109]

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

[0111]

[0112]

[0113] In some embodiments, the isolated nucleic acid is a linear nucleic acid. In some embodiments, the isolated nucleic acid is a vector such as a plasmid vector, adenovirus vector, adeno-associated virus vector, viral vector, retroviral vector (such as a gamma-retroviral vector), or lentiviral vector. In some embodiments, the isolated nucleic acid, or a contiguous portion of the isolated nucleic acid including, for example, a binding domain, a transmembrane domain, and one or more signaling and / or costimulatory endodomains, is integrated into the genome of a host cell, such as a host γδT cell. In an exemplary embodiment, the isolated nucleic acid is a retroviral vector.

[0114] γδT cells: Aspects of the present invention include γδT cells that functionally express the isolated nucleic acids described herein, thereby expressing CARs on the surface of the γδT cells.

[0115] Aspects of the present invention may optionally or additionally include γδT cells having in vitro or in vivo cytotoxic activity against solid tumor cells exhibiting cell surface expression of tumor-associated antigens (TAAs). In some cases, the cytotoxic activity is innate. In some cases, the cytotoxicity is at least partially, significantly (>25%), or completely due 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, in these γδT cells, the solid tumor cytotoxic activity of the γδT cells is greater than the innate level of in vitro and / or in vivo solid tumor cytotoxic activity in control γδT cells. In some cases, the control γδT cells do not contain a CAR construct. In some cases, the control γδT cells contain a CAR construct lacking the binding domain, hinge region, transmembrane domain, signaling domain, and / or costimulatory endodomain described herein.

[0116] In some cases, cytotoxicity is caused by TyrD or TyrD 369-377 Cytotoxicity is at least partially, significantly (>25%), or completely due to the presence of CAR constructs with binding domains that specifically bind to epitopes within TyrD, such as [examples omitted]. In some cases, cytotoxicity is restricted to HLA (e.g., restricted to class I HLA) and also to TyrD, or TyrD 369-377 Cytotoxicity is present, at least partially, significantly (>25%), or completely, due to the presence of CAR constructs with binding domains that specifically bind to epitopes within TyrD, such as HLA-A2 / TyrD. In some cases, cytotoxicity is due to HLA-A2 / TyrD 369-377 This is due to the presence of a CAR construct having a binding domain that specifically binds to TyrD, at least partially, significantly (>25%), or completely. In some cases, γδT cells functionally express CARs encoded by the isolated nucleic acids described herein that specifically bind to TyrD or its peptide fragment.

[0117] In some embodiments, the γδ T cells described herein may exhibit HLA-restricted (e.g., HLA class I-restricted) cytotoxicity. In other embodiments, little (>50%), substantially all (>90%), or all cytotoxic activity is not HLA-restricted (e.g., HLA class I-restricted). HLA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against HLA (e.g., HLA class I) (null) tumor cell lines with in vitro cytotoxicity against HLA+ (e.g., HLA class I+) tumor cell lines. In some embodiments, HLA-restricted cytotoxic activity is provided at least partially, significantly (>25%), or completely by the use of a T cell receptor-like binding domain. A T cell receptor, such as a binding domain, is a binding domain that specifically recognizes an antigen when presented on the surface of a cell in a complex with an MHC molecule. T cell receptor-like binding domains are further described, for example, in WO2016 / 199141.

[0118] The γδT cells described herein may exhibit potent and / or sustained solid tumor cytotoxic activity. In some cases, solid tumor cytotoxic activity may persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with solid tumor cells. In some cases, the solid tumor cytotoxic activity of the γδT cells or their progeny described herein may persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with solid tumor cells or administration of the γδT cells described herein. This sustained solid tumor cytotoxic activity may be exhibited in vitro, in vivo, or both in vitro and in vivo.

[0119] Aspects of the present invention may optionally or additionally include γδT cells that proliferate in response to contact with cells exhibiting cell surface expression or overexpression of tumor-associated antigens (TAAs). 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 innate activity. In some cases, proliferation is at least partially, significantly (>about 20% or >about 25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to TAA expressed on the cell surface. In some cases, γδT cells exhibit higher levels of proliferation in vitro and / or in vivo compared to control γδT cells. In some cases, control γδT cells do not contain a CAR construct. In some cases, control γδT cells contain a CAR construct lacking the binding domain, hinge region, transmembrane domain, signaling domain, and / or costimulatory endodomain described herein.

[0120] In some cases, proliferation is at least partially, significantly (>20% or >25%), or completely due to the presence of a CAR construct having a binding domain that specifically binds to TyrD or an epitope within TyrD. In some cases, γδT cells that proliferate in response to contact with cells exhibiting cell surface expression of TyrD functionally express TyrD-specific CARs encoded by the isolated nucleic acids described herein.

[0121] The γδT cells described herein are capable of vigorous and / or sustained proliferation in host organisms containing cells exhibiting cell surface expression or overexpression of tumor-associated antigens (TAAs). In some cases, proliferation may persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with cells exhibiting cell surface expression or overexpression of tumor-associated antigens (TAAs) or from the date of administration of γδT cells to the host organism. In some cases, proliferation of the γδT cells or their offspring described herein in host organisms containing cells exhibiting cell surface expression or overexpression of tumor-associated antigens (TAAs) may persist for at least about 6 to 120 days, or at least about 6 to 180 days, from initial contact with cells or from the date of initial administration of γδT cells to the host organism. In some cases, proliferation in the host organism is at least partially, significantly (>about 20% or >about 25%), or completely due to the presence of TyrD or a CAR construct having a binding domain that specifically binds to an epitope within TyrD. In some cases, γδT cells that proliferate in a host organism, including cells exhibiting cell surface expression of TyrD, functionally express TyrD-specific CARs encoded by the isolated nucleic acids described herein.

[0122] In some embodiments, the γδT cells described herein express or sustainably express pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with cells expressing or overexpressing TyrD or its peptide fragments on the cell surface.

[0123] In some embodiments, γδT cells or pharmaceutical compositions containing γδT cells exhibit substantially no graft-versus-host response when introduced into an allogeneic host. In some embodiments, γδT cells or pharmaceutical compositions containing γδT cells exhibit a clinically acceptable level of graft-versus-host response when introduced into an allogeneic host. In some embodiments, a clinically acceptable level is the amount of graft-versus-host response that does not require discontinuation of γδT cell therapy to achieve therapeutically effective treatment. In some embodiments, a clinically acceptable level of graft-versus-host response (GvHD) is an acute response less severe than Grade C on the applicable IBMTR rating scale. The severity of the acute graft-versus-host response is determined by assessing the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of lesions in individual organs are combined to create an overall grade with prognostic significance. Grade I(A) GvHD is considered a mild illness, Grade II(B) GvHD is moderate, Grade III(C) is severe, and Grade IV(D) is life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997;97:855): ●Grade A - No liver or gastrointestinal lesions, only Stage 1 skin lesions (<25% of body area covered by maculopapular rash) ● Grade B - Stage 2 skin lesions, Stage 1 to 2 intestinal or hepatic lesions ●Grade C - Stage 3 lesions in any organ system (generalized erythroderma, bilirubin 6.1-15.0 mg / dL, diarrhea 1500-2000 mL / day) ●Grade D - Stage 4 lesions in any organ system (systemic erythroderma with vesicle formation, bilirubin > 15 mg / dL, diarrhea > 2000 mL / day, or pain or ileus). See also Tables 1 and 2 in Schoemans et al., Bone Marrow Transplantation, volume 53, pages 1401-1415 (2018). This also discloses criteria for evaluating and grading acute GvHD.

[0124] In some embodiments, γδT cells, or pharmaceutical compositions containing γδT cells, when introduced into allogeneic hosts, exhibit a reduced or substantial reduction in graft-versus-host response compared to the graft-versus-host response demonstrated by control αβT cells or a control pharmaceutical composition containing control αβT cells administered to allogeneic hosts. In some cases, the control αβT cells are allogeneic, unmodified control αβT cells. In some cases, the control αβT cells are CAR-free or do not contain the same CAR as the reference γδT cells.

[0125] The γδT cells described herein may be δ1, δ2, δ3, or δ4γδT cells, or a combination thereof. In some cases, the γδT cells are almost (>50%), substantially (>90%), essentially all, or completely δ2. - These are γδT cells. In some cases, γδT cells are almost (>50%), substantially (>90%), essentially all, or completely δ1γδT cells.

[0126] γδT cells are available from allogeneic or autologous donors. γδT cells can be partially or completely purified or unpurified and amplified ex vivo. Methods and compositions for ex vivo proliferation include, but are not limited to, those described in WO2017 / 197347. This proliferation may be carried out before, after, or immediately before or after the introduction of a CAR construct into the γδT cells(s).

[0127] The γδT cells described herein may be stored, for example, by cryopreservation for use in adoptive cell transplantation.

[0128] Methods to inhibit or kill tumor cells One or more non-genetically modified (NGM) γδT cell populations, genetically modified (GM) γδT cell populations, and / or mixtures thereof, which have cytotoxic activity against solid tumor cells, may be administered to a subject in any order or simultaneously. When administered simultaneously, the multiple NGM, genetically modified (GM) γδT cell populations, and / or mixtures thereof of the present invention may be provided in a single unified form, such as intravenous injection, or in multiple forms, such as multiple intravenous infusions, subcutaneous injections, or tablets. The NGM, genetically modified (GM) γδT 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 NGM, genetically modified (GM) γδT cell populations, and / or mixtures thereof of the present invention may be given in multiple doses. When not administered simultaneously, the timing between multiple doses may vary from about one week, one month, two months, three months, four months, five months, six months, or up to about one year. In some cases, the non-genetically modified enriched γδT cell populations, genetically modified enriched γδT cell populations, and / or mixtures thereof of the present invention may proliferate in vivo within the subject's body after administration to the subject. One or more non-genetically modified γδT cell populations, one or more genetically modified γδT cell populations, and / or mixtures thereof can be frozen to provide cells for multiple treatments with the same cell preparation. One or more non-genetically modified γδT cell populations, one or more genetically modified γδT cell populations, and / or mixtures thereof of the present disclosure, and pharmaceutical compositions containing them, may be packaged as kits. The kits may include instructions (such as written instructions) for the use of the non-genetically modified γδT cell populations, genetically modified γδT cell populations, and / or mixtures thereof, and compositions containing them.

[0129] In some cases, a method for treating solid tumors involves administering a therapeutically effective amount of a non-genetically modified γδT cell population, a genetically modified γδT cell population, and / or mixtures thereof to a target, thereby treating the solid tumor. In some embodiments, a therapeutically effective amount of a non-genetically modified γδT cell population, a genetically modified γδT cell population, and / or mixtures thereof is administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, a therapeutically effective amount of a non-genetically modified γδT cell population, a genetically modified γδT cell population, and / or mixtures thereof is administered for at least 1 week. In some embodiments, a therapeutically effective amount of a population of non-genetically modified γδT cells, a population of genetically modified γδT cells, and / or mixtures thereof is administered for at least two weeks.

[0130] The unmodified γδT cell populations, modified γδT cell populations, and / or mixtures thereof described herein can be administered before, during, or after the onset of a disease or symptom, and the timing of administration of the pharmaceutical composition containing the γδT cell population can vary. For example, the γδT cell population can be used as a prophylactic agent and can be administered sequentially to subjects showing symptoms or a tendency toward the disease to reduce the likelihood of the disease or symptom developing. The initial dose may be administered via any practical route, such as by any route described herein using any formulation described herein. In some embodiments, the administration of the γδT cell population of this disclosure is intravenous. One or more doses of the γδT cell population may be administered as soon as feasible after the onset of a solid tumor and for the duration necessary to treat the immunological disease, for example, 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, a single or multiple dose of the γδ T cell population may be administered over several years, both after the onset of cancer and before and after other treatments.

[0131] In some embodiments, a population of γδT cells is administered simultaneously or sequentially in one or more ways that elevate common gamma-chain cytokines. As used herein, “one or more ways that elevate common gamma-chain cytokines” refers to a method or combination of methods that alters the physiological state of a subject so that the level of at least one common gamma-chain cytokine is elevated in the subject. In some embodiments, the method elevates the level of one or more common gamma-chain cytokines selected from the group consisting of IL-2, IL-7, and IL-15, preferably therein, the method elevates the level of IL-15 in the subject. In some embodiments, the method includes lymphocyte depletion. In some embodiments, the method includes administering one or more common gamma-chain cytokines to the subject. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, is administered. In some embodiments, the method includes the secretion of common gamma-chain cytokines from the administered γδT cells, etc. In some cases, IL-2, IL-7, and / or IL-15, preferably IL-15, are secreted.

[0132] In some embodiments, one or more administration methods to increase common gamma chain cytokines include lymphocyte depletion before the introduction of γδT cells. In some embodiments, one or more administration methods to increase common gamma chain cytokines include increasing the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδT cells by administering an effective amount of common gamma chain cytokines simultaneously with or sequentially with the introduction of γδT cells, preferably comprising the administration of IL-2 or one or more mimetic IL-2, and more preferably comprising the administration of IL-15 or one or more mimetic IL-15. Doses of common gamma chain cytokines can increase the proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced γδT cells before and / or after the introduction of γδT cells. The exemplary dose of IL-15 includes, but is not limited to, 0.01 to 10 μg / kg / dose every 24 hours. The exemplary dose of IL-2 includes approximately 3 × 10⁶ doses every 8 to 48 hours. 6 and approximately 22 x 10 6 This includes, but is not limited to, units. For example, the dosing regimen for IL2 in RCC is 600,000 international units / kg (0.037 mg / kg) intravenously, administered over 15 minutes in up to 14 doses, every 8 hours.

[0133] In some embodiments, a method of administering one or more common gamma chain cytokines to increase lymphocyte depletion includes lymphocyte depletion before administration of γδT cells, before simultaneous administration of γδT cell introduction, or before sequential administration of common gamma chain cytokines effective in increasing the proliferation, cytotoxic activity, persistence, or combination thereof of the introduced γδT cells. [Examples]

[0134] Example 1 1x106 Human PBMCs at a concentration of / mL were pre-coated with anti-Vδ1 antibody D1-08 or D1-35 in 24-well plates (Costar) in the presence of IL-2 (100U / mL) for 5 days, and activated in modified culture medium. On day 5, the cell cultures were transduced with a γ-retrovirus construct encoding the anti-TyrD chimeric antigen receptor (SEQ ID NO: 8) in the presence of retronectin. On day 6, the cells were returned to the modified culture medium and further grown by feeding and IL-2 supplementation as needed. On days 17, 18, or 19, the cells were harvested and any remaining αβT cells were depleted using the AutoMACS® kit (Miltenyi Biotec). The purity of the γδ cell population and the transduction efficiency were evaluated by FACS. In parallel, untransduced cell cultures were grown using the same method without the addition of retrovirus supernatant. As shown in Figure 2, the non-transduced proliferating Vδ1 cells express tyrosinase, and Tyr 369-377 The peptide induced some degree of cytotoxicity in 526 and WM266.1-Luc melanoma cell lines, which are known to display peptides. This cytotoxicity was enhanced by the introduction of anti-TyrD CAR. Cytotoxicity was measured by total luminescence assay in a 96-well plate after co-culturing at the indicated E / T ratio for 18 hours and then adding the luminescent substrate D-luciferin (Perkin Elmer).

[0135] Example 2 WM266.4-Luc cells (4 x 10 per animal) 6 The tumor was subcutaneously transplanted into NSG mice (Jackson Labs). The tumor was 100-200 mm. 3 Once it reaches the size, place the animal in 6x10 6 The animals were treated with anti-TyrD CAR+Vδ1 cells. They were also administered IL-2 (60,000 U / dose) three times a week until the end of the study. The results are shown in Figure 3. As shown in Figure 3, the animals treated with anti-TyrD CAR+Vδ1 cells showed strong control of tumor burden.

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

[0137] Example 4 In 24-well plates (Costar) pre-coated with anti-Vδ1 antibody D1-08 or D1-35 in the presence of IL-2 (100 U / mL) for 5 days, 1 x 10⁶ of growth medium was used. 6 Human PBMCs were activated at a concentration of 1 / mL. On day 5, the cell cultures were transduced with a γ-retroviral construct encoding an anti-GPC3 chimeric antigen receptor (SEQ ID NO: 20 (GC33CAR) or SEQ ID NO: 22 (GC33CAR+sIL15 and GC33CAR+CO sIL15)) in the presence of retronectin. GC33CAR is encoded by the nucleic acid sequence of SEQ ID NO: 21. GC33CAR+sIL15 is encoded by the nucleic acid sequence of SEQ ID NO: 23, and GC33CAR+CO sIL15 contains a codon-optimized sIL15 coding region and is encoded by the nucleic acid sequence of SEQ ID NO: 24. On day 6, the cells were returned to the growth medium and further grown by feeding and IL-2 supplementation as needed. On days 17, 18, or 19, the cells were harvested and any remaining αβT cells were depleted using the AutoMACS® kit (Miltenyi Biotec). The purity and transduction efficiency of the γδ cell population were evaluated by FACS (Figure 6). In short, CAR-T cells were stained by incubating them with 1 μg / mL of soluble recombinant biotinylated GPC3 (R&D Systems). Binding detection was performed using streptavidin-PE at the manufacturer's recommended dilution ratio of 1:500.

[0138] In parallel, cell cultures without transduction were grown using the same method without adding retroviral supernatant. The grown cells were tested using in vitro cytotoxicity assays for GPC3-positive cells (HepG2, Hep3B, PLC / PRF / 5). As shown in Figure 7, the proliferated Vδ1 cells without transduction induced some degree of cytotoxicity against liver cancer, which is known to express GPC3. This cytotoxicity was enhanced by the introduction of GPC3CAR, regardless of the presence or absence of the sIL15 cytokine, which was genetically engineered to be expressed in tandem. Cytotoxicity was measured by total luminescence measurement in a 96-well plate after co-culturing for 18 hours at the indicated E / T ratio and then adding the luminescent substrate D-luciferin (Perkin Elmer).

[0139] The foregoing merely illustrates the principles of the present invention. Those skilled in the art will understand that it is possible to devise various configurations that embody the principles of the present invention and fall within its spirit and scope, although these are not explicitly described or illustrated herein. Furthermore, all embodiments and conditional language listed herein are primarily intended to assist the reader's understanding of the principles of the present invention and the concepts to which the inventors contribute to advance the art, and should be interpreted as not being limited to such specifically listed embodiments and conditions. Moreover, all descriptions herein listing the principles, embodiments, and specific embodiments thereof are intended to encompass both structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and those to be developed in the future, i.e., any elements developed to perform the same function regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. SEQUENCE LISTING <110> ADICET BIO, INC. <120> COMPOSITIONS AND METHODS REGARDING ENGINEERED AND NON-ENGINEERED GAMMA-DELTA-T CELLS FOR TREATMENT OF SOLID TUMORS <130> ADC-0006-PCT <140> PCT / US2019 / 054144 <141> 2019-10-01 <150> 62 / 739,826 <151> 2018-10-01 <160> 45 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 1 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 1 5 10 15 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 20 25 30 Asp Phe Ala Cys Asp Ile Tyr 35 <210> 2 <211> 47 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 2 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 35 40 45 <210> 3 <211> 22 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 3 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 1 5 10 15 Val Ile Thr Leu Tyr Cys 20 <210> 4 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 4 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 5 <211> 112 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 5 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 6 <211> 42 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 6 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 7 <211> 48 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 7 Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro 1 5 10 15 Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr 20 25 30 Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro 35 40 45 <210> 8 <211> 487 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Ile His Asn Tyr Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp 100 105 110 Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gln Ile Thr 130 135 140 Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln Thr Leu Thr 145 150 155 160 Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 180 185 190 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 195 200 205 Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 210 215 220 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 225 230 235 240 Lys Asp Tyr Gly Ser Ser Phe Tyr Ala Met His Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485 <210> 9 <211> 1464 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 9 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120 atcacttgca aggcgagtca ggacattcac aactatatag cttggtatca gcagaaacca 180 gggaaagccc ctaagctcct gatccactat acatccactt tgcaaccagg ggtcccatca 240 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 300 gaagatattg caacatatta ctgtctacag tatgataatc tctggacgtt cggtcaaggc 360 accaaggtgg aaatcaaacg gggtggaggt ggatctggag gaggaggatc cggtggagga 420 ggtcagatca ccttgaagga gtctggtcct acgctggtga aacccacaca gaccctcacg 480 ctgacctgca ccttctctgg gttctcactc agcactagtg gaatgggtgt gtcctggatc 540 cgtcagcccc caggaaaggc cctggagtgg cttgcacaca tttattggga tgatgataag 600 cgctacaacc catctctgaa gagcaggctc accatcacca aggacacctc caaaaaccag 660 gtggtcctta caatgaccaa catggaccct gtggacacag ccacatatta ctgtgcacga 720 aaggactacg gtagtagctt ctatgctatg cactactggg gtcaaggaac cctagtcacc 780 gtgtcgagta ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840 cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900 agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020 tatatattca aaaaccatt tatgagacca gtacaacta ctcaagagga agatggctgt 1080 1140 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaag aggagtacga tgttttggac aagacgtg gccgggaccc tgagatgggg 1260 ggaaagccgc agaaaggaa gaaccctcag gaagcctgt acaatgaact gcagaaagat 1320 aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440 atgcaggccc tgccccctcg ctaa 1464 <210> 10 <211> 135 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 10 accaccaccc ctgcaccaag gcccccgact cccgcgccca ccatcgcgtc acagcctctt 60 agcctgcgac cggaagcatg cagaccagct gccggggggg ccgtgcatac gagaggtttg 120 gacttcgcct gcgat 135 <210> 11 <211> 135 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 11 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 12 <211> 21 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 12 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 13 <400> 13 000 <210> 14 <211> 114 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 14 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 1 5 10 15 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 20 25 30 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 35 40 45 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 50 55 60 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 65 70 75 80 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 85 90 95 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 100 105 110 Thr Ser <210> 15 <211> 23 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 15 Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 1 5 10 15 Val Glu Glu Asn Pro Gly Pro 20 <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 16 Arg Ala Lys Arg 1 <210> 17 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 17 Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 18 <211> 649 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 18 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp 35 40 45 Ile His Asn Tyr Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 50 55 60 Lys Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser 85 90 95 Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp 100 105 110 Asn Leu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gln Ile Thr 130 135 140 Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln Thr Leu Thr 145 150 155 160 Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 180 185 190 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 195 200 205 Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr 210 215 220 Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg 225 230 235 240 Lys Asp Tyr Gly Ser Ser Phe Tyr Ala Met His Tyr Trp Gly Gln Gly 245 250 255 Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 325 330 335 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 340 345 350 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 355 360 365 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 370 375 380 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 385 390 395 400 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 405 410 415 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg Arg Ala Lys Arg Ser Gly Ser Gly Ala 485 490 495 Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro 500 505 510 Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu 515 520 525 Leu Leu His Ala Ala Arg Pro Asn Trp Val Asn Val Ile Ser Asp Leu 530 535 540 Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu 545 550 555 560 Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys 565 570 575 Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala 580 585 590 Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser 595 600 605 Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu 610 615 620 Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His 625 630 635 640 Ile Val Gln Met Phe Ile Asn Thr Ser 645 <210> 19 <211> 1950 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 19 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 120 atcacttgca aggcgagtca ggacattcac aactatatag cttggtatca gcagaaacca 180 gggaaagccc ctaagctcct gatccactat acatccactt tgcaaccagg ggtcccatca 240 aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag cctgcagcct 300 gaagatattg caacatatta ctgtctacag tatgataatc tctggacgtt cggtcaaggc 360 accaaggtgg aaatcaaacg gggtggaggt ggatctggag gaggaggatc cggtggagga 420 ggtcagatca ccttgaagga gtctggtcct acgctggtga aacccacaca gaccctcacg 480 ctgacctgca ccttctctgg gttctcactc agcactagtg gaatgggtgt gtcctggatc 540 cgtcagcccc caggaaaggc cctggagtgg cttgcacaca tttattggga tgatgataag 600 cgctacaacc catctctgaa gagcaggctc accatcacca aggacacctc caaaaaccag 660 gtggtcctta caatgaccaa catggaccct gtggacacag ccacatatta ctgtgcacga 720 aaggactacg gtagtagctt ctatgctatg cactactggg gtcaaggaac cctagtcacc 780 gtgcgagta ccaccacccc tgcaccaagg cccccgactc ccgcgcccac catcgcgtca 840 cagcctctta gcctgcgacc ggaagcatgc agaccagctg ccgggggc cgtgcatacg 900 agaggtttgg acttcgcctg cgatatctac atctgggcgc ccttggccgg gacttgtggg 960 gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020 tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080 agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140 agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200 ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260 ggaaagccgc agagaagga gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1440. ccgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ccgcgcgag cgatcaggca gcggggcgac aaatttcagc cttctgaaac aagcaggcga cgtggaaga aaccccggtc caatggcctt accagtgacc gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccgaactg ggtgaatgta 1620 ataagtgatt tgaaaaaaat tgaagatctt attcaatcta tgcatattga tgctacttta father gtgatgttca ccccagttgc aaagtaacag caatgaagtg ctttctcttg gagttacaag ttatttcact tgagtccgga gatgcaagta ttcatgatac agtagaaaat ctgatcatcc tagcaaacaa cagtttgtct tctaatggga atgtaacaga atctggatgc aaagaatgtg aggaactgga ggaaaaaaat attaaagaat ttttgcagag ttttgtacat 1920 attgtccaaa tgttcatcaa cacttcttga 1950 <210> 20 <211> 485 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 20 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr 65 70 75 80 Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr 85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu 145 150 155 160 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170 175 Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln 180 185 190 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 195 200 205 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 225 230 235 240 Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255 Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 325 330 335 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 340 345 350 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 355 360 365 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 385 390 395 400 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415 Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 21 <211> 1458 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 21 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 caagtgcagc tggtccagag cggcgccgag gtgaaaaagc ctggcgccag cgtgaaggtg 120 tcctgcaagg cctctggcta caccttcacc gactacgaga tgcactgggt gcggcaggcc 180 cctggacagg gcctggaatg gatgggcgct ctggacccca agaccggcga caccgcttat 240 agccagaagt tcaagggcag agtgaccctg acagctgata agagcacaag caccgcctac 300 atggaactga gcagcctgac cagcgaggac accgccgtgt actactgcac cagattctac 360 agctacacct actggggcca ggggaccctg gtgacagtgt ctagcggtgg aggtggatct 420 ggaggaggag gatccggtgg aggaggtgat gtggtgatga cccagagccc tctgagcctg 480 cctgtgaccc ctggagagcc tgccagcatc agctgcagaa gcagccaatc tctggtgcac 540 agcaaccgga acacatacct gcactggtac ctgcagaaac ctggccagag cccccagctg 600 ctgatctaca aggtgtccaa cagattcagc ggcgtgcctg atagattcag cggatctggc 660 agcggcaccg acttcaccct gaagatctct agagtggaag ccgaggacgt gggcgtgtac 720 tactgcagcc agaacaccca cgtgcccccc accttcggcc agggcacaaa gctggaaatc 780 aagaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccacgc gtcgcagcccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggggg 900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccggacttg tggggtcctt 960 ctcctgtcac tggttatcac cctttactgc aaacggggca gaagaaact cctgtatatata 1020 ttcaacaac cattttagag accagtacaa actactcaag aggagatgg ctgtagctgc 1080 cgatttccag agagaga aggagatgt gaacgagag tgagttcag caggagcgca 1140 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctca tctaggacga 1200 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat gggggaaag 1260 ccgcagagaa ggaagaaccc tcaggaggc ctgtacaatg aactgcagaa agatagatg 1320 gcggaggcct acagtgagat tgggatgaa ggcgagcgcc ggaggggca ggggcacgat 1380 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440 gccctgcccc ctcgctaa 1458 <210> 22 <211> 642 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 22 Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr 1 5 10 15 Asp Ala Arg Cys Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 20 25 30 Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr 35 40 45 Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr 65 70 75 80 Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr 85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu 145 150 155 160 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 165 170 175 Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln 180 185 190 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 195 200 205 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 210 215 220 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 225 230 235 240 Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr 245 250 255 Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys 325 330 335 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 340 345 350 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 355 360 365 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 370 375 380 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 385 390 395 400 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 405 410 415 Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 485 490 495 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Pro Val 500 505 510 Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro 515 520 525 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 530 535 540 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 545 550 555 560 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 565 570 575 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu 580 585 590 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val 595 600 605 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 610 615 620 Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 625 630 635 640 Thr Ser <210> 23 <211> 1929 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 23 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 caagtgcagc tggtccagag cggcgccgag gtgaaaaagc ctggcgccag cgtgaaggtg 120 tcctgcaagg cctctggcta caccttcacc gactacgaga tgcactgggt gcggcaggcc 180 cctggacagg gcctggaatg gatgggcgct ctggacccca agaccggcga caccgcttat 240 agccagaagt tcaagggcag agtgaccctg acagctgata agagcacaag caccgcctac 300 atggaactga gcagcctgac cagcgaggac accgccgtgt actactgcac cagattctac 360 agctacacct actggggcca ggggaccctg gtgacagtgt ctagcggtgg aggtggatct 420 ggaggaggag gatccggtgg aggaggtgat gtggtgatga cccagagccc tctgagcctg 480 cctgtgaccc ctggagagcc tgccagcatc agctgcagaa gcagccaatc tctggtgcac 540 agcaaccgga acacatacct gcactggtac ctgcagaaac ctggccagag cccccagctg 600 ctgatctaca aggtgtccaa cagattcagc ggcgtgcctg atagattcag cggatctggc 660 agcggcaccg acttcaccct gaagatctct aggtggaag ccgaggacgt gggcgtgtac 720 tactgcagcc agacaccca cgtgcccccc accttcggcc agggcacaaa gctggaaatc aagaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcgggggg gcgcagtgca cacgagggggg900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960 1020. ctcctgtcac tggttatcac cctttactgc aaacggggca gaaagaact cctgtatata ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca gacgccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaag 1260 ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380 ggcctttacc agggtctcag tacagccacc aggacacct acgacgccct tcacatgcag 1440 gccctgcccc ctcgcggtag cggggctacg aacttctccc ttcttaaaca agcgggagac 1500 gtgaagaaa atcccggacc tatggcctta ccagtgaccg ccttgctcct gccgctggcc 1560 ttgctgctcc acgccgccag gccgaactgg gtgaatgtaa taagtgattt gaaaaaaaatt 1620 gaagatctta ttcaatctat gcatattgat gctactttat atacggaaag tgatgttcac 1680 cccagttgca aagtaacagc aatgaagtgc tttctcttgg agttacaagt tatttcactt 1740 gagtccggag atgcaagtat tcatgataca gtagaaaatc tgatcatcct agcaaacaac 1800 agtttgtctt ctaatgggaa tgtaacagaa tctggatgca aagaatgtga ggaactggag 1860 gaaaaaaata ttaaagaatt tttgcagagt tttgtacata ttgtccaaat gttcatcaac 1920 acttcttga 1929 <210> 24 <211> 1929 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 24 atgtccgtgc ctacccaggt gctgggcctg ctgctgctgt ggctgaccga cgccagatgc 60 caagtgcagc tggtccagag cggcgccgag gtgaaaaagc ctggcgccag cgtgaaggtg 120 tcctgcaagg cctctggcta caccttcacc gactacgaga tgcactgggt gcggcaggcc 180 cctggacagg gcctggaatg gatgggcgct ctggacccca agaccggcga caccgcttat 240 agccagaagt tcaagggcag agtgaccctg acagctgata agagcacaag caccgcctac 300 atggaactga gcagcctgac cagcgaggac accgccgtgt actactgcac cagattctac 360 agctacacct actggggcca ggggaccctg gtgacagtgt ctagcggtgg aggtggatct 420 ggaggaggag gatccggtgg aggaggtgat gtggtgatga cccagagccc tctgagcctg 480 cctgtgaccc ctggagagcc tgccagcatc agctgcagaa gcagccaatc tctggtgcac 540 agcaaccgga acacatacct gcactggtac ctgcagaaac ctggccagag cccccagctg 600 ctgatctaca aggtgtccaa cagattcagc ggcgtgcctg atagattcag cggatctggc 660 agcggcaccg acttcaccct gaagatctct agagtggaag ccgaggacgt gggcgtgtac 720 tactgcagcc agaacaccca cgtgcccccc accttcggcc agggcacaaa gctggaaatc 780 aagaccacga cgccagcgcc gcgaccacca acaccggcgc ccaccatcgc gtcgcagccc 840 ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgaggggg 900 ctggacttcg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960 ctcctgtcac tggttatcac cctttactgc aaacggggca gaagaaact cctgtatatata 1020 ttcaacaac cattttagag accagtacaa actactcaag aggagatgg ctgtagctgc 1080 cgatttccag agagaga aggagatgt gaacgagag tgagttcag caggagcgca 1140 gacgcccccg cgtaccagca gggccagaac cagctctata acgagctca tctaggacga 1200 agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat gggggaaag 1260 ccgcagagaa ggaagaaccc tcaggaggc ctgtacaatg aactgcagaa agatagatg 1320 gcggaggcct acagtgagat tgggatgaa ggcgagcgcc ggaggggca ggggcacgat 1380 ggcctttacc agggtctcag tacaccacc aaggacaccct acgaccccct tcacaccag 1440 gccctgcccc ctcgcggtag cggggctacg aacttctccc ttcttaaca agcgggac 1500 gtggaagaaa atcccggacc tatggcctta ccagtgaccg ccttgctcct gccgctggcc 1560 ttgctgctcc acgccgccag gccgaactgg gtgaatgtga tcagcgatct gaagaagatc 1620 gaggatctga tccagtccat gcacatcgat gccaccctgt ataccgagag cgatgtgcac 1680 cccagctgca aggtgaccgc catgaagtgc tttctgctgg agctgcaggt gatctccctg 1740 gagtccggag atgccagcat ccacgatacc gtggagaatc tgatcatcct ggccaacaac 1800 agcctgtcct ccaatggcaa tgtgaccgag tcgggatgca aggagtgcga ggagctggag 1860 gagaagaata tcaaggagtt tctgcagagc tttgtacata ttgtccaaat gttcatcaac 1920 acttcttga 1929 <210> 25 <211> 22 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 25 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro Gly Pro 20 <210> 26 <211> 48 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 26 Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 <210> 27 <211> 19 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 27 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 1 5 10 15 Pro Gly Pro <210> 28 <211> 23 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 28 Val Lys Gln Thr Leu Asn Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 1 5 10 15 Val Glu Ser Asn Pro Gly Pro 20 <210> 29 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 29 Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser 1 5 10 15 Asn Pro Gly Pro 20 <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 30 Glu Gly Arg Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly 1 5 10 15 Pro <210> 31 <211> 553 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 31 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat 60 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct 120 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg 180 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aaacgtct gtagcgaccc 240 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg 300 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg 360 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga 420 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt 480 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttccttttgaa 540 aaacacgatg ata 553 <210> 32 <211> 461 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 32 agcaggtttc cccaactgac acaaaacgtg caacttgaaa ctccgcctgg tctttccagg 60 tctagagggg taacactttg tactgcgttt ggctccacgc tcgatccact ggcgagtgtt 120 agtaacagca ctgttgcttc gtagcggagc atgacggccg tgggaactcc tccttggtaa 180 caaggaccca cggggccaaa agccacgccc acacgggccc gtcatgtgtg caaccccagc 240 acggcgactt tactgcgaaa cccactttaa agtgacattg aaactggtac ccacacactg 300 gtgacaggct aaggatgccc ttcaggtacc ccgaggtaac acgcgacact cgggatctga 360 gaaggggact ggggcttcta taaaagcgct cggtttaaaa agcttctatg cctgaatagg 420 tgaccggagg tcggcacctt tcctttgcaa ttactgacca c 461 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 33 Thr Ser Gly Met Gly Val Ser 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 34 His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 35 Lys Asp Tyr Gly Ser Ser Phe Tyr Ala Met His Tyr 1 5 10 <210> 36 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Lys Ala Ser Gln Asp Ile His Asn Tyr Ile Ala 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Tyr Thr Ser Thr Leu Gln Pro 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Leu Gln Tyr Asp Asn Leu Trp Thr 1 5 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Asp Tyr Glu Met His 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Gly Tyr Thr Phe Thr Asp Tyr Glu Met His 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Phe Tyr Ser Tyr Thr Tyr 1 5 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Arg Ser Ser Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr Leu His 1 5 10 15 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 45 Ser Gln Asn Thr His Val Pro Pro Thr 1 5

Claims

1. A δ1γδ T cell, a) A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR is in the order 5' to 3': (i) A binding domain that specifically binds to GPC3 on the surface of solid tumor cells; (ii) CD8α hinge domain containing the sequence of Sequence ID No. 2 (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY); (iii) CD8α transmembrane domain containing the sequence of Sequence ID No. 3 (IWAPLAGTCCGVLLLLSLVITLYC); (iv) The 4-1BB costimulatory signaling region containing the sequence of Sequence ID No. 6 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL); and (v) comprising a CD3ζ signaling domain containing the sequence of Sequence ID No. 4 (RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRRDPEMGGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRRGKGHDGLYQGLSTAKDTYDALHMQALPPR); or b) comprising a polypeptide containing a CAR containing the amino acid sequence encoded by the nucleic acid of a), The δ1γδT cells functionally express the CAR encoded by the polypeptide binding domain or the nucleic acid on the surface of the δ1γδT cells. δ1γδT cells.

2. The binding domain is the following CDR: CDRH1 containing the sequence of sequence number 39 (DYEMH); CDRH2 containing the sequence of sequence number 41 (ALDPKTGDTAYSQKFKG); CDRH3 containing the sequence of sequence number 42 (FYSYTY); CDRL1 containing the sequence of sequence number 43 (RSSQSLVHSNRNTYLH); CDRL2 containing the sequence of sequence number 44 (KVSNRFS); and CDRL3 containing sequence number 45 (SQNTHVPPT) The δ1γδT cell according to claim 1, comprising:

3. The δ1γδT cell according to claim 1, wherein the nucleic acid encodes a polypeptide containing the sequence of SEQ ID NO:

20.

4. The δ1γδT cell according to claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO:

21.

5. The δ1γδT cell according to claim 1 or 2, wherein the nucleic acid further encodes secreted IL-15.

6. The secreted IL-15 is operably linked to a secretion signal sequence containing the sequence of Sequence ID 12 (MALPVTALLLPLAALLHAARP) and Sequence ID 14 (NWVNVISDLKKKIEDLIQSMHIDATLYT ESSDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIIILANNSLSSSNGNVTESGCKECEEELEEKNIKEFLQSFVHIVQMFI The δ1γδT cell according to claim 5, comprising the sequence of NTS.

7. The δ1γδT cell according to claim 5 or 6, wherein the nucleic acid further encodes a P2A cleavage sequence at the amino terminus of secreted IL-15 that includes the sequence of SEQ ID NO: 25 (GSGATNFSLLKQAGDVEENPGP).

8. The δ1γδT cell according to claim 1, wherein the nucleic acid encodes a polypeptide containing the sequence of SEQ ID NO:

22.

9. The δ1γδT cell according to claim 1, wherein the nucleic acid comprises the sequence of SEQ ID NO:

24.

10. A δ1γδ T cell, a) A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR is in the order 5' to 3': (i) A binding domain that specifically binds to GPC3 on the surface of solid tumor cells, the following CDR: CDRH1 containing the sequence of sequence number 39; CDRH2 containing the sequence of sequence number 41; CDRH3 containing the sequence of sequence number 42; CDRL1 containing the sequence of sequence number 43; CDRL2 containing the sequence of sequence number 44; and CDRL3 containing the sequence of sequence number 45 A combined domain including; (ii) CD8α hinge domain containing the sequence of Sequence ID No. 2; (iii) CD8α transmembrane domain containing the sequence of Sequence ID No. 3; (iv) The 4-1BB costimulatory signaling region containing the sequence of Sequence ID No. 6; (v) CD3ζ signaling domain containing the sequence of Sequence ID No. 4; (vi) P2A cleavage sequence containing the sequence of sequence number 25; (vii) Secretory signaling sequences including the sequence of sequence number 12; and (viiii) containing secreted IL-15 containing the sequence of sequence number 14; or b) comprising a polypeptide containing a CAR containing the amino acid sequence encoded by the nucleic acid of a), The δ1γδT cells functionally express the CAR encoded by the polypeptide binding domain or the nucleic acid on the surface of the δ1γδT cells. δ1γδT cells.

11. A population of cells comprising a plurality of δ1γδT cells according to any one of claims 1 to 10.

12. The cell population according to claim 11, comprising at least 10⁸ δ¹γδT cells.

13. The cell population according to claim 12, comprising 108 δ1γδT cells to 1011 δ1γδT cells.

14. The cell population according to any one of claims 11 to 13, comprising at least 60% δ1γδT cells.

15. The cell population according to claim 14, comprising 60% to 95% δ1γδT cells.

16. A pharmaceutical composition comprising a therapeutically effective amount of δ1γδT cells according to any one of claims 1 to 10 or a cell population according to any one of claims 11 to 15, and a pharmaceutically acceptable excipient.

17. δ1γδT cells according to any one of claims 1 to 10, a cell population according to any one of claims 11 to 15, or a pharmaceutical composition according to claim 16, for use in the treatment of a target cancer.

18. A δ1γδT cell, a population of δ1γδT cells, or a pharmaceutical composition for use according to claim 17, wherein the cancer is liver cancer.

19. A δ1γδT cell, a δ1γδT cell population, or a pharmaceutical composition for use according to claim 17 or 18, wherein the treatment comprises multiple administrations of δ1γδT cells, a δ1γδT cell population, or a pharmaceutical composition, and the interval between such multiple administrations is at least one week.

20. A δ1γδT cell, a population of δ1γδT cells, or a pharmaceutical composition for use according to claim 19, wherein multiple administrations of δ1γδT cells are performed no more than once every six months.

21. The use of a δ1γδT cell, a δ1γδT cell population, or a pharmaceutical composition according to claim 17, further comprising introducing one or more agents that increase common gamma chain cytokines to the target, either simultaneously or sequentially, during the treatment to the target of the δ1γδT cell, δ1γδT cell population, or pharmaceutical composition.

22. The use of a δ1γδT cell, a δ1γδT cell population, or a pharmaceutical composition according to claim 17, further comprising introducing one or more agents that increase common gamma chain cytokines before and / or after introduction to the subject of the δ1γδT cell, δ1γδT cell population, or pharmaceutical composition.

23. A δ1γδT cell, a population of δ1γδT cells, or a pharmaceutical composition for use according to claim 21 or 22, wherein one or more agents that increase common gamma chain cytokines deplete lymphocytes before the introduction of δ1γδT cells.

24. A δ1γδT cell, a population of δ1γδT cells, or a pharmaceutical composition for use according to claim 21 or 22, wherein one or more agents that increase common gamma chain cytokines induce the secretion of one or more common gamma chain cytokines from the introduced δ1γδT cells.

25. A δ1γδT cell, a population of δ1γδT cells, or a pharmaceutical composition for use according to any one of claims 21 to 24, wherein the common gamma chain cytokine(s) are IL-2 and / or IL-15.

26. A method for producing δ1γδT cells according to any one of claims 1 to 10, or a cell population according to any one of claims 11 to 15, the method comprising transfecting δ1γδT cells (or more) by gamma retroviral transduction using the nucleic acid described in any one of claims 1 to 10.

Images