Chimeric antigen receptor

Abstract

A chimeric antigen receptor (CAR) containing the NGK2D ectodomain is provided. Compositions, cells, and cell therapies containing the same are provided. Treatment methods are further provided. [Solution] A chimeric antigen receptor (CAR) is provided, comprising an NKG2D ectodomain, a transmembrane domain, a 4-1BB costimulatory domain, and a signaling domain including a CD3-zeta signaling domain.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 192,296, filed on May 24, 2021, entitled "Chimeric Antigen Receptor", which is hereby incorporated by reference in its entirety.

[0002] (Field of the Invention) The present disclosure relates to the field of cell therapy, and more specifically, to NKG2D chimeric antigen receptors.

Background Art

[0003] Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic transformation into abnormal cancer cells. By doing so, cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's natural immune system to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells such as T and B lymphocytes from normally targeting cancer cells.

[0004] Current T-cell therapies are based on human T cells enriched or modified to target and kill a patient's cancer cells. To increase the ability of T cells to target and kill specific cancer cells, methods have been developed to genetically engineer T cells to express constructs that direct them to specific target cancer cells. Chimeric antigen receptors (CARs) and genetically engineered T cell receptors (TCRs) containing binding domains that can interact with specific tumor antigens enable T cells to target and kill cancer cells that express specific tumor antigens. However, some tumor types, particularly solid tumors, are resistant to T-cell immunotherapy, and the development of next-generation enhancement strategies to target tumor-specific resistance mechanisms to T-cell immunotherapy is needed. NKG2D ligands are expressed on most types of tumors, and these demonstrate relative selectivity of ligand expression on tumor cells compared to healthy cells, representing targets for enhancing traditional T-cell therapies. [Overview of the project]

[0005] A chimeric antigen receptor (CAR) comprising an NKG2D ectodomain, a transmembrane domain, a 4-1BB costimulatory domain, and a signaling domain comprising a CD3-zeta signaling domain are disclosed. In embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence described in SEQ ID NO: 33. In embodiments, the CD3-zeta signaling domain comprises the amino acid sequence described in SEQ ID NO: 27. In embodiments, the CAR further comprises a CD8-alpha-hinge domain. In embodiments, the CD8-alpha-hinge domain comprises the amino acid sequence described in SEQ ID NO: 15. In embodiments, the NKG2D ectodomain comprises the amino acid sequence described in SEQ ID NO: 3. In embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In embodiments, the CD28 transmembrane domain comprises the amino acid sequence described in SEQ ID NO: 21. In embodiments, the signaling domain further comprises a CD3-epsilon signaling domain. In embodiments, the CD3-epsilon signaling domain comprises the amino acid sequence described in SEQ ID NO: 31.

[0006] Disclosed nucleic acids encoding CARs and vectors containing them are disclosed. In embodiments, the recombinant vector or nucleic acid further comprises nucleic acid encoding a modified T cell receptor (TCR) specific to a tumor antigen. In embodiments, the recombinant vector or nucleic acid is specific to a tumor antigen. The embodiment further comprises nucleic acids encoding a specific second CAR. In this embodiment, the tumor antigens are HPV-16 E6, HPV-16 E7, alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, and CS1. Includes EGFR, the EGFR family including ErbB2 (HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-llRα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEM, or VEGFRII.

[0007] Host cells transformed with nucleic acids or recombinant vectors are disclosed. In embodiments, host cells are transformed with the disclosed nucleic acids or recombinant vectors and nucleic acids or recombinant vectors encoding tumor antigen-specific manipulated T cell receptors (TCRs) or tumor antigen-specific second CARs. In this embodiment, the tumor antigens include HPV-16E6, HPV-16E7, alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2(HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, and EpC The composition includes AM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-llRα, IL-13Rα2, lambda, Lewis-Y, kappa, mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEM, or VEGFRII. In embodiments, the host cell includes induced pluripotent stem cells (iPSCs), T cells, or NK cells. A pharmaceutical composition comprising disclosed T cells and / or NK cells is disclosed. A method for treating a disease in a patient requiring treatment of the disease is disclosed, comprising administering disclosed T cells and / or NK cells or a pharmaceutical composition to the patient. In embodiments, the host cells are allogeneic to the patient. [Modes for carrying out the invention]

[0008] term

[0009] To facilitate understanding of this disclosure, certain terms are first defined below. Further definitions of these terms and other terms are provided throughout this specification.

[0010] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise.

[0011] Where used herein, unless otherwise specified or evident from the context, the term “or” is understood to be inclusive and encompasses both “or” and “and.”

[0012] As used herein, the terms "and / or" mean 2, with or without the other. Each of the specified features or components should be interpreted as a specific disclosure. Accordingly, the term "and / or" as used in phrases such as "A and / or B" in this specification is intended to include A and B, A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0013] Where used herein, the term “for example” is used merely as an example and is not intended to be limiting, and should not be construed as referring only to items explicitly listed herein.

[0014] Terms such as "greater than or equal to," "at least," and "greater than," for example, "at least one," are not limiting, but they mean at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. ,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88 ,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,1 It is understood to include 31, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction in between is also included.

[0015] Conversely, the term "less than or equal to" includes each value smaller than the listed value. For example, "100 or fewer nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 5 This includes 3, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any fewer number or fraction in between is also included.

[0016] Terms such as "multiple," "at least two," "two or more," and "at least the second" are not limiting, but include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 ,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,1 04, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 13 It is understood to include 7, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction in between is also included.

[0017] Throughout this specification, the word "comprising" or "comprises" Alternatively, variations such as "comprising" are understood to mean including the element, integer, or process, or group of elements, integers, or processes, that is described, but not to mean excluding any other element, integer, or process, or group of elements, integers, or processes. Whenever an aspect is described herein using the phrase "comprising," it is understood that other similar aspects described using the terms "consisting of" and / or "consisting essentially of" are also presented.

[0018] Unless specifically stated or evident from the context, the term “about” refers to a value or composition that falls within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and this depends to some extent on how that value or composition is measured or determined, i.e., on the limits of the measurement system. For example, “about” or “essentially from” may mean within a range of 1 or more than 1 standard deviation by the practice of the art. “About” or “essentially from” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term may mean a value of up to one order of magnitude or up to five times the stated value. Where specific values ​​or compositions are presented in this disclosure, unless otherwise specified, the meaning of “approximately” or “essentially consisting of” should be assumed to be within an acceptable margin of error for those specific values ​​or compositions.

[0019] As described herein, any range of concentration, percentage, ratio, or integer should be understood to include any integer values ​​within the listed ranges, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise specified.

[0020] The units, prefixes, and symbols used herein are presented in the format accepted by the Systeme International de Unites (SI). Numerical ranges include the number that defines the range.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this disclosure relates. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2 nded., (2001), CRC Press, “The Dictionary of Cell & Molecular Biology”, 5 th ed., (2013), Academic Press, and “The Oxford Dictionary Of Biochemistry And Molecular Biology”, Cammack et al. eds., 2 nd The ed. (2006), Oxford University Press, provides a general dictionary to those skilled in the art for many of the terms used in this disclosure.

[0022] "Administering" refers to the physical delivery of a drug, such as the modified T cells disclosed herein, to a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by injection or infusion. The term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intratracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal, or mucosal administration routes, such as intranasal, intravaginal, rectal, sublingual, or topical. The administration may also be carried out, for example, once, multiple times, and / or over a longer period of time.

[0023] The terms “activated” and “activated” refer to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In one embodiment, activation may also relate to induced cytokine production and detectable effector function. The term “activated T cell” refers, among other things, to a proliferating T cell. Signals generated solely via the TCR may be insufficient for complete T cell activation, and one or more secondary or co-stimulatory signals may also be required. Thus, T cell activation includes a primary stimulatory signal mediated by the TCR / CD3 complex and one or more secondary co-stimulatory signals. Co-stimulation may be demonstrated by proliferation and / or cytokine production by T cells that have received a primary activating signal, such as stimulation mediated by the TCR / CD3 complex.

[0024] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are incorporated into more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of Ab can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, human antibodies are tetramers of about 150 kD, consisting of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other in a structure commonly referred to as a "Y-shape". The heavy and light chains are linked or connected to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, resulting in the dimers being linked to each other to form a tetramer. Naturally produced antibodies, for example, C H Glycosylation occurs on two domains.

[0025] The term "human antibody" is intended to include antibodies having variable and constant domain sequences generated, assembled, or induced from human immunoglobulin sequences or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) have amino acid sequences that contain residues or elements not encoded by human germline immunoglobulin sequences. For example, an antibody may be considered “human” even if it includes mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. The term “humanized” is intended to include antibodies that have a variable domain having a sequence derived from a variable domain of a non-human species (e.g., mouse) and have been modified to be more similar to a human germline encoding sequence. In some embodiments, a “humanized” antibody includes one or more framework domains having substantially the amino acid sequence of a human framework domain and one or more complementarity-determining regions having substantially the amino acid sequence of a non-human antibody. In some embodiments, a humanized antibody includes at least a portion of the immunoglobulin constant region (Fc), generally of the human immunoglobulin constant domain. In some embodiments, a humanized antibody includes the C of the human heavy chain constant domain. H 1. Hinge, C H 2, C H 3, and optionally, C H It may include 4 regions.

[0026] Antibodies include, for example, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to as "antibody conjugates" in this specification), heteroconjugate antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies, or single-chain Fv (single-chain antibodies). Examples include Fv, scFv), camelized antibodies, aphibodies, Fab fragments, F(ab')2 fragments, disulfide-bound Fv(sdFv), anti-idiotype (anti-Id) antibodies (e.g., including anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetic”), and any of the antigen-binding fragments described above. In certain embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies may also include, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single-chain Fv, polypeptide-Fc fusions, single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or its fragments, and human heavy-chain antibodies (UniAb)), single-chain or tandem diabodies (TandAb®), camelid antibodies, Anticalins®, Nanobodies® minibodies, BiTE®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®.

[0027] Immunoglobulins may originate from any of the generally known isotypes, including but not limited to IgA, secretory IgA, IgG, IgE, and IgM. The IgG subclass is also well known to those skilled in the art and includes, but is not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass encoded by a heavy chain constant region gene (e.g., IgM or IgG1). The term "antibody" includes, by example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, totally synthetic antibodies, and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods that reduce their immunogenicity in humans. Unless expressly stated or the context indicates otherwise, the term "antibody" includes any antigen-binding fragment or antigen-binding moiety of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or moieties, and single-chain antibodies.

[0028] "Antigen-binding molecule," "antigen-binding portion," "antigen-binding fragment," or "antibody fragment" The term "antigen-binding domain" refers to any molecule that contains an antigen-binding portion of the molecule. In one example, the antigen-binding molecule is an antibody, or a part thereof such as an scFv. In one example, the antigen-biding molecule is a part of a TCR that binds to an antigen, and may be the antigen-binding portion of the TCR alpha chain and / or the antigen-binding portion of the TCR alpha chain. In one example, the antigen-biding molecule may be a part of an NKG2D that binds to an NKG2D ligand. The antigen-binding molecule may contain an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing peptide-binding domains) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on tumor cells. In some embodiments, the antigen-binding molecule binds to an antigen on cells involved in hyperproliferative diseases, or to a viral or bacterial antigen. In some embodiments, the antigen-binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In certain embodiments, the antigen-binding molecule or domain is an antibody fragment that specifically binds to an antigen and includes one or more of its complementarity-determining regions (CDRs). In further embodiments, the antigen-binding molecule is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding molecule or domain includes or consists of an avimer.

[0029] In some examples, the CDR is substantially identical to that found in the sequence of the reference antibody (e.g., the antibody of this disclosure) and / or the CDR provided in this disclosure. In some embodiments, the CDR is substantially identical to the reference CDR in that it is sequence-identical to the reference CDR, or in that it contains one, two, three, four, or five (e.g., one to five) amino acid substitutions. In some embodiments, the CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) relative to the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that, compared to the reference CDR, one amino acid in the CDR is deleted, added, or substituted, but at the same time, the CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the CDR is substantially identical to the reference CDR in that, compared to the reference CDR, two, three, four, or five (e.g., two to five) amino acids in the CDR are deleted, added, or substituted, but at the same time, the CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In various embodiments, the antigen-binding fragment binds to the same antigen as the reference antibody. In various embodiments, the antigen-binding fragment cross-competes with the reference antibody and, for example, binds to a substantially identical or identical epitope as the reference antibody.

[0030] Antigen-binding fragments can be produced by any means. For example, in some embodiments, antigen-binding fragments can be produced enzymatically or chemically by fragmentation of intact antibodies. In some embodiments, antigen-binding fragments can be produced by recombination (e.g., by expression of an engineered nucleic acid sequence). In some embodiments, antigen-binding fragments can be produced entirely or partially by synthesis. In some embodiments, antigen-binding fragments may have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, or 190 amino acids, and in some embodiments, they may have a length of at least about 200 amino acids (e.g., 50-100, 50-150, 50-200, or 100-200 amino acids).

[0031] The terms “variable region” and “variable domain” are used interchangeably. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically the 110–120 amino acids at the amino-terminus of the mature heavy chain and approximately 90–115 amino acids of the mature light chain, which vary significantly in sequence between antibodies and are used for the binding and specificity of a particular antibody to a particular antigen. Sequence variability is concentrated in a region called the complementarity-determining region (CDR), while more highly conserved regions within the variable domain are called the framework region (FR). While we do not wish to be bound to any particular mechanism or theory, the CDRs of the light and heavy chains are considered to be primarily responsible for the antibody’s interaction with and specificity of the antigen. In certain embodiments, the variable region is the human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In embodiments, the variable region is the primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes a rodent or mouse CDR and a primate (e.g., non-human primate) framework region (FR).

[0032] The terms "VL" and "VL domain" are used interchangeably to refer to the variable region of the light chain of an antibody or its antigen-binding molecule.

[0033] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or its antigen-binding molecule.

[0034] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is a compromise between the two, used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on the analysis of available composite crystal structures.

[0035] TIFF2026082962000001.tif58163

[0036] The term "Kabat numbering" is recognized in the art and refers to a system for numbering amino acid residues in the heavy and light chain variable regions of antibodies or their antigen-binding molecules. In certain embodiments, the CDR of an antibody can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDR within the antibody heavy chain molecule is typically amino acid positions 31-35 (optionally including one or two additional amino acids following 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50-65 (CDR2). , and are located at amino acid positions 95-102 (CDR3). Using the Kabat numbering system, the CDRs within the antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), amino acid positions 50-56 (CDR2), and amino acid positions 89-97 (CDR3). In certain embodiments, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0037] In certain embodiments, the CDR of an antibody can be determined according to a Chothia numbering scheme that points to the position of the immunoglobulin structural loop (e.g., Chothia C See & Lesk AM, (1987), J Mol Biol 196:901-917, Al-Lazikani B et al., (1997) J Mol Biol 273:927-948, Chothia C et al., (1992) J Mol Biol 227:799-817, Tramontano A et al., (1990) J Mol Biol 215(1):175-82, and U.S. Patent No. 7,709,226. Typically, when using Kabat numbering rules, the Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34, the Chothia CDR-H2 loop is located at heavy chain amino acids 52-56, and the Chothia CDR-H3 loop is located at heavy chain amino acids 95-102, while Chothia The CDR-L1 loop is located at light chain amino acids 24–34, the Chothia CDR-L2 loop is located at light chain amino acids 50–56, and the Chothia CDR-L3 loop is located at light chain amino acids 89–97. When numbered using the Kabat numbering rules, the termination of the Chothia CDR-HI loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop terminates at 32; if only 35A is present, the loop terminates at 33; and if both 35A and 35B are present, the loop terminates at 34). In certain embodiments, the CDR of the antibodies described herein is determined according to the Chothia numbering scheme.

[0038] The terms "constant region" and "constant domain" are interchangeable and have common meanings in the art. The constant region is the antibody portion, specifically the carboxyl-terminal portions of the light and / or heavy chains, which, for example, do not directly participate in the binding of the antibody to the antigen but can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain.

[0039] When used in reference to antibodies, the term “heavy chain” may refer to any different type, for example, alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the constant domain, and these types give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including the subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0040] When used in relation to antibodies, the term “light chain” may refer to any different type, for example, kappa (κ) or lambda (λ) based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0041] "Antigen" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in a human or animal, including compositions (such as those containing tumor-specific proteins) that are injected or absorbed into a human or animal. Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens. "Target antigen" or "target antigen of interest" is substantially not found on the surface of other normal (desired) cells and is intended herein to be used in relation to the specified purpose. An antigen is an antigen designed to bind to the binding domain of a TCR or CAR. Those skilled in the art will readily understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Antigens may be expressed endogenously, i.e., by genomic DNA, or by recombination. Antigens may be specific to certain tissues, such as cancer cells, or they may be expressed broadly. Furthermore, fragments of larger molecules can act as antigens. The “target” is any molecule to which a binding motif, CAR, TCR, or antigen-binding agent, such as an antibody, is bound.

[0042] “Antigen-specific targeting region” (ASTR) is a This refers to a region of a CAR or TCR that targets a heteroantigen. The targeting region on the CAR or TCR is extracellular. In some embodiments, the antigen-specific targeting region includes an antibody or its functional equivalent or a fragment or derivative thereof, each of which targets a different antigen. The targeting region may include a full-length heavy chain, a Fab fragment, a single-chain Fv(scFv) fragment, a bivalent single-chain antibody, or a diabody, each of which is specific to the target antigen. However, there are many alternatives, such as linked cytokines (leading to recognition by cells carrying cytokine receptors), aphibodies, naturally occurring receptor-derived ligand-binding domains such as NKG2D, soluble protein / peptide ligands for receptors (e.g., on tumor cells), peptides, and vaccines that stimulate an immune response, each of which may be used in various embodiments of this disclosure. In fact, as will be understood by those skilled in the art, almost any molecule that binds to a given antigen with high affinity can be used as an antigen-specific targeting region.

[0043] "Antigen-presenting cells" or "APCs" refer to cells that process and present antigens to T cells. Exemplary APCs include dendritic cells, macrophages, B cells, certain activated epithelial cells, and other cell types capable of TCR stimulation and appropriate T cell co-stimulation.

[0044] "Antitumor effect" refers to a biological effect that may be presented as a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall survival or progression-free survival, an extension of life expectancy, or an improvement in various physiological symptoms associated with tumors. Antitumor effect may also refer to the prevention of tumor development.

[0045] Two events or entities are "associated" with each other if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, an entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a disease, disorder, or condition if its presence, level, and / or form correlates (e.g., across a relevant population) with the incidence and / or susceptibility of the disease, disorder, or condition. For example, two or more entities are physically "associated" with each other if they interact directly or indirectly such that they are physically proximate to each other (e.g., bind, etc.) and / or remain physically proximate. In a further example, two or more entities that are physically associated with each other are covalently linked or connected to each other or non-covalently associated with each other, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0046] The term "autologous" refers to any material derived from the same individual that is re-introduced later. For example, the methods of engineered autologous cell therapy (eACT™) described herein include the collection of lymphocytes from a patient, which are then engineered, for example, to express a CAR construct, and then administered to the same patient.

[0047] "Binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured and / or expressed in many ways known in the art, including, but not limited to, the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A ). K D is k off / kon It is calculated from the quotient of, while K A is, k on / k off It is calculated from the quotient of k. on k refers, for example, to the association rate constant of an antibody against an antigen. off This refers, for example, to the dissociation of an antibody against an antigen. on and k off This can be determined by techniques known to those skilled in the art, such as BIACORE® or KinExA.

[0048] The term "KD" (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment that binds to an antigen. D There is an inverse relationship between and binding affinity, therefore, K D The smaller the value, the higher the affinity, i.e., the stronger it is. Therefore, the terms "higher affinity" or "stronger affinity" indicate a higher ability to form an interaction, and thus a smaller K. D Regarding the values, conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction, and therefore a higher K. D Regarding the value: In some situations, the higher binding affinity (or K) of a particular molecule (e.g., antibody) to its interaction partner molecule (e.g., antigen X) is compared to the binding affinity of the molecule (e.g., antibody) to another interaction partner molecule (e.g., antigen Y). D ) is greater than K D (Lower or weaker affinity) with a smaller K D It can be expressed as a binding ratio determined by dividing by (a higher or stronger affinity), and may, for example, be expressed as a binding affinity that is 5 or 10 times greater in some cases.

[0049] "k d The term (seconds - 1 or 1 / s) refers to the dissociation rate constant of a specific binding pair, such as an antibody-antigen interaction, or the dissociation rate constant of a binding pair such as an antibody or antibody-binding fragment. The value is k 0i It is also called the r-value.

[0050] "k a The term (M-1 × sec-1 or 1 / M) refers to the association rate constant of a specific binding pair, such as an antibody-antigen interaction, or the association rate constant of a specific binding pair, such as an antibody or antibody-binding fragment.

[0051] "K A The term (M-1 or 1 / M) refers to the association equilibrium constant of a specific binding pair, such as an antibody-antigen interaction, or the association equilibrium constant of a binding pair such as an antibody or antibody-binding fragment. The association equilibrium constant is k a to k d It is obtained by dividing by [a certain factor].

[0052] The term "binding" generally refers to a non-covalent association between two or more entities. Direct binding involves physical contact between entities or parts. "Indirect" binding includes physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be evaluated in any of the following contexts, for example, when the interacting entities or parts are studied individually or in more complex systems (for example, while they are associated with a carrier entity by covalent bonds or other means, and / or within a biological system such as a cell).

[0053] The terms “immunely binding,” “immunely recognizing,” “specifically binding,” and “specifically recognizing” are similar terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex) in a manner that is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other peptides or polypeptides with generally lower affinity, as determined by, for example, immunoassays, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a particular embodiment, a molecule that specifically binds to one antigen may bind to another antigen. K when it comes together AK is at least 2log, 2.5log, 3log, 4log, or greater than that. A The binding domain, antibody, or antigen-binding system binds to the antigen. The binding may involve preferential association of the binding domain, antibody, or antigen-binding system with the target of the binding domain, antibody, or antigen-binding system compared to association of the binding domain, antibody, or antigen-binding system with a non-target entity (i.e., a non-target). In some embodiments, the binding domain, antibody, or antigen-binding system selectively binds to the target if the binding of the binding domain, antibody, or antigen-binding system to the target is more than 2 times, more than 5 times, more than 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more than 100 times compared to binding of the binding domain, antibody, or antigen-binding system to a non-target. In some embodiments, the binding domain, antibody, or antigen-binding system has a binding affinity of approximately 10 -5 Less than M, approximately 10 -6 Less than M, approximately 10 -7 Less than M, approximately 10 -8 Less than M, or about 10 -9 If the value is less than M, it selectively binds to the target.

[0054] In another embodiment, the molecule that specifically binds to the antigen is approximately 1 × 10⁶ -7 The dissociation constant (K) of M d ) binds. In some embodiments, the antigen-binding molecule is K d is approximately 1 x 10 -9 M ~ approx. 5×10 -9 If M, it binds specifically to the antigen with "high affinity". In some embodiments, the antigen-binding molecule is K d is 1 x 10 -10 M ~ approx. 5×10 -10 When M is present, it binds specifically to the antigen with "very high affinity". In one embodiment, the antigen-binding molecule is 10 -9 M's K d It has the following characteristics: In one embodiment, the dissociation rate is approximately 1 × 10⁻⁶. -5 It is less than.

[0055] In certain embodiments, the Specified herein provides an antibody or antigen-binding molecule that binds to a target human antigen and, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay, binds to the target antigen with an affinity of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or higher than that to another species of target antigen. In certain embodiments, the antibody or antigen-binding molecule described herein that binds to a target human antigen binds to another species of target antigen with an affinity of less than 10%, 15%, or 20% of that of the antibody or antigen-binding molecule to the human antigen, as measured by, for example, radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.

[0056] "Cancer" refers to a broad group of diseases characterized by the uncontrolled proliferation of abnormal cells in the body. Unregulated cell division and proliferation can lead to the formation of malignant tumors, which may invade adjacent tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors. In some embodiments, the methods of this disclosure may be used to treat, for example, prostate cancer, 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 carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL). , primary mediastinal large B cell lymphoma, PMBC, diffuse large B cell lymphoma (DL) BCL), follicular lymphoma (FL), transformed follicular lymphoma , splenic marginal zone lymphoma (SMZL), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system cancer Neoplasms of the CNS system, primary CNS lymphoma, tumor angiogenesis, axial vertebral tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, multiple myeloma, and tumor size of tumors resulting from combinations of these cancers. This can reduce the risk. Certain cancers may be responsive to chemotherapy or radiotherapy, or certain cancers may be refractory. Refractory cancers are those that are not suitable for surgical intervention, and are either unresponsive to chemotherapy or radiotherapy from the outset, or become unresponsive over time.

[0057] Chemokines are a type of cytokine that mediates chemotaxis or directional movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokines (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1α (MIP-1α, MIP-1a), MIP-1β (MIP-1b), gamma-inducible protein 10 (IP-10), and thymic and activation-regulating chemokines (TARC or CCL17).

[0058] A “chimeric antigen receptor” or “CAR” refers to a molecule engineered to include a binding domain and means for activating immune cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, NK cells, or combinations thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR comprises a binding domain, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR T cells. Similarly, NK cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR NK cells.

[0059] "Decrease," "lower," "mitigate," "reduce," or "weaken" generally refers to the ability of a composition intended herein to produce, induce, or cause a lower physiological response (i.e., a downstream effect) compared to the response caused by either the vehicle alone (i.e., the active portion) or the control molecule / composition. The amount "decreased" or "reduced" is typically a "statistically significant" amount and may include a decrease of 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30 times or more (e.g., 500 times, 1000 times) (including all integers and decimals above 1, such as 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the control composition vehicle (reference response).

[0060] The “extracellular domain” (or “ECD”) refers to the portion of the polypeptide that, when the polypeptide is located on the cell membrane, is understood to be located outside the cell membrane, in the extracellular space. The term “ectodomain” may be used interchangeably with “extracellular domain” in this specification.

[0061] As used herein, the term “extracellular ligand-binding domain” refers to a ligand, such as an oligonucleotide or polypeptide capable of binding to a cell surface molecule. For example, an extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a specific disease condition (e.g., cancer). Examples of cell surface markers that can act as ligands include those associated with viruses, bacterial and parasitic infections, autoimmune diseases, and cancer cells.

[0062] A "spacer" or "hinge" may follow the binding domain of a CAR, which refers to a region that moves the antigen-binding domain away from the effector cell surface, enabling proper cell-to-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). The hinge region in a CAR is generally located between the transmembrane (TM) domain and the binding domain. In certain embodiments, the hinge region is an immunoglobulin hinge region, which may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region, such as the Igg4 hinge. Other exemplary hinge regions used in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8 alpha, CD4, CD28, and CD7, which may be or may be modified wild-type hinge regions of these molecules.

[0063] The “transmembrane” region or domain is the portion of the CAR that fixes the extracellular binding portion to the plasma membrane of an immunoeffector cell and facilitates the binding of the binding domain to the target antigen. The transmembrane domain may be the CD3 zeta transmembrane domain, however, other transmembrane domains that may be used include those derived from CD8 alpha, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, NKG2D, 2B4, and CD154. In certain embodiments, the transmembrane domain is synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine.

[0064] The term “intracellular signaling domain” or “signaling domain” refers to a portion of a chimeric antigen receptor protein that is involved in inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells or other cellular responses induced by antigen binding to extracellular CAR domains, by transmitting effective CAR messages that bind to target antigens into the interior of immune effector cells. The term “effector function” refers to a specific function of a cell. The effector function of a T cell may be, for example, assistance or activity including cytolytic activity or cytokine secretion. Therefore, the terms “intracellular signaling domain” or “signaling domain,” as used interchangeably herein, refer to a portion of a protein that transmits effector function signals and directs cells to perform specific functions. Usually, the entire intracellular signaling domain may be used, but in many cases, it is not necessary to use the entire domain. Insofar as a cleavage portion of an intracellular signaling domain is used, such a cleavage portion may be used in place of the entire domain, insofar as it translates the effector function signal. The term “intracellular signaling domain” means that it includes any cleavage portion of an intracellular signaling domain sufficient to transmit effector function signals. Intracellular signaling domains are also known as "signaling domains" and are typically derived from portions of the human CD3 or FcRy chain.

[0065] It is known that signals generated solely through T cell receptors are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via T cell receptors (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). Co-stimulatory cytoplasmic signaling sequences may contain signaling domains known as immunoreceptor tyrosine-based activation domains or ITAMs.

[0066] Examples of ITAM-containing primary cytoplasmic signaling sequences particularly useful in this disclosure include those derived from DAP10, DAP12, TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0067] As used herein, the terms “co-stimulus signaling domain” or “co-stimulus domain” refer to the portion of the CAR of a co-stimulus molecule that includes the intracellular domain. These are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such co-stimulatory molecules include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, 2B4, CD137, DAP12B7-H2, and CD83. Thus, this disclosure provides exemplary co-stimulatory domains derived from CD28, CD3-epsilon, and 4-1BB, but other co-stimulatory domains are intended for use with CARs as described herein. Inclusion of one or more co-stimulatory signaling domains may enhance the efficacy and proliferation of T cells and NK cells expressing CAR receptors. Intracellular signaling and co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order.

[0068] CARs engineered to contain signaling domains derived from CD3 or FcR gamma have been shown to deliver potent signals for T cell activation and effector function, but they are not sufficient to induce signals that promote T cell survival and proliferation in the absence of accompanying co-stimulatory signals. Other CARs containing binding domains, hinges, transmembrane and signaling domains derived from CD3 zeta or FcR gamma, along with one or more co-stimulatory signaling domains (e.g., intracellular co-stimulatory domains derived from 4-1BB, CD28, CD137, CD134, and CD278), may more effectively direct antitumor activity and increased cytokine secretion, lytic activity, survival, and proliferation in CAR-expressing T cells in vitro, as well as in animal models and cancer patients (Milone). et al., Molecular Therapy, 2009;17:1453-1464, Zhong et al., Molecular Therapy, 2010;18:413-420, Carpenito et al., PNAS, 2009;106:3360-3365).

[0069] "Co-stimulatory signals" refer to signals that, in combination with primary signals such as TCR / CD3 ligation, bring about T cell responses such as proliferation and / or upregulation or downregulation of important molecules, though not limited to these signals.

[0070] "Co-stimulatory ligands" include molecules on antigen-presenting cells that specifically bind to homologous costimulatory molecules on T cells. The binding of costimulatory ligands provides signals that mediate T cell responses, such as proliferation, activation, and differentiation, though this is not limited to these processes. In addition to the primary signals provided by stimulating molecules, costimulatory ligands induce signals, for example, through the binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules. Co-stimulatory ligands are not limited to, but include 3 / TR6, 4-1BB ligand, agonists or antibodies that bind to Toll ligand receptors, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT)3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), and B7-H3. Heterogenetically binding ligands, lymphotoxin beta receptors, MHC class I chain-related protein A (MICA), MHC class I chain-related proteins Protein B (MHC class I chain-related protein B, MICB), OX40 ligand, P Examples include D-L2 or programmed death (PD) L1. Co-stimulatory ligands are not limited to antibodies that specifically bind to costimulatory molecules present on T cells, such as, but are not limited to, antibodies that specifically bind to 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, and CD83. Examples of binding ligands include lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0071] A "costimulatory molecule" is a congenital junction partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates a costimulatory response by T cells, such as proliferation, but is not limited to this.Co-stimulatory molecules include 4-1BB / CD137, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD33, and CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8 alpha, CD8 beta, CD9, CD96 (Tactile), CDl-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CDl This includes, but is not limited to, la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptors, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavage, or combinations thereof.

[0072] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR or within the framework region of its antibody or its antigen-binding molecule may be replaced with amino acid residues having a similar side chain. Generally, two sequences are considered "substantially similar" if they contain conserved amino acid substitutions at corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids and / or having "polar" or "nonpolar" side chains. Substitutions of different amino acids of the same type may be considered conserved substitutions. Exemplary amino acid classifications are shown in Table 2 below. This is summarized in Table 3.

[0073] TIFF2026082962000002.tif102151

[0074] TIFF2026082962000003.tif30142

[0075] "Combination therapy" refers to situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more treatment portions). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "dose" of the first regimen are administered before any dose of the second regimen); and in some embodiments, such drugs are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy may include the administration of one or more drugs or modalities to a subject receiving administration of other drugs or modalities in combination. For clarity, combination therapy does not require that individual drugs be administered together (or necessarily simultaneously) in a single composition; however, in some embodiments, two or more drugs or their active portions may be administered together in a combination composition, or even as part of a combination compound (e.g., a single chemical complex or covalent entity).

[0076] The term "corresponds to" can be used to indicate the position / identity of a structural element in a molecule or composition through comparison with a suitable reference molecule or composition. For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as the "corresponding" residue in a suitable reference polymer. For example, for the purpose of simplification, a residue in a polypeptide may be designated using a standard numbering system based on the reference-related polypeptide, and as a result, for example, the amino acid "corresponding to" the residue at position 100 does not actually have to be the 100th amino acid in the amino acid chain, insofar as it corresponds to the residue found at position 100 in the reference polypeptide. For example, various sequence alignment strategies are available, including software programs such as BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which may be used to identify the "corresponding" residues in polypeptides and / or nucleic acids according to this disclosure.

[0077] If the interaction between an antigen and a first antigen-binding molecule blocks, limits, inhibits, or otherwise reduces the ability of a reference binding molecule to interact with the antigen, then the antigen-binding molecule, such as an antibody, its antigen-binding fragment, CAR, or TCR, "cross-competes" with the reference binding molecule, such as the antibody or its antigen-binding fragment. Cross-competition can be complete, for example, the binding of the antigen-binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind to the antigen, or it can be partial, for example, the binding of the antigen-binding molecule to the antigen reduces the ability of the reference antigen-binding molecule to bind to the antigen. In certain embodiments, the antigen-binding molecule cross-competing with the reference antigen-binding molecule binds to the same or overlapping epitopes as the reference antigen-binding molecule. In other embodiments, the antigen-binding molecule cross-competing with the reference antigen-binding molecule binds to a different epitope than the reference antigen-binding molecule. Many types of competitive binding assays can be used to determine whether one antigen-binding molecule competes with another: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (ENZI). Immunossay (EIA), sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (Kirkland et al., 1986, J.Immunol.137:3614-3619); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct labeling RIA using 1-125 labeling (Morel et al., 1988, Molec.Immunol.25:7-15); solid-phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct labeling RIA (Moldenhauer et al. al., 1990, Scand. J. Immunol. 32:77-82).

[0078] Cytokines are non-antibody proteins released by one cell in response to contact with a specific antigen, and cytokines interact with a second cell to mediate the response in that second cell. Cytokines can be endogenously expressed by cells or administered to a target. Cytokines can be released by immune cells such as macrophages, B cells, T cells, and mast cells to propagate the immune response. Cytokines can induce a variety of responses in recipient cells. Cytokines may include homeostatic cytokines, chemokines, pro-inflammatory cytokines, effectors, and acute-phase proteins. Examples of homeostatic cytokines include interleukin (IL) 7 and IL-15. Homeostatic cytokines promote the survival and proliferation of immune cells, while pro-inflammatory cytokines can stimulate inflammatory responses. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of pro-inflammatory cytokines include IL-1a, IL-1b, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF)2, granulocyte macrophage colony-stimulating factor (GM-CSF), and soluble intercellular adhesion molecules. 1 (soluble intercellular adhesion molecule 1, sICAM-1), soluble intercellular adhesion molecule 1, Examples include soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF), but are not limited to these. Not defined. Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute-phase proteins include C-reactive proteins. Examples include protein (CRP) and serum amyloid A (SAA). , but not limited to these.

[0079] The term “domain” refers to a part of an entity. In some embodiments, a “domain” is associated with the structural and / or functional features of an entity, for example, so that the domain substantially or completely retains structural and / or functional features when the domain is physically separated from the rest of its parent entity. In some embodiments, a domain may include a part of an entity that, when separated from its (parent) entity and linked or connected to a different (recipient) entity, substantially retains and / or confers to one or more structural and / or functional features characterized in the parent entity to the recipient entity. In some embodiments, a domain is a part of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a part of a polypeptide. In some such embodiments, a domain is characterized by structural elements (e.g., amino acid sequence or sequence domain, α-helix properties, β-sheet properties, coiled-coil properties, random-coil properties, etc.) and / or functional features (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0080] The term “dosage form” may be used to refer to physically distinct units of an active agent (e.g., an antigen-binding system or antibody) for administration to a subject. Generally, each such unit contains a predetermined amount of the active agent. In some embodiments, such an amount is a unit dose (in whole) appropriate for administration according to a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to the population in question. The total amount of the therapeutic composition or drug to be administered to a subject is determined by one or more physicians and may include administrations of two or more dosage forms.

[0081] The term “dosage regimen” may be used to refer to a set of one or more unit doses administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosage regimen which may include one or more doses. In some embodiments, a dosage regimen includes multiple doses, each separated in time from the others. In some embodiments, a dosage regimen includes multiple doses, with consecutive doses separated by periods of equal length, and in some embodiments, a dosage regimen includes multiple doses, with consecutive doses separated by periods of at least two different lengths. In some embodiments, all doses in a dosage regimen are the same unit dose amount. In some embodiments, different doses in a dosage regimen are different amounts. In some embodiments, a dosage regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosage regimen is periodically adjusted to achieve a desired or beneficial outcome.

[0082] "Effector cells" refer to immune system cells that express one or more Fc receptors and mediate one or more effector functions. In some embodiments, effector cells may include, but are not limited to, one or more of the following: monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, macrogranule lymphocytes, Langerhans cells, natural killer (NK) cells, T lymphocytes, and B lymphocytes. Effector cells may be from any organism, including, but are not limited to, humans, mice, rats, rabbits, and monkeys.

[0083] "Effector function" refers to the biological consequences of the interaction between the antibody Fc region and the Fc receptor or ligand. Effector function includes, but is not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector function can be antigen-binding dependent, antigen-binding independent, or both. ADCC refers to the lysis of antibody-bound target cells by immune effector cells. While we do not wish to be bound by any theory, ADCC generally refers to the lysis of antibody-bound target cells by the Fc receptor (Fc receptor, Fc Effector cells, including R), are understood to be involved in recognizing antibody-coated target cells (e.g., cells that express the antigen to which the antibody is bound on their surface) and subsequently killing them. ADCC-mediated effector cells may include, but are not limited to, one or more of the following immune cells: natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0084] The term "engineered autologous cell therapy," also known as adoptive cell transfer and sometimes abbreviated as "eACT(trademark)," is a process in which a patient's own T cells are harvested and subsequently genetically modified to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignant tumor cells. T cells or NK cells can be engineered to express, for example, a chimeric antigen receptor (CAR) and / or T cell receptor (TCR). In some examples, CAR-positive (+) T cells or NK cells are engineered to express an extracellular single-strand variable fragment (scFv) specific to a particular tumor antigen, linked to an intracellular signaling region containing at least one costimulatory domain and at least one activation domain. In some examples, CAR-positive (+) T cells or NK cells are engineered to express an extracellular domain of NKG2D specific to the NKG2D antigen, linked to an intracellular signaling region containing at least one costimulatory domain and at least one activation domain. The co-stimulatory domains may originate from naturally occurring co-stimulatory domains or variants thereof, for example, variants having a truncated hinge domain ("THD"), and the activating domains may originate from, for example, CD3-zeta and / or CD3-epsilon. In certain embodiments, the CAR is designed to have two, three, four, or more co-stimulatory domains.

[0085] In some embodiments, the CAR is manipulated so that the co-stimulatory domain is expressed as a separate polypeptide chain. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Applications Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated in their entirety by reference. “Adoptive cell therapy” or “AC” "T" includes targeting immune cells with antitumor activity, for example, transplanting them into cancer patients. In some embodiments, ACT is a therapeutic approach that includes the use of lymphocytes with antitumor activity (e.g., engineered lymphocytes).

[0086] An "epitope" refers to a localization region of an antigen to which an antibody can specifically bind. An epitope may be, for example, a sequence of amino acids in a polypeptide (linear or continuous epitope), or it may be a combination of, for example, two or more discontinuous regions of a polypeptide (conformational, nonlinear, discontinuous, or discontinuous epitopes). In certain embodiments, the epitope to which the antibody binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenic mapping (e.g., site-directed mutagenic mapping). In the case of X-ray crystallography, crystallization is performed in the art. This can be achieved using any of the known methods (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt4):339-350, McPherson A (1990) Eur J Biochem 189:1-23, Chayen NE (1997) Structure 5:1269-1274, McPherson A (1976) J Biol Chem 251:6300-6303). Antibody-antigen crystals can be studied using well-known X-ray diffraction techniques, such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al, U.S. Patent Application Publication No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60, Bricogne G (1997) Meth Enzymol 276A:361-423, ed The results can be refined using computer software such as Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10):1316-1323. Mutagenic mapping studies can be achieved using any method known to those skilled in the art. For example, for a description of mutagenic techniques, including alanine scanning mutagenic techniques, see Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085.

[0087] With respect to genes, proteins, and / or nucleic acids, "endogenous" refers to the natural presence of those genes, proteins, and / or nucleic acids in cells such as immune cells.

[0088] "Exogenous" refers to the introduction of drugs, such as nucleic acids, genes, or proteins, into cells from an external source, for example. A nucleic acid introduced into a cell is considered exogenous even if it codes for a protein naturally found in the cell. Such exogenous introduction of protein-coding nucleic acids can be used to increase protein expression beyond levels naturally found in the cell under similar conditions, for example, without the introduction of exogenous nucleic acids.

[0089] The term "excipient" refers to a substance that may be included in a composition to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0090] The “fragments” or “parts” of materials or entities described herein have a structure that includes, for example, a physical entity or an abstract entity, which is a separate part of the whole. In some embodiments, the fragment lacks one or more parts that are present in the whole. In some embodiments, the fragment consists of or includes characteristic structural elements, domains, or parts that are present in the whole. In some embodiments, the polymer fragment contains or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) found throughout the polymer. The material or entire entity may be referred to as the "parent" of the fragments in some embodiments.

[0091] The terms “fusion polypeptide” or “fusion protein” generally refer to a polypeptide comprising at least two segments. Generally, a polypeptide comprising at least two such segments is considered a fusion polypeptide if the two segments are (1) a portion not inherently present in the same peptide and / or (2) a portion not pre-linked or connected to one another in a single polypeptide and / or (3) a portion linked or connected to one another through the action of the human hand. In embodiments, CAR is a fusion protein. In embodiments, TCR is a fusion protein.

[0092] The terms "gene product" or "expression product" generally refer to RNA transcribed from a gene (before and / or after treatment), or polypeptides encoded by RNA transcribed from a gene (before and / or after modification).

[0093] The terms “genetically modified” or “manipulated” refer to methods of altering a cell’s genome, including, but not limited to, deleting coding regions or non-coding regions or parts thereof, or inserting coding regions or parts thereof. In some embodiments, the modified cells are lymphocytes, e.g., T cells or NK cells, which can be obtained from either a patient or a donor. In some embodiments, the modified cells are induced pluripotent stem cells (iPSCs) that can differentiate into lymphocytes such as T cells or NK cells. Cells may be modified to express exogenous constructs, e.g., chimeric antigen receptors (CARs) or T cell receptors (TCRs), which are incorporated into the cell’s genome. Other gene editing can also be performed, for example, to reduce rejection and / or enhance cytocompatibility. Manipulation generally involves human manipulation. For example, a polynucleotide is considered “manipulated” if it is manipulated by human hands so that two or more sequences that are not linked or connected in their natural order are directly linked or connected to each other in the manipulated polynucleotide. In the context of cell manipulation using molecular biology techniques, a cell or organism is considered “manipulated” if it is manipulated in such a way that its genetic information is altered (e.g., new genetic material that was not previously present is introduced, for example, by transformation, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, for example, by substitution or deletion mutation, or by other protocols). In some embodiments, the binding agent is a modified lymphocyte, e.g., a T cell or NK cell, which can be obtained from either a patient or a donor. The manipulated cell may be modified to express an exogenous construct, e.g., a chimeric antigen receptor (CAR) or T cell receptor (TCR), which is incorporated into the cell’s genome. The offspring of a manipulated polynucleotide or binding agent are generally referred to as “manipulated,” even if the actual manipulation was performed on the previous entity. In some embodiments, “manipulated” refers to a designed and manufactured entity.The term “designed” refers to a drug whose structure is (i) selected or chosen by human hands, (ii) produced by a process requiring human hands, and / or (iii) different from natural substances and other known drugs.

[0094] "T cell receptors" or "TCRs" refer to antigen-recognizing molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to produce a wide variety of TCR proteins. TCR-based T cell therapy Examples are disclosed in International Applications PCT / US2013 / 059608 and PCT / US2015 / 033129, which are incorporated herein by reference in their entirety.

[0095] The term "heterogeneous" means that a sequence originates from any source other than those found in nature. For example, a heterogeneous sequence included as part of a co-stimulatory protein is an amino acid that does not exist naturally as part of the wild-type human co-stimulatory protein, i.e., it does not align with the wild-type human co-stimulatory protein. For example, a heterogeneous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human co-stimulatory protein coding sequence.

[0096] The term "identity" refers to the overall relationship between polymer molecules, for example, between nucleic acid molecules (e.g., DNA and / or RNA molecules), and / or polypeptide molecules. Methods for calculating the identity percentage between two given polypeptide sequences are known. For example, the calculation of the identity percentage between two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). Then, nucleotides or amino acids at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The identity percentage between two sequences is optionally a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison or alignment of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms such as BLAST (a basic local alignment search tool). In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are identical by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0097] To calculate the percentage of identity, the sequences being compared are typically aligned in a way that gives the greatest match between them. An example of a computer program that can be used to determine the percentage of identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which the percentage of sequence identity is to be determined. The sequences are aligned for the best possible matching of each amino acid or nucleotide ("matched span" as determined by the algorithm). In certain embodiments, standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM250 comparison matrix, and Henikoff et al., 1992, Proc.Natl.Acad.Sci.USA89:10915-10919 for the BLOSUM62 comparison matrix) are also used in the algorithm. Other algorithms are available for comparing amino acid sequences or nucleic acid sequences, including those available in commercially available computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. An example of such a program is Altschul, et al., Basic local alignment search tool, J.Mol.Biol., 215(3):403-410, 1990, Altschul These methods are described in *Methods in Enzymology* by chul, et al., *Gapped BLAST and PSI-BLAST: a new generation of protein database search programs*, Nucleic Acids Res. 25:3389-3402, 1997, *Baxevanis, et al.*, *Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins*, Wiley, 1998, and *Misener, et al.*, (eds.), *Bioinformatics Methods and Protocols* (*Methods in Molecular Biology*, Vol. 132), Humana Press, 1999. In addition to identifying similar sequences, these programs generally provide an indicator of similarity. In some embodiments, two sequences are considered substantially similar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, or more of the corresponding residues are similar and / or identical across a relevant stretch of residues (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the relevant stretch is a complete sequence.In some embodiments, a related sequence consists of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, and at least 500 or more residues. Sequences having substantial sequence similarity may be homologous to one another.

[0098] The term "substantially identical" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity, as measured by any well-known algorithm of sequence identity such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity with respect 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.

[0099] When applied to polypeptides, the term “substantially similar” means that two peptide sequences share at least 95% sequence identity, and more preferably at least 98% or 99%, when optimally aligned by a programmed method such as GAP or BESTFIT using default gap weights. Preferably, non-identical residue positions are distinguished by conserved amino acid substitutions.

[0100] The terms “improve,” “increase,” “inhibit,” and “reduce” refer to values ​​relative to a baseline or other reference baseline. In some embodiments, appropriate baseline measurements may include measurements in a particular system (e.g., in a single individual) under otherwise equivalent conditions, either in the absence of a drug or treatment (e.g., before and / or after) or in the presence of a suitable equivalent reference drug. In some embodiments, appropriate baseline measurements are relevant This may include measurements in equivalent systems known or expected to respond in an equivalent manner in the presence of a drug or treatment.

[0101] "Immune response" refers to the action of immune system cells (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules (including Ab, cytokines, and complement) produced by either these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination from the body of a vertebrate of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmune or pathological inflammation, normal human cells or tissues.

[0102] The term "immunotherapy" refers to the treatment of individuals who are suffering from a disease or are at risk of developing or relapsing from a disease, by means of methods including inducing, enhancing, suppressing, or otherwise modifying the immune response. Examples of immunotherapy include, but are not limited to, NK cell and T cell therapies. T cell therapies include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, and manipulation. This may include autologous cell therapy (eACT®) and allogeneic T cell transplantation. However, those skilled in the art will understand that the pre-treatment methods disclosed herein enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapies are described in U.S. Patent Applications Publications 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and International Publication 2008 / 081035. Examples of TCR-based T cell therapies are disclosed in International Applications PCT / US2013 / 059608 and PCT / US2015 / 033129, which are incorporated herein by reference in their entirety.

[0103] T cells or NK cells for immunotherapy may be derived from any source known in the art. For example, T cells and NK cells can be differentiated in vitro from hematopoietic stem cell populations (e.g., iPSCs) or obtained from a subject. T cells and NK cells may be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. Furthermore, T cells may be derived from one or more T cell lines available in the art. T cells may also be obtained from blood units taken from a subject using various techniques known to those skilled in the art, such as FICOLL® isolation and / or apheresis. Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0104] The term "in vitro" refers to events that occur in an artificial environment, such as a test tube, reaction vessel, or cell culture, rather than within a multicellular organism. The term "in vitro cell" refers to any cell cultured ex vivo. In particular, in vitro cells may include T cells or NK cells. The term "in vivo" refers to events that occur within a multicellular organism, such as a human or non-human animal.

[0105] The term “isolated” means (1) a substance that has been separated from at least some components to which the substance had previously associated or which the substance would have otherwise associated, and / or (2) a substance present in a composition containing one or more known or unknown contaminants in limited or defined amounts or concentrations. In some embodiments, the isolated substance is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., The substance may be isolated from 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%. In certain cases, a substance is isolated if it is present in a composition containing the same or similar types of molecules in limited or reduced amounts or concentrations. For example, in certain cases, nucleic acid, DNA, or RNA substances are isolated if they are present in a composition containing nucleic acid, DNA, or RNA molecules that are not the substance in limited or reduced amounts or concentrations. For example, in certain cases, polypeptide substances are isolated if they are present in a composition containing polypeptide molecules that are not the substance in limited or reduced amounts or concentrations. In certain embodiments, the amount may be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, the limited amount may be an amount that is 100% or less of the amount of the substance in the composition, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or less of the amount of the substance in the composition (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain examples, the composition is pure or substantially pure with respect to the selected substance. In some embodiments, the isolated substance has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). A substance is "pure" if it is substantially free of other components or impurities. In some embodiments, a substance may still be considered "isolated" or "pure" even after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), in which case the isolation or purity percentage of the substance is calculated without including such carriers or excipients.

[0106] A “linker” (L), “linker domain,” or “linker region” refers to an oligo or polypeptide region of approximately 1 to 100 amino acids in length that links together, for example, one of the domains / regions of a CAR, TCR, and / or scFv, or many of these polypeptides. Linkers may consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Longer linkers may be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. Linkers may be cleavable or incleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or their functional equivalents, and combinations thereof. In some embodiments, linkers include picornavirus 2A-like linkers, porcine swaensis virus (P2A), the CHYSEL sequence of virus (T2A) (SEQ ID NO: 1), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence results in a cleavage between 2A glycine and 2B proline, as in Asp-Val / Ile-Glu-X-Asn-Pro-Gly (2A) -Pro (2B)It may include a domain (SEQ ID NO: 2). Other linkers will be apparent to those skilled in the art and may be used in connection with this disclosure. A linker may be part of a multi-component agent that connects different elements to one another. For example, a polypeptide containing two or more functional or structural domains may include stretches of amino acids between such domains that link them together. In some embodiments, a polypeptide containing a linker element has a general overall structure of form S1-L-S2, where S1 and S2 may be the same or different and represent two domains linked to one another by the linker. The linker may connect or link together either a CAR or a TCR domain / region. In some embodiments, the polypeptide linker has an amino acid length of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1 The amino acid lengths are ~70, 1~80, 1~90, 1~100, 10~20, 10~30, 10~40, 10~50, 10~60, 10~70, 10~80, 10~90, or 10~100. In some embodiments, the linker tends not to take a rigid three-dimensional structure, but instead provides flexibility to the polypeptide. In another example, it may be used to link to an expressed or many polypeptides, such as CARs and / or TCRs.

[0107] Other linkers include non-cuttable linkers. To realize the present invention, a number of linkers, including "flexible linkers," are used. The latter are rich in glycine. Klein et al., Protein Engineering, Design & Selection Vol.27, No.10, pp.325-330, 2014; Priyanka et al., Protein Sci., 2013 Feb;22(2):153-167.

[0108] In some embodiments, the linker is a synthetic linker. The synthetic linker may have a length of about 10 to about 200 amino acids, for example, 10 to 25 amino acids, 25 to 50 amino acids, 50 to 75 amino acids, 75 to 100 amino acids, 100 to 125 amino acids, 125 to 150 amino acids, 150 to 175 amino acids, or 175 to 200 amino acids. The synthetic linker may have a length of 10 to 30 amino acids, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The synthetic linker may have a length of 30 to 50 amino acids, for example, 30 to 35 amino acids, 35 to 40 amino acids, 40 to 45 amino acids, or 45 to 50 amino acids.

[0109] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in both glycine and serine residues.

[0110] The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that represents the components of the genetic immune system. NK cells reject tumor and virus-infected cells. They act through the process of apoptosis or programmed cell death. They are called "natural killers" because they do not require activation to kill cells. T cells play a role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) differentiate from other lymphocyte types. The thymus, a differentiated organ of the immune system, is primarily responsible for the maturation of T cells. There are six types of T cells: helper T cells (e.g., CD4+ cells), cytotoxic T cells (TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or also known as killer T cells), and memory T cells ((i) stem memory T cells). SCMThe cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting numerous functional attributes unique to memory cells), (ii) Central Memory T CM The cells express L-selectin and CCR7 and secrete IL-2, but they do not secrete IFNγ or IL-4. (iii) Effector Memory T EM Cells include regulatory T cells (Treg, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells (which do not express L-selectin or CCR7 and produce effector cytokines such as IFNγ and IL-4). B cells, on the other hand, play a role in humoral immunity (involving antibodies). B cells produce antibodies and antigens and act as antigen-presenting cells (APCs). After activation through antigen interaction, they transform into memory B cells. In mammals, immature B cells are formed in the bone marrow, and the name originates from this location.

[0111] The term "neutralize" refers to an antigen-binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological action of that ligand. In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand or alters the ligand's ability to bind through indirect means (such as structural or energetic changes to the ligand). In some embodiments, the antigen-binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it is bound from performing its biological function.

[0112] "Nucleic acid" refers to any polymer chain of nucleotides. Nucleic acids may be DNA, RNA, or a combination thereof. In some embodiments, nucleic acids comprise one or more native nucleic acid residues. In some embodiments, nucleic acids consist of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of the following: isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, regeneration in recombinant cells or systems, and chemosynthesis. In some embodiments, nucleic acids comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, The lengths are 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues (e.g., 20-100, 20-500, 20-1000, 20-2000, or 20-5000, or more residues). In some embodiments, the nucleic acid is partially or entirely single-stranded, and in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence containing at least one element that codes for a polypeptide or is a complement to a sequence that codes for a polypeptide.

[0113] "Operatively linked" refers to a juxtaposition of described components that is in a relationship that enables them to function in their intended manner. For example, a control element "operatively linked" to a functional element is associated such that the expression and / or activity of the functional element is achieved under conditions compatible with the control element. In embodiments, a promoter is operatively linked to a nucleic acid.

[0114] "Patient" includes any human being suffering from cancer (e.g., multiple myeloma). The terms "Subject" and "Patient" are used interchangeably herein.

[0115] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, of which there are many types, commonly referred to in the art as proteins. A “polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include native peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0116] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or whose benefits to the recipient outweigh any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and must not be harmful to the recipient or whose benefits to the recipient outweigh any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulation material, that is involved in the transport or delivery of a drug from one part of the body to another (for example, from one organ to another). Each carrier present in a pharmaceutical composition must be compatible with the other components of the formulation and "acceptable" in the sense that it is not harmful to the patient, or whose benefits to the recipient outweigh any harmful effects. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, suitable substances used in pharmaceutical formulations.

[0117] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant subjects or population. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, but is not limited to, forms adapted for: oral administration, e.g., liquid (aqueous or non-aqueous or suspension), tablets, e.g., buccal, sublingual, and those targeting systemic absorption, bolus, powder, granules, paste for application to the tongue; parenteral administration, e.g., sterile solution or suspension, or as a sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; topical administration, e.g., as a cream, ointment, or sustained-release patch or spray applied to the skin, lungs, or oral cavity; intravaginal or rectal, e.g., as a pessary, cream, or foam; sublingual; ocular; transdermal; or transnasal, lung, and other mucosal surfaces.

[0118] The term “proliferation” refers to an increase in cell division, whether symmetric or asymmetric. In some embodiments, “proliferation” refers to the symmetric or asymmetric division of T cells. “Increased proliferation” occurs when the number of cells in a treated sample increases compared to the number of cells in an untreated sample.

[0119] The term “reference” describes the standard or control on which the comparison is performed. For example, in some embodiments, the drug, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control which is a drug, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a reference or control from the past that is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation. Similarity is determined by the selected reference or If sufficient to justify the dependence on and / or comparison with the control.

[0120] Regulatory T cells ("Treg," "Treg cells," or "Treg") refer to a lineage of CD4+ T lymphocytes involved in regulating specific immune activities, such as responses to autoimmunity, allergies, and infections. Regulatory T cells can modulate the activity of T cell populations and can also influence certain innate immune cell types. Tregs can be identified by the expression of biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically constitute about 5-10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells) can constitute as much as 20-30% of the total CD4+ T lymphocyte population.

[0121] The term “sample” generally refers to an aliquot of material obtained from or derived from the source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may include cells or organisms such as a cell population, tissue, or animal (e.g., human). In some embodiments, the source of interest may include biological tissue or fluid. In some embodiments, the biological tissue or fluid may include amniotic fluid, aqueous humor, ascites, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, ovoid, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, ascites, pleural fluid, pus, catarrhal secretions, saliva, sebum, semen, serous fluid, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous fluid, vomit, and / or combinations or components thereof. In some embodiments, the biological fluid may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid may include plant exudates. In some embodiments, the biological tissue or specimen may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral swab, nasal swab, skin swab, or vaginal swab), scraping, surgery, or lavage (e.g., bronchoalveolar, tubal, nasal cavity, eye, oral cavity, uterus, vagina, or other lavage). In some embodiments, the biological specimen includes cells obtained from an individual. In some embodiments, the specimen is a “primary specimen” obtained directly from the source of interest by any suitable means. In some embodiments, as is evident from the context, the term “specimen” refers to a preparation obtained by processing a primary specimen (e.g., by removing one or more components of the primary specimen and / or by adding one or more agents to the primary specimen). Such “processed samples” may include, for example, nucleic acids or proteins extracted from a sample, or obtained by subjecting a primary sample to one or more techniques such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components.

[0122] A "single-chain variable fragment," "single-chain antibody variable fragment," or "scFv" antibody refers to an antibody form that contains variable regions consisting only of the heavy and light chains linked by a linker peptide.

[0123] The term “cancer staging” refers to a qualitative or quantitative assessment of the level of cancer progression. In some embodiments, the criteria used to determine cancer staging may include, but are not limited to, one or more of the following: where the cancer is located in the body, tumor size, whether the cancer has spread to lymph nodes, and whether the cancer has spread to one or more different parts of the body. In some embodiments, staging may be performed using the so-called TNM system, in which T refers to the size and extent of the main tumor, usually called the primary tumor; N refers to the number of nearby lymph nodes with cancer; and M refers to whether the cancer has metastasized. In some embodiments, cancer is staging stage 0 (abnormal cells are present without spreading to nearby tissue, also called carcinoma in situ or CIS, CIS is not cancer but can become cancer), stages I-III (cancer is present, the larger the number, the larger the tumor and the more it has spread to nearby tissue), or stage IV (cancer has spread to distant parts of the body). It may be referred to as (r). In some embodiments, cancer may be assigned to a stage selected from the group consisting of: in situ, localized (cancer is confined to where it began and there are no signs of it spreading), local (cancer has spread to nearby lymph nodes, tissues, or organs); distal (cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the stage).

[0124] "Stimulus" refers to the primary response induced by the binding of a stimulating molecule to its homologous ligand, and this binding mediates a signaling event. A "stimulating molecule" is a molecule on T cells, such as the T cell receptor (TCR) / CD3 complex, that specifically binds to homologous stimulating ligands present on antigen-presenting cells. A "stimulating ligand," when present on antigen-presenting cells (e.g., APCs, dendritic cells, B cells, etc.), specifically binds to stimulating molecules on T cells, thereby mediating primary T cell responses, including, but not limited to, activation, initiation of an immune response, and proliferation. Stimulating ligands include, but are not limited to, anti-CD3 antibodies (such as OKT3), peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0125] The term "therapeutic agent" can refer to any agent that, when administered to a living organism, induces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, the suitable population may be a model organism or a population of human subjects. In some embodiments, the suitable population may be defined by various criteria, such as a specific age group, sex, genetic background, or pre-existing clinical status, depending on the presence or absence of biomarkers. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is an agent that has been or needs to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent that requires a medical prescription for administration to humans.

[0126] The “therapeutic effective dose,” “effective dose,” “effective amount,” or “therapeutic effective dosage” of a therapeutic agent, such as engineered CAR T cells or NK cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disability or impairment resulting from the onset of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems to predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0127] The terms “transduction” and “transduced” refer to the process by which foreign DNA is introduced into a cell via a viral vector (see Jones et al., “Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, DNA vector, RNA vector, adenovirus vector, baculovirus vector, Epstein-Barr virus vector, papovavirus vector, vaccinia virus vector, herpes simplex virus vector, adenovirus-associated vector, lentiviral vector, or any combination thereof.

[0128] "Transformation" refers to any process in which exogenous DNA is introduced into a host cell. Transformation can occur naturally or artificially using a variety of methods. Transformation occurs in prokaryotic host cells. Alternatively, this can be achieved using any known method for inserting an exogenous nucleic acid sequence into a eukaryotic host cell. In some embodiments, several transformation methodologies are selected based on the host cell to be transformed and / or the nucleic acid to be inserted. Transformation methods may include, but are not limited to, viral infection, electroporation, and lipofection. In some embodiments, the “transformed” cell is stably transformed in such a way that the inserted DNA can replicate autonomously as part of a plasmid or host chromosome. In some embodiments, the transformed cell may express the introduced nucleic acid.

[0129] "Treatment" or "treating" a subject means any type of intervention or process performed on the subject, or administration of an activator to the subject, with the aim of reversing, alleviating, improving, inhibiting, slowing or preventing the onset, progression, development, severity or relapse of symptoms, complications or conditions, or biochemical signs associated with the disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment may be the treatment of a subject that does not show signs of the related disease, disorder and / or condition, and / or a subject that shows only initial signs of the disease, disorder and / or condition. In some embodiments, such treatment may be the treatment of a subject that shows one or more definitive signs of the related disease, disorder and / or condition. In some embodiments, treatment may be the treatment of a subject that has been diagnosed with the related disease, disorder and / or condition. In some embodiments, treatment may be the treatment of a subject that is known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the related disease, disorder and / or condition.

[0130] The term “vector” refers to a recipient nucleic acid molecule that contains or has been modified to incorporate a provided nucleic acid sequence. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can autonomously replicate in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors contain sequences that direct the expression of the inserted gene into which they are operably linked. Such vectors may be referred to herein as “expression vectors.” Standard techniques can be used for vector manipulation and are found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference.

[0131] The term “sequence” refers to a nucleotide sequence of any length, which may be DNA or RNA, and may be linear, circular, or branched, and may be single-stranded or double-stranded. The term “donor sequence” refers to a nucleotide sequence to be inserted into the genome. Donor sequences may be of any length, for example, 2 to 10,000 nucleotides (or any integer between or greater than that), preferably about 100 to 1,000 nucleotides (or any integer between those), and more preferably about 200 to 500 nucleotides.

[0132] For the purposes of this disclosure, “gene” includes the DNA region that codes for a gene product (see below) and all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding sequence and / or transcription sequence. Thus, a gene includes promoter sequences, terminators, ribosome binding sites and internal ribosome entry sites, etc. This includes, but is not limited to, translation regulatory sequences, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus regulatory regions.

[0133] A “transmembrane domain” is a polypeptide domain that, when present in the corresponding endogenous polypeptide expressed in mammalian cells, includes at least one consecutive amino acid sequence that traverses the lipid bilayer. For example, a transmembrane domain may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acid sequences, each traversing the lipid bilayer, when present in the corresponding endogenous polypeptide expressed in mammalian cells. A transmembrane domain may include, for example, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) consecutive amino acid sequences having an α-helix secondary structure in the lipid bilayer (traversing the lipid bilayer when present in the corresponding endogenous polypeptide expressed in mammalian cells). In some embodiments, a transmembrane domain may include two or more consecutive amino acid sequences that form a β-barrel secondary structure in the lipid bilayer (each traversing the lipid bilayer when present in the corresponding endogenous polypeptide expressed in mammalian cells). Non-limiting examples of transmembrane domains are described herein. Further examples of transmembrane domains are known in the art.

[0134] When the phrase "extracellular space of the plasma membrane" is used to describe the location of a polypeptide, it means that the polypeptide comprises at least one transmembrane domain that traverses the plasma membrane and at least one domain located in extracellular space (e.g., at least one antigen-binding domain).

[0135] This disclosure may utilize methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, which are within the scope of the art of the art, unless otherwise indicated, many of which are described below for illustrative purposes. Such techniques are fully described in the literature, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001), Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated). July 2008), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience, Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985), Anand, Techniques for the Analysis of Complex See monographs in journals such as Genomes (Academic Press, New York, 1992), Transcription and Translation (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide to Molecular Cloning (1984), Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology (QEColigan, AMKruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0136] This disclosure provides antigen receptors (CARs) comprising a portion of the extracellular domain of NKG2D that can bind to one or more NKG2D ligands, referred to herein as NKG2D CARs. In particular, this disclosure provides methods and compositions useful for the treatment of cancer and / or initiation or modulation of immune responses. In some embodiments, the NKG2D CAR is expressed together with a TCR specific to one or more tumor antigens, and / or one or more additional CARs specific to one or more tumor antigens.

[0137] Various embodiments of the Disclosure provide vectors encoding NKG2D CARs, e.g., vectors encoding NKG2D CARs. Various embodiments of the Disclosure also provide vectors encoding TCRs or one or more additional CARs (e.g., CARs that bind to a different target than NKG2D CAR), e.g., vectors encoding NKG2D CAR and TCR or one or more additional CARs. In some embodiments, NKG2D CAR is encoded in a separate vector from the vector encoding TCR or one or more additional CARs. In some embodiments, NKG2D CAR is encoded in the same vector encoding TCR or one or more additional CARs.

[0138] Various embodiments of this disclosure provide cells encoding or expressing NKG2D CAR, for example, induced pluripotent stem cells (iPSCs), T cells, or NK cells engineered to encode or express NKG2D CAR. Various embodiments of this disclosure provide cells encoding or expressing NKG2D CAR and TCR or one or more additional CARs, for example, T cells or NK cells engineered to encode or express NKG2D CAR and TCR or one or more additional CARs. This disclosure provides immune cells genetically modified with incorporated genes, for example, a nucleotide sequence of interest (e.g., constitutive expression constructs and / or inducible expression constructs containing such nucleotide sequences). In embodiments, the immune cells are further engineered to express TCR or one or more additional CARs. In some embodiments, this disclosure provides a method for treating a subject having a tumor, comprising administering the subject an NKG2D CAR therapy as described herein. In some embodiments, the method further comprises the administration of one or more additional therapies (e.g., a second conjugate (e.g., CAR-T cells, CAR-NK cells, TCR-T cells, TIL cells, allogeneic NK cells, and autologous NK cells), an antibody-drug conjugate, an antibody, a bispecific antibody, a T cell-associated bispecific antibody, an engineered antibody, and / or a polypeptide as described herein).

[0139] Natural killer cells preferentially express several calcium-dependent (C-type) lectins involved in regulating NK cell function. NKG2D (NCBI Gene ID: 22914, updated March 7, 2021, incorporated herein by reference) is a transmembrane protein belonging to the NKG2 family of C-type lectin-like receptors. The NKG2 gene family is located within the NK complex, a region containing several C-type lectin genes that are preferentially expressed in NK cells. NKG2D is a recognition receptor for the detection and elimination of transformed and infected cells, as its ligands are induced during cellular stress, either as a result of infection or genomic stress, such as in cancer. NKG2D binds to a diverse family of ligands, including MHC class I chain-related A and B proteins and UL-16 binding proteins. Surface expression of these ligands is crucial for the recognition of cells stressed by the immune system; therefore, this protein and its ligands are therapeutic targets for the treatment of immune diseases and cancer.

[0140] NKG2D ligand is absent or present only at low levels on the surface of normal cells, but is overexpressed in infected cells, transformed cells, senescent cells, and stressed cells. These are induced autoproteins. Their expression is regulated at different stages (transcription, mRNA and protein stabilization, cleavage from the cell surface) by various stress pathways. NKG2D ligands are homologous to MHC class I molecules and are divided into two families: MIC and RAET1 / ULBP. The human MIC gene is located within the MHC locus and consists of seven members (MICA-G), of which only MICA and MICB produce functional transcripts. Of the 10 known human RAET1 / ULBP genes, six encode functional proteins: RAET1E / ULBP4, RAET1G / ULBP5, RAET1H / ULBP2, RAET1 / ULBP1, RAET1L / ULBP6, and RAET1N / ULBP3.

[0141] Chimeric antigen receptors (CARs) are engineered receptors that can direct or redirect T cells or NK cells (e.g., patient or donor T or NK cells) to a selected target. CARs may be engineered to recognize a target (such as an antigen and, in the case of a disclosed NKG2D CAR, an NKG2D ligand) and, upon binding to that target, activate immune cells to attack and destroy the target-carrying cells. If these targets are present on tumor cells, immune cells expressing CARs can target and kill the tumor cells. CARs generally include an extracellular binding domain that mediates antigen binding (e.g., an NKG2D ectodomain), a transmembrane domain that crosses or is understood to cross the cell membrane if the CAR is present on the cell surface or cell membrane, and an intracellular (or cytoplasmic) signaling domain.

[0142] From at least one non-limiting viewpoint, there are at least three “generations” of CAR compositions. In the first generation CAR, a binding domain (e.g., a single-strand fragment variable binding domain) is linked or connected to a signaling domain (e.g., CD3ζ) via a transmembrane domain that optionally includes a hinge domain and one or more spacers. In the second generation CAR, a co-stimulatory domain (CM1 such as CD28, 4-1BB, or OX-40) is introduced together with the signaling domain (e.g., CD3ζ). In the third generation CAR, a second co-stimulatory domain (CM2) is included.

[0143] The transmembrane chain (TCR) is a heterodimer composed of an α-chain and a β-chain. TCR signaling requires the recruitment of signaling proteins that generate immune synapses. In addition, the localization of the TCR in the plasma membrane depends on the CD3 complex expressed in T cells. Manipulated single-stranded TCRs can be generated, for example, using the transmembrane and signaling domains of CAR constructs, known methods and constructs (e.g., sTCR and TCR-CAR molecules, e.g., fusions of the TCRβ chain with CD28™ and CD28 and CD3ζ signaling modules).

[0144] The NKG2D CARs of this disclosure may include an extracellular NKG2D domain that binds to an NKG2D ligand. In some embodiments, the antigen-binding system further includes a costimulatory domain and / or an extracellular domain (e.g., a "hinge" or "spacer" region), and / or a transmembrane domain, and / or an intracellular (signaling) domain, CD3-zeta, and / or a CD3-epsilon-activating domain.

[0145] In certain embodiments, NKG2D CAR includes an NKG2D ectodomain (extracellular domain) polypeptide, which refers to a polypeptide having at least 75% sequence identity to SEQ ID NO: 3 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNT YICMQRTV (SEQ ID NO: 3). In embodiments, the NKG2D ectodomain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: TTATTCAACCAAGAAGTCCAGATTCCCTTGACCGAAAGTTACTGCGGCCCATGTCCGAAAAACTGGATATGTTATAAAAATAACTGTTACCAGTTCTTCGATGAATCTAAAAACTGGTATG AGAGCCAGGCATCTTGTATGTCTCAAAATGCCAGCCTGCTCAAAGTATACAGCAAGGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCACTGGATGGGATTGGTACACATTCCCACAAATGGATCTTGGCAGTGGGAAGAC GGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGCGCACTCTATGCATCGAGCTTTAAAGGTTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTA (SEQ ID NO: 4).In embodiments, the NKG2D ectodomain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: TTATTCAACCAAGAAGTCCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGTTACCAATTCTTCGATGAAAGTAAAAACTGGTATG AGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAGGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGAC GGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCATCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTG (SEQ ID NO: 5).In embodiments, the NKG2D ectodomain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: TTATTCAACCAAGAAGTCCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAGAACTGGATATGTTACAAAAATAACTGTTACCAATTCTTCGATGAATCTAAGAA TTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTTAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGG CAGTGGGAAGACGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCATCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATATATTTGCATGCAAAG GACTGTG (SEQ ID NO: 55).

[0146] In some embodiments, the NKG2D CAR of the present disclosure may include an antigen-binding system comprising one or more or all of the following: a leader peptide (P), an NKG2D ectodomain (B), a hinge (E), a transmembrane domain (T), a costimulatory domain (C), a second costimulatory domain (C'), and an activating domain (A). In some examples, the NKG2D CAR is It is configured according to the BETA below. In certain examples, the activation domain includes one or more activation domains. In certain embodiments, the activation domain includes CD3ζ, CD3ε, or both CD3ζ and CD3ε. In some examples, the NKG2D CAR is configured according to the PBETA below. In some examples, the NKG2D CAR is configured according to the BETCA below. In some examples, the NKG2D CAR is configured according to the PBETCA below. In some examples, the NKG2D CAR is configured according to the BETC C' A below. In some examples, the NKG2D CAR is configured according to the PBETC C' A below.

[0147] In certain embodiments, the CARs contemplated herein may include linker residues added for proper spacing conformation of the molecule between various domains. The CARs contemplated herein may include one, two, three, four, or five or more linkers. In some embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linkers have an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more.

[0148] Examples of linkers include glycine polymer (G)n and glycine-serine polymer (G 1~5 S 1~5Examples include )n (wherein n is an integer of at least 1, 2, 3, 4, or 5), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between domains of fusion proteins such as CARs described herein. Glycine has even more access to the phi-psy space than alanine and is far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Other linkers considered herein include the Whitlow linker (see Whitlow, Protein Eng. 6(8):989-95 (1993)). Those skilled in the art will recognize that the design of a CAR in some embodiments may include a fully or partially flexible linker, thereby the linker may include a flexible linker and one or more parts that impart a less flexible structure to provide a desired CAR structure. In one embodiment, any of the constructs described herein may include a "GS" linker. In another embodiment, any of the constructs described herein may include a "GSG" linker. In one example, the glycine-serine linker includes or comprises the amino acid sequence GS (SEQ ID NO: 6), which may be encoded by the nucleic acid sequence ggatcc (SEQ ID NO: 7) or gggtcc (SEQ ID NO: 8). In one example, the glycine-serine linker includes or comprises the amino acid sequence GGGSGGGS (SEQ ID NO: 9), which may be encoded by the nucleic acid sequence gggggtggaagcggaggaggttcc (SEQ ID NO: 10). In another embodiment, the CAR described herein includes an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 11 (GSTSGSGKPGSGEGSTKG(SEQ ID NO: 11) (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).In one embodiment, the linker is encoded by a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence described below (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%): gggagcactagcggctctggcaaacctggatctggcgagggatct. accaagggc (SEQ ID NO: 12), gggagcacaagcggctctggcaaacctggatctggcgagggatctaccaagggc (SEQ ID NO: 13), or gggagcacaagcggctctggcaaacctggatccggcgagggatctaccaagggc (SEQ ID NO: 14).

[0149] The binding domain of a CAR can generally be followed by one or more "hinge domains," which play a role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain. The hinge domains can originate from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions.

[0150] In some embodiments, the CARs described herein may include a hinge that is an immunoglobulin-like hinge domain, derived from an immunoglobulin-like hinge domain, or derived from an immunoglobulin-like hinge domain (e.g., including all or fragments of an immunoglobulin-like hinge domain). In some embodiments, the hinge domain is from or derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinges or fragments thereof of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM. The hinges may be derived from natural or synthetic sources. Hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may be or may be modified wild-type hinge regions of these molecules. The hinges may be derived from natural or synthetic sources. In some embodiments, the antigen-binding system of the present disclosure is CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8α, CD8β, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD6 6b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex-associated alpha chain), CD79B (B cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1),CD158D(KIRDL4), CD158F1(KIR2DL5A), CD158F2(KIR2DL5B), CD158K(KIR3DL2), CD160(BY55), CD162(SELPLG), CD226(DNAM1), CD229(SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), C D319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), Inducible T-cell Costimulatory Factor (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R Beta, IL-2R Gamma, IL-7R Alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP, -76(LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, or Toll ligand receptor, or hinges that are fragments or combinations thereof, or are derived from or are derived from them (e.g., including all or fragments thereof).

[0151] The polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, the polynucleotide encoding the hinge domain contains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identical nucleotide sequences to known nucleotide sequences. In some embodiments, the polypeptide sequence of the hinge domain contains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the same polypeptide sequence as a known polypeptide sequence.

[0152] In embodiments, the hinge domain includes a CD8α hinge region. In embodiments, the CAR described herein includes a CD8α-derived hinge domain having an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 15TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 15) (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In embodiments, the hinge domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ACCACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 16). In embodiments, the hinge domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ACAACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGAGGCCTGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCTTGTGAC (Sequence ID 17).In embodiments, the hinge domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ACCACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGCGCCCCGAGGCGTGCCGGCCAGCGGCG. GGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 18). In embodiments, the hinge domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ACCACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGAGGCCTGAAGCGTGCCGGCCAGCGGCGGGCGGCGCAGTGCACACGAGAGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 56).

[0153] In embodiments, the hinge domain includes a cleaved CD28 hinge region (CD28T) hinge region, as disclosed in International Application PCT / US2017 / 025351, filed on March 31, 2017 (which is incorporated herein by reference in its entirety). In embodiments, the CAR described herein includes a CD28T hinge domain having an amino acid sequence having at least 75% sequence identity with respect to Sequence ID No. 230 with respect to Sequence ID No. 19 (LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(Sequence ID No. 19)) (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In this embodiment, the CD28T hinge domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the following sequence: CTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (Sequence ID 20).

[0154] Generally, a “transmembrane domain” (e.g., in an antigen-binding system) refers to a domain that, when present in a molecule on the cell surface or cell membrane, has the attribute of being intramembrane (e.g., spanning part or all of the cell membrane). The costimulatory domains of antigen-binding systems of this disclosure may further include transmembrane domains and / or intracellular signaling domains. Not all amino acids in a transmembrane domain need to be intramembrane. For example, in some embodiments, a transmembrane domain is characterized by a designated sequence or portion of a protein being substantially intramembrane. The amino acid sequence or nucleic acid sequence can be analyzed using various algorithms for predicting the intracellular localization (e.g., transmembrane localization) of a protein. The programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0155] The type of transmembrane domain included in the antigen-binding system described herein is not limited to any particular type. In some embodiments, a transmembrane domain that naturally associates with the binding domain and / or intracellular domain is selected. In some examples, the transmembrane domain includes one or more amino acid modifications (e.g., deletions, insertions, and / or substitutions) to minimize interaction with other members of the receptor complex, for example, by avoiding the binding of such domain to the transmembrane domain of the same or different surface membrane protein.

[0156] Transmembrane domains may originate from either natural or synthetic sources. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Exemplary transmembrane domains include the alpha, beta, or zeta chains of T cell receptors, 2B4, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, and C. D7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, C D276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DAP-12, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITG A4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligands that bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), P It may be derived from SGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1;CD150;IPO-3), SLAMF4 (CD244;2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof (for example, it may include at least a transmembrane domain).In some embodiments, the transmembrane domain may be synthetic (and may include primarily hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is contained at both ends of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked to or attached to the cytoplasmic domain. In some embodiments, a short oligo or polypeptide linker (e.g., 2-10 amino acid length) may form a link between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.

[0157] The polynucleotide and polypeptide sequences of transmembrane domains provided herein are known. In some embodiments, the polynucleotide encoding the transmembrane domain contains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the same nucleotide sequence as known nucleotide sequences. In some embodiments, the polypeptide sequence of the transmembrane domain contains at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) the same polypeptide sequence as a known polypeptide sequence. Optionally, short spacers may form links between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0158] In the embodiment, the NKG2D CAR described herein is sequence number 21 (FWVLVV It contains a CD28-derived TM domain having an amino acid sequence that has at least 75% sequence identity (at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to VGGVLACYSLLVTVAFIIFWV (Sequence ID 21). In embodiments, the TM domain derived from CD28 is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 22). In embodiments, the TM domain derived from CD28 is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: TTTTGGGTATTGGTAGTAGTGGGCGGAGTCCTGGCTTGCTATAGTCTGCTAGTAACAGTGGCTTTTATTATATTTTGGGTG (SEQ ID NO: 23). In embodiments, the TM domain derived from CD28 is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (Sequence ID 24).

[0159] In embodiments, the CAR described herein includes a CD8α-derived TM domain having an amino acid sequence that has at least 75% sequence identity with respect to SEQ ID NO: 25 (IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO: 25)) (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In embodiments, the TM domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTATTGC (SEQ ID NO: 26). In embodiments, the TM domain derived from CD8α is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 57).

[0160] Intracellular signaling domains capable of transmitting signals upon antigen binding to immune cells are known, and any of them may be included in the antigen-binding systems of this disclosure. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signaling after TCR binding to an antigen (e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0161] In some embodiments, the CARs contemplated herein include an intracellular signaling domain. “Intracellular signaling domain” refers to a portion of the CAR that transmits effective CAR messages binding to target antigens into the interior of immune effector cells, inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors to CAR-bound target cells or other cellular responses induced by antigen binding to the extracellular CAR domain. In some embodiments, the signaling domain and / or activation domain includes an immune receptor tyrosine-based activation domain (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010), Smith-Garvin). See et al., Annu. Rev. Immunol. 27:591-619 (2009). In certain embodiments, preferred signaling domains include, but are not limited to, 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276(B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96(Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT. AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligands that bind to CD83, LIGHT, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA- 1. Includes CD1-1a / CD18), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavage, or combinations thereof.

[0162] The term "effector function" refers to a specific function of a cell. The effector function of a T cell may include, for example, assisting or activating cytolytic activity or cytokine secretion. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transmits effector function signals and directs the cell to perform its specific function. While the entire intracellular signaling domain may be used, it is often not necessary to use the entire domain. Insofar as a cleavage portion of an intracellular signaling domain is used, such a cleavage portion may be used in place of the entire domain, provided it translates the effector function signal. The term "intracellular signaling domain" means including any cleavage portion of an intracellular signaling domain sufficient to transmit effector function signals.

[0163] It is known that signals generated solely via the TCR are insufficient for complete T cell activation, and that secondary or co-stimulatory signals may also be required. Therefore, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains (e.g., the TCR / CD3 complex) that initiate primary activation via the TCR in an antigen-dependent manner, and co-stimulatory signaling domains that act antigen-independently to provide secondary or co-stimulatory signals. In some embodiments, the CARs contemplated herein include an intracellular signaling domain comprising one or more "co-stimulatory signaling domains" and "primary signaling domains."

[0164] Exemplary examples of ITAMs containing primary signaling domains useful in this disclosure include those derived from TCRζ, FcRγ, FcRβ, DAP12, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order. In one embodiment, the CAR comprises a CD3ζ domain having an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 27 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 27).In this embodiment, the CD3ζ domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below:CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGGCAGAACCAACTCTATAACGAGCTCAATCTAGGAAGGAGA GAAGAGTACGATGTTCTAGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCACGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTACAAAAAGATAAAATGGCGGAGGCCTACAGT GAGATTGGCATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGCCTCAGTACAGCCACCAAGGACACCTATGACGCCCTTCACATGCAAGCTCTGCCCCCTCGC (SEQ ID NO: 28).In this embodiment, the CD3ζ domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGCATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 29).

[0165] In the embodiment, the CD3ζ domain is used for nucleic acids having the sequence described below, at least It is also encoded by nucleic acids having 75% sequence identity (at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%): CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGGCGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGA TGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 30). In this embodiment, the CD3ζ domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAAGCTCTGCCCCCTCGCTGA (SEQ ID NO: 58).In this embodiment, the CD3ζ domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: CTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGA GGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGA GATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGA (SEQ ID NO: 59).In this embodiment, the CD3ζ domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGCTCTATAACGAGCTCAA TCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG. CAAGCTCTGCCCCCTCGCTGA (Sequence No. 60).

[0166] In some embodiments, the CAR comprises a CD3ζ signaling domain, a CD3ε signaling domain, and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order. In embodiments, the CAR has a CD3ε domain having an amino acid sequence having at least 75% sequence identity with that of SEQ ID NO: 31 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGL (SEQ ID NO: 31). In this embodiment, the CAR has a CD3ε domain having an amino acid sequence that has at least 75% sequence identity with respect to SEQ ID NO: __ (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 61).

[0167] In this embodiment, the CD3ε domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AAGAACCGAAAAGCAAAAGCCAAGCCTGTTACAAGAGGAGCAGGGGCAGGAGGCCGACAGAGAGGGCAAAACAAAGAAAGGCCCCCGCCCGTCCCAAACCCGGATTATGAGCCAATTAGGAAGGGTCAGAGAGACCTGTATTCTGGGCTC (Sequence ID 32). In embodiments, the CD3ε domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AAGAACCGCAAAGCAAAGGCAAAACCCGTCACACGAGGAGCGGGCGCAGGGGGACGACAACGCGGTCAGAATAAGGAACGCCCGCCTCCAGTACCAAATCCAGATTATGAACCAATTCGGAAGGGACAACGCGATCTCTACTCCGGTCTCAATCAGAGGCGAATT (SEQ ID NO: 62).

[0168] The CARs contemplated herein comprise one or more costimulatory signaling domains for enhancing the efficacy and proliferation of T cells expressing CAR receptors. As used herein, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the intracellular signaling domain of a costimulatory molecule. In some embodiments, the costimulatory molecules are DAP-10, DAP-12, CD27, CD28, CD137(4-IBB), OX40(CD134), CD30, CD40, PD-I, ICOS(CD278), CTLA4, LFA-1, CD2, CD7, LIGHT, TRIM, LCK3, SLAM, DAPIO, LAG3, HVEM, B7-H3, NKD2C, GITR, CD5, ICAM-I, CDI This may include la, Lck, TNFR-I, TNFR-II, FasR, NKG2C, B7-H3, and CD83.

[0169] In this embodiment, the CAR has at least 75% sequence identity to sequence number 33 (for example, 85-90%, 85-95%, 85-100%, 90-95%, etc.). It includes a 4-1BB co-stimulatory domain having an amino acid sequence having 90-100% or 95-100% sequence identity. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE (SEQ ID NO: 33). In embodiments, CAR has a 4-1BB co-stimulatory domain having an amino acid sequence having at least 75% sequence identity to SEQ ID NO: __ (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 63). In this embodiment, the 4-IBB costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AAACGAGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACAACTCAGGAGGAGGATGGCTGTAGCTGCCGATTCCCGGAAGAAGAAGAAGGTGGCTGTGAA (SEQ ID NO: 34). In this embodiment, the 4-IBB costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA (Sequence ID 35).In this embodiment, the 4-IBB costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA (Sequence ID 36). In this embodiment, the 4-IBB costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AAGAGAGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTG (SEQ ID NO: 64).

[0170] In this embodiment, the CAR includes a CD28 costimulatory domain containing an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 37 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 37). In embodiments, the CD28 costimulatory domain is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: AGGAGTAAGAGGAG CAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 38).

[0171] The manipulated NKG2D CAR described herein may also contain an N-terminal signal peptide or tag at the N-terminus of the NKG2D ectodomain. In one embodiment, a heterologous signal peptide may be used. The antigen-binding domain may be fused to a leader or signal peptide that guides the nascent protein into the endoplasmic reticulum and subsequently to the cell surface. When a polypeptide containing a signal peptide is expressed on the cell surface, it is understood that the signal peptide is generally proteolytically removed during the processing of the polypeptide in the endoplasmic reticulum and its transition to the cell surface. Therefore, polypeptides such as the CAR constructs described herein are generally expressed on the cell surface as mature proteins lacking a signal peptide, although the precursor form of the polypeptide contains a signal peptide. Any suitable signal sequence known in the art may be used. Similarly, any known tag sequence known in the art may also be used.

[0172] In embodiments, the signal sequence is a CD8α signal sequence. In embodiments, the NKG2D CAR described herein includes a CD8α signal sequence having an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 39 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%); MALPVTALLLPLALLLHAARP (SEQ ID NO: 39). In this embodiment, the CD8α signal sequence is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ATGGCTCTTCCTGTGACTGCACTACTGCTGCCCCTGGCCTTACTTCTTCATGCTGCGCGTCCT (SEQ ID NO: 40). In this embodiment, the CD8α signal sequence is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCT (SEQ ID NO: 65).

[0173] In one embodiment, the signal sequence is a CSF2RA signal sequence. In an embodiment, the NKG2D CAR described herein includes a CSF2RA signal sequence having an amino acid sequence that has at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) with respect to MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 41), MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 42), or MALPVTALLLPLALLLHAARP (SEQ ID NO: 43). In this embodiment, the CSF2RA signal sequence is encoded by a nucleic acid having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the nucleic acid having the sequence described below: ATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCT (SEQ ID NO: 44).

[0174] The components of a CAR may be replaced or “swapped” with equivalent components using routine biotechnology techniques. In some non-limiting and partial examples, the CARs of this disclosure may include a binding domain provided herein in combination with the hinge and co-stimulatory domains provided herein. In certain examples, the CARs of this disclosure may include a leader sequence provided herein, together with the binding domain provided herein, in combination with the hinge and co-stimulatory domains provided herein.

[0175] In one embodiment described herein, the NKG2D CAR construct has an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 45 (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 45). In this embodiment, the NKG2D CAR-bound CAR has at least 75% sequence identity to nucleic acids having the sequences described below (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%).Encoded by a nucleic acid having: or 95-100%): ATGGCTCTTCCTGTGACTGCACTACTGCTGCCCCTGGCCTTACTTCTTCATGCTGCGCGTCCTTTATTCAACCAAGAAGTCCAGATTCCCTTGACCGAAAGTTACTGCGGCCCATGTCCGAAAAACTGGATATGTTATAAAAATAACTGTTACCAGTTCTTCGATGAATCTAAAAACTGGTATGAGAGCCAGGCATCTTGTATGTCTCAAAATGCCAGCCTGCTCAAAGTATACAGCAAGGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCACTGGATGGGATTGGTACACATTCCCACAAATGGATCTTGGCAGTGGGAAGACGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGCGCACTCTATGCATCGAGCTTTAAAGGTTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTAACAACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGAGGCCTGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCTTGTGACTTTTGGGTATTGGTAGTAGTGGGCGGAGTCCTGGCTTGCTATAGTCTGCTAGTAACAGTGGCTTTTATTATATTTTGGGTGAAACGAGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACAACTCAGGAGGAGGATGGCTGTAGCTGCCGATTCCCGGAAGAAGAAGAAGGTGGCTGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGGCAGAACCAACTCTATAACGAGCTCAATCTAGGAAGGAGAGAAGAGTA, CGATGTTCTAGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCACGAAGGAAACCCTCAGGAAGGGCCTGTACAACGAACTACAAAAAGATAAAATGGCGGAGGCCTACAGTGAGATTGGCATGAAAGGCGAGCGCGGAGGGGCAAGGGCCACGATGGCCTTTACCAGGGCCTCAGTACAGCCACCAGGACACCTATGACGCCCTTCACATGCAAGCTCTGCCCCTCGC (SEQ ID NO: 46).

[0176] AAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGCCTCAGTACAGCCACCAAGGACACCTATGACGCCCTTCACATGCAAGCTCTGCCCCCTCGC (SEQ ID NO: 48).

[0177] GGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAAGGAAGAACCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGCATGAAAGGCGAGCGCCGGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCAGGACACCCTACGACGCCCTTCACATGCAGGCCCTGCCCCTCGC (SEQ ID NO: 50).

[0178] In the embodiment, the NKG2D CAR construct has an amino acid sequence having at least 75% sequence identity with SEQ ID NO: 51 (for example, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRG RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 51). In embodiments, the NKG2D CAR-bound CAR has at least 75% sequence identity to nucleic acids having the sequences described below (for example, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%).Encoded by a nucleic acid having: TTATTCAACCAAGAAGTCCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGTTACCAATTCTTCGATGAAAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAGGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGACGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCATCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATACATCTGCATGCAAAGGACTGTGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAAAAGAACCGAAAAGCAAAAGCCAAGCCTGTTACAAGAGGAGCAGGGGCAGGAGGCCGACAGAGAGGGCAAAACAAAGAAAGGCCCCCGCCCGTCCCAAACCCGGATTATGAGCCAATTAGGAAGGGTCAGAGAGACCTGTATTCTGGGCTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACG, or 95 - 100% CCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTG CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGCATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 52).

[0179] This disclosure envisions the use of the NKG2D CAR described herein in conjunction with engineered T cell receptors (TCRs) used in T cell immunotherapy. A library of TCRs may be screened for their selectivity for target antigens. In this way, native TCRs with high binding affinity and reactivity to target antigens may be selected, cloned, and subsequently introduced into a T cell population used in adoptive immunotherapy. T cells or NK cells having engineered TCRs that also express the NKG2D CAR described herein can target specific antigens not only due to TCR specificity, but also cells expressing the NKG2D ligand. Therefore, combining the NKG2D CAR described herein with TCRs may provide a method for maintaining or enhancing the therapeutic effect of adoptive T cell or NK cell immunotherapy. In embodiments, the NKG2D CAR described herein is co-expressed with a TCR.

[0180] In one embodiment described herein, T cells or NK cells are modified by introducing a polynucleotide encoding a TCR subunit capable of forming a TCR that confers specificity to the T cells or NK cells against tumor cells expressing a target antigen and / or NKG2D ligand. In some embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to a naturally occurring subunit, insofar as the subunit retains the ability to form a TCR conferred to transfected T cells and NK cells, the ability to home to target cells, and is involved in immunologically relevant cytokine signaling. The engineered TCR may also bind to target cells that express relevant tumor-associated peptides with high binding activity and optionally mediate the efficient death of target cells that present relevant peptides in vivo.

[0181] The nucleic acids encoding the engineered TCR may be isolated from their natural context on the (naturally occurring) chromosomes of T cells and incorporated into a suitable vector, as described elsewhere herein. Both the nucleic acids and the vectors containing them may be transferred to cells, which may be T cells. The modified T cells can then express one or more strands (and in some embodiments, two strands) of the transduced nucleic acid or the TCR encoded by the nucleic acid. In some embodiments, the engineered TCR is an exogenous TCR, since it is introduced into T cells that do not normally express the transduced TCR. An essential aspect of the engineered TCR is that it has high avidity to tumor antigens presented by the major histocompatibility complex (MHC) or similar immunological components. In contrast to the engineered TCR, a CAR is engineered to bind to a target antigen in an MHC-independent manner.

[0182] The nucleic acid-encoded proteins described herein may be expressed with additional polypeptides attached to the amino-terminus or carboxyl-terminus of the α- or β-chain of the TCR, provided that the attached additional polypeptides do not interfere with the α- or β-chain's ability to form a functional T cell receptor and with MHC-dependent antigen recognition.

[0183] Antigens recognized by the manipulated TCRs intended herein include, but are not limited to, cancer antigens, including those of both hematological cancers, solid tumors, and virus-induced cancers. No. TCR therapy for the treatment of HPV-induced cervical cancer is a promising area of ​​interest. Therefore, the oncolytic proteins HPV-16 E6 and HPV-16 E7 may be potential target antigens for use with TCR (see, for example, International Application PCT / US2015 / 033129). Other exemplary antigens include HPV-16 HPV oncoproteins including E6 and HPV-16 E7, alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family including ErbB2(HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD 3. Examples include, but are not limited to, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, MAGA-A3, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NY-ES0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TACI, TEM, and VEGFRII.

[0184] Combining any TCR construct described herein with the NKG2D CAR of this disclosure may restore, maintain, or enhance the therapeutic effect of TCR therapy. Accordingly, in one embodiment described herein, the NKG2D CAR is co-expressed in T cells or NK cells with a TCR directed to HPV. In another embodiment described herein, the NKG2D CAR is co-expressed in T cells or NK cells with a TCR directed to the HPV-16 E6 protein. In yet another embodiment described herein, the NKG2D CAR is co-expressed in T cells or NK cells with a TCR directed to the HPV-16 E7 protein.

[0185] T cells or NK cells may also be genetically engineered with vectors designed to express a second CAR (in addition to the NKG2D CAR) that redirects cytotoxicity to tumor cells. In some embodiments, the CAR is a molecule that combines antibody-based specificity against a target antigen (e.g., a tumor antigen) with an activated intracellular domain to produce a chimeric protein exhibiting specific anti-tumor cell immune activity. This disclosure intends to use the NKG2D CAR described herein with one or more additional CARs. Similar to the use of TCRs, co-expression of the NKG2D CAR with one or more additional CARs may facilitate, enhance, protect, and, in some cases, restore the expansion of CAR therapy. In embodiments, the NKG2D CAR is co-expressed with one or more additional CARs.

[0186] One or more additional CARs contemplated herein include an extracellular domain, a transmembrane domain, and an intracellular signaling domain that bind to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain). A key feature of one or more additional CARs is their ability to redirect immunoeffector cell specificity, thereby triggering the production of molecules that can mediate cell death of target antigen-expressing cells in a manner independent of proliferation, cytokine production, phagocytosis, or major histocompatibility (MHC), and utilizing the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0187] In some embodiments, one or more additional CARs include extracellular binding domains that specifically bind to a target antigen, including, but not limited to, an antibody or its antigen-binding fragment, a tether ligand, or the extracellular domain of a coreceptor. In non-limiting examples, target antigens may include: HPV tumors, including HPV-16 E6 and HPV-16 E7. Sex proteins, alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE 1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-IIRα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEM, and VEGFRII; In the embodiments described herein, CAR binds to tumor antigens including BCMA, CLL-1, CD19, CD20, CD22, CD28, CD137(4-1BB), Glypican-3 (GPC3), PSCA, PSMA, or TACI.

[0188] In some embodiments, one or more additional CARs contemplated herein include an extracellular binding domain that specifically binds to a target polypeptide expressed on tumor cells, such as a target antigen. As used herein, the terms “binding domain,” “extracellular domain,” “extracellular binding domain,” “antigen-specific binding domain,” “antigen-binding domain,” and “extracellular antigen-specific binding domain” are interchangeable and provide a CAR having the ability to specifically bind to a target antigen of interest. The binding domain may include any protein, polypeptide, oligopeptide, or peptide having the ability to specifically recognize and bind to a biological molecule (e.g., cell surface receptors or tumor proteins, lipids, polysaccharides, or other cell surface target molecules or components thereof). The binding domain includes a binding partner for the biological molecule of interest, which may be naturally occurring, synthetically, semi-synthetically, or recombinantly produced.

[0189] In some embodiments, one or more additional extracellular binding domains of the CAR include its antibody or antigen-binding fragment. “Antibody” refers to a binder which is a polypeptide containing at least a light-chain or heavy-chain immunoglobulin variable region that specifically recognizes and binds to an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by immune cells. The antibody includes its antigen-binding fragment. This term also includes genetically modified forms such as chimeric antibodies (e.g., humanized mouse antibodies), hetero-conjugate antibodies (e.g., bispecific antibodies), and their antigen-binding fragments. See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0190] In some embodiments, the target antigen is the following epitopes: HPV oncoproteins including HPV-16 E6 and HPV-16 E7, alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137(4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family including ErbB2(HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, F AP, fetal AchR, FRa, GD2, GD3, glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0 -1, HLA-A3+NY-ES0-1, IL-llRα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, TACI, and VEGFRII polypeptide. In one embodiment described herein, CAR binds to a tumor antigen epitope comprising BCMA, CLL-1, CD19, CD20, CD22, CD28, CD137 (4-1BB), Glypican-3 (GPC3), PSCA, PSMA, or TACI.

[0191] In certain embodiments, one or more additional CARs contemplated herein may include linker residues added between various domains, for example, between the VH domain and the VL domain, for proper interspersed conformation of the molecule. The CARs contemplated herein may include one, two, three, four, or five or more linkers. In some embodiments, the length of the linker is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker has an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more.

[0192] Exemplary examples of linkers include glycine polymers (G)n, glycine-serine polymers (G1~5S1~5)n (wherein n is an integer of at least 1, 2, 3, 4, or 5), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between domains of fusion proteins such as CARs described herein. Glycine has even more access to the phi-psy space than alanine and is far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Other linkers contemplated herein include the Whitlow linker (see Whitlow, Protein Eng. 6(8):989-95 (1993)). Those skilled in the art will recognize that the design of a CAR in some embodiments may include a fully or partially flexible linker, thereby the linker may include a flexible linker and one or more parts that impart a less flexible structure to provide a desired CAR structure. In one embodiment, any of the constructs described herein may include a "GS" linker. In another embodiment, any of the constructs described herein may include a "GSG" linker. In another embodiment, the CAR described herein includes an amino acid sequence having at least 75% sequence identity with respect to SEQ ID NO: 40 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). GSTSGSGKPGSGEGSTKG (SEQ ID NO: 40).

[0193] In other embodiments, the CAR further comprises an scFv containing a variable region linking sequence. The "variable region linking sequence" is an amino acid sequence that links the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two subbinding domains so that the resulting polypeptide maintains specific binding affinity to the same target molecule as an antibody containing the same light chain and heavy chain variable regions. In one embodiment, the variable region linking sequence has an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more.

[0194] In other embodiments, the CAR binding domain is followed by one or more "spacer domains" which move the antigen-binding domain away from the effector cell surface. This refers to a region that enables appropriate cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains may originate from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, a spacer domain is part of an immunoglobulin, which includes, but is not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. Spacer domains may include amino acid sequences of naturally occurring or modified immunoglobulin hinge regions.

[0195] The binding domain of a CAR can generally be followed by one or more "hinge domains," which play a role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain. The hinge domains can originate from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions.

[0196] If the expression of two or more polypeptides is desired, the encoding polynucleotide sequences may be separated by an IRES sequence. In another embodiment, the two or more polypeptides may be expressed as a fusion protein containing one or more self-cleaving polypeptide sequences, such as a T2A polypeptide. In yet another embodiment, they may be expressed from different promoters and present in two or more vectors. In some embodiments, the NKG2D CAR is encoded in the same vector as the TCR and / or one or more non-NKG2D CARs and is operably linked to the same promoter, with the sequence separated by an IRES sequence. In some embodiments, the NKG2D CAR is encoded in the same vector as the TCR and / or one or more non-NKG2D CARs and is operably linked to the same promoter, with the sequence separated by a cleavable linker. In some embodiments, the NKG2D CAR is encoded in the same vector as the TCR and / or one or more non-NKG2D CARs, and the NKG2D CAR is operably linked to a different promoter than the TCR and / or one or more non-NKG2D CARs. In some embodiments, the NKG2D CAR is encoded in a vector different from the TCR and / or one or more non-NKG2D CARs.

[0197] "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used according to their conventional meanings, i.e., as sequences of amino acids, unless otherwise specified. A polypeptide is not limited to a specific length and may include, for example, a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, and other modifications known in the art, both spontaneously and unspontaneously. In various embodiments, the polypeptides contemplated herein include a signal (or leader) sequence at the N-terminus of the protein, which directs the movement of the protein during or after translation.

[0198] Polypeptides include “polypeptide variants.” Polypeptide variants may differ from naturally occurring polypeptides in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the polypeptide sequences described above. For example, in some embodiments, it may be desirable to improve the binding affinity and / or other biological properties of the manipulated NKG2D CAR by introducing one or more substitutions, deletions, additions, and / or insertions. Preferably, the polypeptides of the Disclosure comprise polypeptides having at least about 50%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity thereto. The variants include those having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the reference sequences described herein (see, for example, the sequence listing), and typically the variants maintain at least one biological activity of the reference sequence. Polypeptides include "polypeptide fragments." A polypeptide fragment refers to a polypeptide that may be a monomer or polymer having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment may include an amino acid chain of at least 5 to about 500 amino acids in length. In certain embodiments, it will be understood that the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long.

[0199] Polypeptides can also be in-frame fused or conjugated to linkers or other sequences to facilitate the synthesis, purification, or identification of polypeptides (e.g., poly-His) or to enhance the binding of polypeptides to solid supports. As described above, the polypeptides of this disclosure can be modified in a variety of ways, including amino acid substitution, deletion, cleavage, and insertion. Methods for such operations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in DNA. Methods for mutagenesis and alteration of nucleotide sequences are well known in the art. For example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods (in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J. Det. al., (Molecular Biology of See the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. For guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein, see Dayhoff et al., (1978) Atlas This can be found in the model of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0200] In certain embodiments, the variant includes a conservative substitution. A “conservative substitution” is one in which an amino acid is substituted for another amino acid having similar properties, such that those skilled in peptide chemistry would expect the secondary structure and hydroxyl properties of the polypeptide to remain substantially unchanged. Modifications can be made to the structures of the polynucleotides and polypeptides of this disclosure, and functional molecules encoding variants or derivative polypeptides with desirable properties can still be obtained.

[0201] Polypeptide variants further include glycosylated forms, agglomerative conjugates with other molecules, and covalent conjugates having unrelated chemical moieties (e.g., pegylated molecules). Covalent variants can be prepared by linking functional groups found in amino acid chains or N- or C-terminal residues, as is known in the art. Variants also include allelic variants, species variants, and mutains. Cleavage or deletion of regions that do not affect the functional activity of a protein are also variants.

[0202] The polypeptides of this disclosure include fusion polypeptides. In some embodiments, fusion polypeptides Polynucleotides encoding lipeptides and fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. Fusion polypeptides are typically C-terminus to N-terminus, but they can also be C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein may be in any order or a specific order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired transcriptional activity of the fusion polypeptide is preserved. Fusion polypeptides may be produced by chemical synthesis or by chemical bonding between two parts, and may generally be prepared using other common techniques. The linked DNA sequence containing the fusion polypeptide is operably linked to a suitable transcriptional or translational regulatory element, as discussed elsewhere in this specification.

[0203] In one embodiment, the fusion partner includes a sequence that helps express the protein (expression enhancer) in a higher yield than the naturally occurring recombinant protein. Other fusion partners may be selected to increase the solubility of the protein, or to enable the protein to be targeted to a desired intracellular compartment, or to facilitate the transport of the fusion protein across the cell membrane.

[0204] The fusion polypeptide may further contain polypeptide cleavage signals between each of the polypeptide domains described herein. Furthermore, the polypeptide moieties may be inserted into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, e.g., protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (deFelipe and Ryan, 2004. Traffic, 5(8); 616-26). Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J Gener. Viral. 78:699-722, and Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, those of potivirus Nia protease (e.g., tobacco ecchi virus protease), potivirus HC protease, potivirus Pl(P35) protease, biovirus Nia protease, biovirus RNA-2-coding protease, aftovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picoma 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungrovirus) 3C-like protease, PYVF (parsnip yellow spot virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, the TEV (tobacco etch virus) protease cleavage site may be used. In other embodiments, the self-cleaving peptide may include polypeptide sequences obtained from pochivirus and cardiovirus 2A peptide, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine tesshou virus.In other embodiments, the self-cleaving polypeptide site includes 2A or a similar site, sequence, or domain of 2A (Donnelly et al., 2001. J Gen.Viral. 82:1027-1041).

[0205] In general, it is understood that any suitable viral vector or vector may be used for transduction of the manipulated constructs described herein. In one embodiment described herein, cells (e.g., T cells, NK cells, or iPSCs) are transduction of a retroviral vector, e.g. The organism is transduced using a lentiviral vector. As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Exemplary retroviruses suitable for use in some embodiments include, but are not limited to, Moloney murine leukemia virus (M- MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine mammary tumor virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (feline leukemia virus) Viruses (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentiviruses.

[0206] As used herein, the term “lentivirus” refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV types 1 and HIV type 2), visna-maedi virus (VMV), caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0207] The term “vector” is used herein to refer to a nucleic acid molecule capable of moving or transporting another nucleic acid molecule. The transferred nucleic acid is generally ligated to the vector nucleic acid molecule, for example, by insertion into the vector nucleic acid molecule. A vector may contain a sequence that directs autonomous replication within a cell, or a sequence sufficient to enable integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses.

[0208] As will be apparent to those skilled in the art, the term “viral vector” is widely used to refer to either a nucleic acid molecule (e.g., a transfer plasmid) containing a virus-derived nucleic acid element that facilitates the movement or integration of nucleic acid molecules into a cell’s genome, or a viral particle that mediates nucleic acid movement. Viral particles typically contain various viral components and sometimes host cell components in addition to nucleic acids.

[0209] The term "viral vector" can refer to either a virus or viral particle capable of transferring nucleic acids to cells, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or a portion thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or a portion thereof, primarily derived from lentiviruses, including LTRs. The term "hybrid vector" refers to a vector, LTR, or other nucleic acid containing both retroviruses, e.g., lentiviruses, sequences, and non-retroviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid containing retroviruses, e.g., lentiviruses, sequences for reverse transcription, replication, integration, and / or packaging.

[0210] In some embodiments, the terms “lentiviral vector” and “lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. Where elements such as cloning sites, promoters, regulatory elements, and heterologous nucleic acids are referred to herein, it should be understood that the sequences of these elements are present in RNA form in the lentiviral particles of this disclosure and in DNA form in the DNA plasmids of this disclosure. In one embodiment described herein, the expression vector is a lentiviral expression vector.

[0211] Proviruses have structures at both ends called "long terminal repeats" or "LTRs." The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the ends of retroviral DNA, which, in their natural sequence configuration, are direct repeats and contain U3, R, and U5 regions. LTRs generally provide fundamental functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain numerous regulatory signals, including transcriptional regulatory elements, polyadenylation signals, and sequences necessary for replication and integration of the viral genome. Viral LTRs are divided into three regions called U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence. The R (repeat) region is adjacent to the U3 and U5 regions. LTRs consist of the U3, R, and U5 regions and appear at both the 5' and 3' ends of the viral genome. The sequences adjacent to the 5'LTR are necessary for reverse transcription of the genome (tRNA primer binding sites) and for efficient packaging of viral RNA into particles (Psi sites).

[0212] As used herein, the terms “packaging signal” or “packaging sequence” refer to a sequence located within the retroviral genome necessary for the insertion of viral RNA into the viral capsid or particle; see, for example, Clever et al., 1995. J of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors utilize a minimal packaging signal (also referred to as the psi['P] sequence) necessary for capsid formation of the viral genome. Therefore, as used herein, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “'P” are used in relation to non-coding sequences necessary for capsid formation of the retroviral RNA strand during viral particle formation.

[0213] In various embodiments, the vector comprises a modified 5'LTR and / or 3'LTR. Either or both LTRs may contain one or more modifications, including but not limited to one or more deletions, insertions, or substitutions. Modifications to the 3'LTR are often made to improve the safety of lentiviral or retroviral systems by making the virus's replication defective. As used herein, the term “defective replication” refers to a virus that cannot replicate completely and effectively so as not to produce infectious virions (e.g., a defective replication lentiviral offspring). The term “replicable” refers to a wild-type or mutant virus that is capable of replication so as to enable the viral replication of the virus to produce infectious virions (e.g., a replicable lentiviral offspring).

[0214] Self-inactivating (SIN) vectors are known as the U3 region. This refers to a replication-defective vector, such as a retroviral or lentiviral vector, in which a certain right (3')LTR enhancer-promoter region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This means that during viral replication, the right (3')LTR U3 region is used as a template for the left (5')LTR U3 region, and therefore, without the U3 enhancer-promoter, viral transcription would not occur. This is because a copy cannot be made. In further embodiments of the present disclosure, the 3'LTR is modified so that the U5 region is replaced, for example, with an ideal poly(A) sequence. It should be noted that modifications to the LTR, such as modifications to the 3'LTR, 5'LTR, or both the 3' and 5'LTR, are also contemplated herein.

[0215] Safety is further enhanced by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. An example of a heterologous promoter that can be used is, for example, that used in viral Simian virus 40. ,SV40) (for example, early or late stage), cytomegalovirus (C) Examples include MV (e.g., immediate early stage), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical examples include Heterogeneous promoters can drive high levels of transcription in a Tat-independent manner. Because the complete U3 sequence is absent in the viral production system, this substitution reduces the likelihood of recombinant, reproducible viruses being produced. In certain embodiments, heterogeneous promoters offer additional advantages in controlling the manner in which the viral genome is transcribed. For example, a heterogeneous promoter may be inducible so that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more chemical compounds or physiological conditions such as the temperature or pH under which the host cells are cultured.

[0216] In some embodiments, the viral vector includes a TAR element. The term "trans-activation response, TAR" refers to the R region of a lentivirus (e.g., HIV) LTR. This refers to the "trans-activation response" genetic element located in the region. This element interacts with the lentiviral trans-activator (TAT) genetic element, causing multiple viruses to multiply. To increase production.

[0217] The "R region" refers to a region within the retroviral LTR that begins at the start of the capping group (i.e., transcription start) and ends immediately before the start of the polyA tract. The R region is also defined as being adjacent to the U3 and U5 regions. During reverse transcription, the R region plays a role in enabling the movement of nascent DNA from one end of the genome to the other.

[0218] As used herein, the term “FLAP element” refers to nucleic acids whose sequences include the central polypurine tract and central termination sequences (cPPT and CTS) of retroviruses, e.g., HIV-I or HIV-2. Preferred FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. During HIV-I reverse transcription, the central start of the positive-strand DNA in the central polypurine tract (cPPT) and the central termination in the central termination sequence (CTS) result in the formation of a triple-stranded DNA structure: the HIV-I central DNA flap. While we do not wish to be bound by any theory, the DNA flap may act as a cis-activity determinant of lentiviral genome nuclear translocation and / or increase viral titer.

[0219] In one embodiment, a retroviral or lentiviral transfer vector comprises one or more export elements. The term “export element” refers to a cis-acting post-transcriptional regulatory element that modulates the transport of RNA transcripts from the cell nucleus to the cytoplasm. Examples of RNA export elements include the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J Virol. 65:1053 and Cullen et al., 1991. Cell 58:423), and B Examples include, but are not limited to, post-transcriptional regulatory elements (HPREs) of hepatitis B virus. Generally speaking, RNA export requirements The element is located within the 3'UTR of a gene and can be inserted as one or more copies.

[0220] In other embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and optionally, transcription termination signals into the vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE, Zufferey et al., 1999, J Virol., 73:2886), the post-transcriptional regulatory element present (HPRE) in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864), and others (Liu et al., 1995, Genes Dev., 9:1766).

[0221] In some embodiments, the vector may include a regulatory oligonucleotide having transcriptional or translational regulatory activity. Such oligonucleotides can be used in various gene expression constructs to modulate the control of expression. Transcriptional regulatory oligonucleotides can increase (enhance) or decrease (silence) the expression level of recombinant expression constructs. Regulatory oligonucleotides can selectively regulate expression in context-specific ways, including, for example, for constitutive or inducible expression, including tissue-specific, developmental stage-specific, or similar expression of polynucleotides. The regulatory oligonucleotides of this disclosure may also be recombinant nucleic acid molecules, including a component of an expression vector or a regulatory oligonucleotide operably linked to an expressible polynucleotide. The regulatory elements may be of varying lengths, from a few nucleotides to several hundred nucleotides.

[0222] Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In some embodiments, the vector contains a 3' polyadenylation sequence of the polynucleotide encoding the polypeptide to be expressed. As used herein, the terms “polyA site” or “polyA sequence” refer to a DNA sequence that induces both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can enhance mRNA stability by adding a polyA tail to the 3' end of the coding sequence and thus contribute to increased translation efficiency. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking a polyA tail are unstable and rapidly degrade. Exemplary examples of polyA signals that may be used in the vectors of this disclosure include ideal polyA sequences (e.g., AATAAA, ATTAAA, AGTAAA), bovine growth hormone polyA sequences (BGHpA), rabbit β-globin polyA sequences (rβgpA), or other suitable polyA sequences known in the art. Examples include species or endogenous polyA sequences.

[0223] This specification also describes “codon-optimized” nucleic acids. “Codon-optimized” nucleic acids refer to nucleic acid sequences modified so that codons are optimized for expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence may be optimized for expression in mammalian cells or a particular mammalian species (such as human cells) by replacing at least one, two or more, or a significant number of codons in the native sequence with codons that are more frequently or most frequently used in the genes of that species. Codon optimization does not alter the amino acid sequence of the encoding protein.

[0224] Codon-optimized nucleotide sequences can exhibit improved properties related to expression efficacy. In some embodiments, the transcribed DNA sequence is transcribed more efficiently and The DNA sequence may be optimized to / or facilitate translation. In some embodiments, the DNA sequence may be optimized with respect to cis-regulatory elements (e.g., TATA boxes, termination signals, and protein-binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase sliding sites, and / or other elements related to transcription. The DNA sequence may also be optimized with respect to cryptic splice sites, mRNA secondary structure, and mRNA DNA sequences may be optimized with respect to stable free energy, repetitive sequences, RNA instability domains, and / or other factors related to mRNA processing and stability. DNA sequences may also be optimized with respect to codon use bias, codon compatibility, internal chi sites, ribosome binding sites (e.g., IRES), immature poly-A sites, Shine-Dalgarno (SD) sequences, and / or other factors related to translation, and / or DNA sequences may be optimized with respect to codon context, codon-anticodon interactions, translation pause sites, and / or other factors related to protein folding.

[0225] A vector may have one or more LTRs, each LTR comprising one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. A vector may further comprise one or more accessory elements for increasing other factors that increase transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., Psi(Ψ) packaging signal, RRE), and / or therapeutic gene expression (e.g., poly(A) sequence), and may optionally comprise WPRE or HPRE. Those skilled in the art will understand that many other different embodiments can be formed from existing embodiments of this disclosure.

[0226] "Host cells" include cells that have been transfected, infected, or transduced in vivo, ex vivo, or in vitro with the recombinant vector or polynucleotide of the Disclosure. Host cells may include packaging cells, producer cells, and cells infected with the viral vector. In some embodiments, host cells infected with the viral vector of the Disclosure are administered to a subject in need of treatment. In certain embodiments, the term "target cells" is used interchangeably with "host cells" and refers to transfected, infected, or transduced cells of a desired cell type. In some embodiments, target cells are T cells.

[0227] In many cases, large-scale viral particle production is required to achieve a reasonable viral titer. Viral particles are produced by transfecting a transfer vector into a packaging cell line containing viral structural genes and / or accessory genes, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0228] As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and contains polynucleotides encoding one, two, three, four or more viral structures and / or accessory genes. Typically, packaging vectors are contained in packaging cells and introduced into cells via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art. The retroviral / lentiviral transfer vectors of this disclosure may be introduced into packaging cell lines via transfection, transduction, or infection to produce producer cells or cell lines. The packaging vectors of this disclosure may be introduced into human cells or cell lines by common methods including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into cells with a dominant selectable marker such as neomycin, hygromycin, puromycin, blastosidine, zeosin, thymidine kinase, DHFR, Gln synthetase, or ADA, and then selected and isolated clones in the presence of the appropriate drug. Selectable Mark The Kerr gene can be physically bound to the gene it encodes by a packaging vector, for example, by IRES or self-cleaving viral peptides.

[0229] The viral envelope protein (env) determines the range of host cells that can ultimately be infected and transformed by recombinant retroviruses generated from a cell line. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, the env proteins include gp41 and gp120. In some embodiments, the viral env proteins expressed by the packaging cells of this disclosure are encoded on a vector separate from the viral gag and pol genes, as described above.

[0230] Exemplary examples of retroviral env genes that may be used in the embodiments described herein include, but are not limited to, the following: MLV envelope, IOAI envelope, BAEV, FeLV-B, RDI 14, SSAV, Ebola, Sendai, FPV (tripest virus), and influenza virus envelope. Similarly, genes encoding envelopes derived from RNA viruses (e.g., the RNA virus families Picomaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Bimaviridae, and Retroviridae) and DNA viruses (the families Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) can be used. Typical examples include FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BL V, EBV, CAEV, SNV, ChTL V, STLV, MPMV SMRV, RAV, FuSV, MH2, AEV, AMV, CTIO, and EIAV.

[0231] In other embodiments, the envelope protein for pseudotyping the viruses of this disclosure includes, but is not limited to, any of the following viruses: influenza A virus, e.g., H1N1, H1N2, H3N2, and H5N1 (avian influenza); influenza B virus; influenza C virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; hepatitis D virus; hepatitis E virus; rotavirus; any virus of the Norwalk virus group; enterovirus; parvovirus; dengue virus; monkeypox; and the order Mononegavirals. Lyssaviruses, such as rabies virus, Lagos bat virus, Mokola virus, Dubenhaji virus, European bat virus 1 and 2, Australian bat virus, ephemerovirus, vediclovirus, vesicular stomatitis virus (VSV), herpesviruses, such as herpes simplex virus types 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), and human herpesvirus (HHV). ), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, murine gamma herpesvirus, arenavirus, such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridae, such as cri. Mia-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever viruses that cause renal syndrome, Rift Valley fever virus, Filoviridae (filoviruses) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Kaysanur forest disease Viruses, including omsc hemorrhagic fever virus and the virus that causes tick-borne encephalitis, are among the flaviviruses. Virididae (Flaviviridae), Paramyxoviridae (Paramyxoviridae) For example, Hen Doravirus, Nipah virus, smallpox major and minor, alphaviruses, e.g., Venezuelan horse encephalitis virus, Eastern horse encephalitis virus, Western horse encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile A virus, or a virus that causes any type of encephalitis.

[0232] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus in which the viral envelope protein has been replaced with that of another virus having other characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein, whereby HIV can infect a broader range of cells because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4+ presenting cells.

[0233] As used herein, the term "packaging cell line" is used with respect to a cell line that does not contain a packaging signal but stably or transiently expresses viral structural proteins and replication enzymes (e.g., gag, pol, and env) necessary for proper packaging of viral particles. Any suitable cell line can be used to prepare the packaging cells of the present disclosure. Generally, the cells are mammalian cells. In another embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used to generate a packaging cell line include, for example, CHO cells, BHK cells, MDCK cells, C3H 10Tl / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC1 cells, BSC40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HTI080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0234] As used herein, the term "producer cell line" refers to a cell line capable of producing recombinant retroviral particles containing a transcription vector construct that includes a packaging cell line and a packaging signal. The production of infectious virus particles and virus stocks can be carried out using conventional techniques. Methods for preparing virus stocks are known in the art; see, for example, Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N.R. Landau et al. (1992) J Virol. 66:5110-5113. Infectious virus particles can be collected from packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis or collection of the supernatant of cell cultures, as is known in the art. Optionally, the collected virus particles may be purified. Suitable purification techniques are well known to those skilled in the art.

[0235] Delivery of a gene or other polynucleotide sequence using a retroviral or lentiviral vector by viral infection rather than transfection is referred to as "transduction." In one embodiment, a retroviral vector is transduced into a cell via infection and proviral integration. In certain embodiments, a target cell, e.g., a T cell or NK cell, is "transduced" if it contains a gene or other polynucleotide sequence delivered to the cell by infection using a virus or retroviral vector. In some embodiments, transduced cells contain one or more genes or other polynucleotide sequences delivered by a retroviral or lentiviral vector in the cell genome.

[0236] Host cells expressing one or more of the constructs of the disclosure are disclosed. The host cells contain nucleic acid sequences encoding one or more polypeptides that express engineered TCRs and / or CARs Transduction can be performed using one or more viral vectors. Other methods relating to the use of viral vectors in gene therapy that may be utilized according to certain embodiments of this disclosure include, for example, Kay, MA (1997) Chest 111 (6 Supp.): 138S-142S; Ferry, N. and Heard, JM (1998) Hum. Gene Ther. 9: 1975-81; Shiratory, Y. et al., (1999) Liver 19: 265-74; Oka, K. et al., (2000) Curr. Opin. Lipidol. 11: 179-86; Thule, PM and Liu, JM (2000) Gene Ther. 7: 1744-52; Yang, NS (1992) Crit. Rev. Biotechnol. 12: 335-56; Alt, M. (1995) J Hepatol. 23: 746-58; Brody, SLand It can be found in Crystal, RG (1994) Ann. NY Acad. Sci. 716:90-101, Strayer, DS (1999) Expert Opin. Investig. Drugs 8:2159-2172, Smith-Arica, JRand Bartlett, JS (2001) Curr. Cardiol. Rep. 3:43-49, and Lee, HC et al., (2000) Nature 408:483-8.

[0237] The compositions described herein may include one or more polynucleotides, polypeptides, vectors comprising them, and T cell compositions and NK compositions, as intended herein. One embodiment described herein is a composition comprising modified T cells expressing NKG2D CAR. Another embodiment described herein is a composition comprising modified NK cells expressing NKG2D CAR. Compositions include, but are not limited to, pharmaceutical compositions. "Pharmaceutical composition" means a composition formulated into a pharmaceutically acceptable or physiologically acceptable solution for administration to cells or animals, either alone or in combination with one or more other therapeutic agents. It will also be understood that, as needed, the compositions of this disclosure may be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmacoactive agents. Other components that may be included in a composition are not substantially limited, as long as the additional agents do not adversely affect the composition's ability to deliver the intended therapy.

[0238] The term "pharmaceutically acceptable" is used herein to mean these compounds, materials, compositions, and / or dosage forms that are within the bounds of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit-to-risk ratio.

[0239] As used herein, “pharmaceutically acceptable carriers, diluents or excipients” includes, but is not limited to, any adjuvants, carriers, excipients, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers approved by the U.S. Food and Drug Administration as acceptable for use in human or animal husbandry. Exemplary pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose, and its derivatives such as carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols such as propylene glycol; glycerin Polyols such as sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol; phosphate buffer; and used in pharmaceutical preparations. This includes, but is not limited to, any other compatible substances used.

[0240] In one embodiment described herein, the composition of the disclosure comprises the amount of modified T cells or NK cells as intended herein. The pharmaceutical composition comprising the T cells or NK cells as intended herein is 10 2 ~10 10 cells / kg body weight, 10 5 ~10 9 cells / kg body weight, 10 5 ~10 8 cells / kg body weight, 10 5 ~10 7 cells / kg body weight, 10 7 ~10 9 Cells / kg body weight, or 10 7~10 8 It can generally be stated that it can be administered in doses of cells / kg body weight (including all integer values ​​within these ranges). The number of cells, as well as the types of cells contained therein, will depend on the final intended use of the composition. (Operated NKG2D) T cells or NK cells modified to express CARs may be administered multiple times in doses within these ranges. The cells may be allogeneic, syngeneic, heterogeneic, or autologous to the patient receiving treatment. If desired, the treatment may also include the administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-7, IL-15, IL-12, TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the engraftment and function of the injected T cells.

[0241] In general, compositions comprising activated and proliferated cells as described herein may be used for the treatment and prevention of diseases occurring in immunocompromised or immunosuppressed individuals. Some compositions comprising modified T cells or NK cells as envisioned herein are used for the treatment of cancer. Modified T cells or NK cells as envisioned herein may be administered alone or in combination with other components such as carriers, diluents, excipients, and / or IL-2, IL-7, and / or IL-15 or other cytokines or cell populations as pharmaceutical compositions. In some embodiments, the pharmaceutical compositions envisioned herein contain a certain amount of genetically modified T cells or NK cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0242] Pharmaceutical compositions comprising modified T cells or NK cells as intended herein may further comprise buffers such as neutral buffered saline or phosphate-buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of this disclosure may be formulated for parenteral administration, for example, intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration.

[0243] Liquid pharmaceutical compositions, whether in solution, suspension or other similar form, may contain one or more of the following: sterile diluents such as water for injection; physiological salines such as saline, Ringer's solution, or isotonic sodium chloride; fixing oils such as synthetic mono or diglycerides that can function as a solvent or suspension medium; polyethylene glycol, glycerin, propylene glycol, or other solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral preparations may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials. Sterile pharmaceutical compositions for injection are also included.

[0244] In some embodiments, the compositions contemplated herein comprise an effective amount of a proliferated and modified T cell or NK cell composition, either alone or in combination with one or more therapeutic agents. Thus, the T cell or NK cell composition may be used alone or in conjunction with radiotherapy, chemotherapy, transplantation, or immunotherapy. The composition may be administered in combination with other known cancer treatments, such as hormone therapy and photodynamic therapy. The composition may also be administered in combination with antibiotics and antiviral agents. Such therapeutic agents may be accepted in the art as treatments for disease conditions described herein, such as cancer. In one embodiment, the composition intended herein may also be administered with TGF-β inhibitors, such as the small molecule inhibitor LY55299. Exemplary therapeutic agents intended include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and adjuvants.

[0245] In certain embodiments, the composition comprising T cells or NK cells as intended herein can be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carbocon, metredopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, and trimethylomellamine regimen; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard. Nitrosourea, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., acrasinomycin, actinomycin, ausramycin, azaserin, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, Mycophenolic acid, nogaramycin, olibomycin, peplomycin, pofilomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g. For example, ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenaline, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofsphamide glycoside; aminolevulinic acid; amsacrin, bestrabusil; bisanthren; edatrexate; Defofamine; Demecoltin; Diadiquan; Elformitin; Erliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamin; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK(registered trademark); Lazoxane; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine;Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and Doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum analogs, e.g.; Examples include cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeroda; ibandronate; CPT-11; topoisomerase inhibitor RPS 2000; difluoromethylomitine (DMFO); retinoic acid derivatives such as Targretin (trademark) (bexarotene) and Panretin (trademark) (allitretinoin); ONTAK (trademark) (denileukin difutytox); esperamycin; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of the above. Furthermore, this definition includes, for example, anti-hormone agents that act to modulate or inhibit the hormonal effects on tumors, such as anti-estrogens including tamoxifen, raloxifen, the aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); as well as anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0246] Various additional therapeutic agents may be used in conjunction with the compositions or drugs / therapies described herein. In one embodiment, the composition containing T cells is administered together with an anti-inflammatory agent. Examples of anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mycophenolic acid.

[0247] In some embodiments, the NSAID is selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialates. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol, or propoxifene hydrochloride. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Examples of biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Examples of biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Examples of disease-modifying anti-rheumatic drugs (DMARDs) include... Examples include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0248] In other embodiments, suitable therapeutic antibodies for combination with CAR or TCR-modified T cells or NK cells as intended herein include avagovomab, adecatumumab, aftuzumab, alemtuzumab, altumomab, amatsuximab, anatumomab, alsitumomab, bavituximab, vectumomab, bevacizumab, vibatuzumab, blinatumomab, brentuximab, cantuzumab, catumakisomab, cetuximab, and cy Tatuzumab, sixtumumab, cribatuzumab, konatumumab, daratumumab, dorozitumumab, duligotuzumab, dusigituzumab, detumomab, dasetuzumab, dalotuzumab, eclomeximab, elotuzumab, ensituximab, ertumaxomab, etalacizumab, farietuzumab, ficratuzumab, figitumumab, fura Mbotuzumab, Futuximab, Ganituzumab, Gemtuzumab, Gilentuzumab, Grembatumumab, Ibritumomab, Igobomab, Imatsuzumab, Indatuzumab, Inotuzumab, Intetumumab, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Rorbotuzumab, Lucatumumab, Mapatumumab, Matuzumab, Miratuzumab, Minretsumomab, Mitsumomab, Moxetumomab, Namatumomab, Naptumomab, Necitumumab, Nimotuzumab, Nofetumomab, Okalatuzumab, Ofatumumab, Oralatumumab, Ona Tuzumab, oporutuzumab, oregobomab, panitumumab, pulsatuzumab, patrizumab, pemtumomab, pertuzumab, pintumomab, pritumumab, lacotumomab, radrezumab, rilotumumab, rituximab, lobatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitomab, takatuzumab, tapritumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotzumab, ubrituximab, beltuzumab, borsetuzumab, botumumab, zaru This includes, but is not limited to, zalutumumab, CC49, and 3F8.

[0249] In some embodiments, the compositions described herein are administered together with cytokines. As used herein, “cytokines” refers to a general term for proteins released by a population of cells that act on another cell as intercellular mediators. Examples of cytokines include lymphokines, monokines, chemokines, and conventional polypeptide hormones. Cytokines include growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; Müllerian duct inhibitor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor, such as NGF-beta; platelet growth factor; and transforming growth factor. Factors (TGF), e.g., TGF-alpha and TGF-beta; insulin-like growth factor-I and growth factor-II; erythropoietin (EPO); bone induction factors; interferons, e.g., interferon-alpha, beta, and gamma; colony-stimulating factor (CSF) For example, macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). This includes CSFs; interleukins (ILs), e.g., IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15; tumor necrosis factors, e.g., TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligands (kit ligands, KL). As used herein, the term cytokine includes proteins derived from natural sources or recombinant cell cultures, and biologically active equivalents of naturally occurring cytokines.

[0250] Any cell can be used as a host cell for the polynucleotides, vectors, or polypeptides of this disclosure. In some embodiments, the cell may be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria such as Gram-negative or Gram-positive organisms, e.g., Enterobactehaceae such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis. ;Pseudomonas such as P. aeruginosa; and Streptomyces are included. In some embodiments, the cells are human cells. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, tumor infiltrating lymphocytes (TIL), TCR-expressing cells, natural killer (NK) cells, dendritic cells, granulocytes, innate lymphocytes, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and bone marrow cells. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In certain embodiments, the T cells are tumor infiltrating lymphocytes (TIL), autologous T cells, engineered autologous T cells (eACT™), allogeneic T cells, xenogeneic T cells, or any combination thereof. Different from antibody therapy or standalone NKG2D CAR-modified T cells, the T cells (or any of the cells above).

[0251] Another embodiment described herein is a method of treating cancer in a subject in need thereof, comprising administering an effective amount, such as a therapeutically effective amount, of a composition comprising T cells or NK cells expressing a TCR or CAR described herein. The amount and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, etc., but an appropriate dosage can be determined by clinical trials.

[0252] In other embodiments, methods are provided that include administering to a patient in need thereof a therapeutically effective amount of modified T cells or a composition comprising the same, alone or in combination with one or more therapeutic agents. In certain embodiments, the cells of the present disclosure are used in the treatment of patients at risk of developing cancer. Accordingly, the present disclosure provides methods for the treatment or prevention of cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the modified T cells of the present disclosure.

[0253] Those skilled in the art will recognize that multiple administrations of the compositions of the present disclosure may be required to achieve the desired treatment. For example, the compositions may be administered one, two, three, four, five, six months, one year, two years, five years, ten years, or more, over a span of one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, ten years, or more.

[0254] In certain embodiments, it may be desirable to administer activated T cells to a target, then re-collect (or perform apheresis) the blood, activate T cells from it according to this disclosure, and re-inject these activated and proliferated T cells into the patient. This process may be performed multiple times at intervals of several weeks. In certain embodiments, T cells may be activated from 10cc to 400cc of blood. While not bound by theory, this multiple blood collection / multiple re-injection protocol may be useful in selecting a particular population of T cells.

[0255] The compositions intended herein may be administered by any convenient method, including aerosol inhalation, injection, ingestion, blood transfusion, transplantation, or implantation. In some embodiments, the compositions are administered parenterally. As used herein, the terms “parenteral administration” and “administered parenterally” mean a mode of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and substernal injections and infusions. In one embodiment, the compositions intended herein are administered to a target by direct injection into a tumor, lymph node, or site of infection.

[0256] In one embodiment, a subject requiring it is administered an effective amount of the composition to increase the cellular immune response against cancer in the subject. The immune response may include a cellular immune response mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses that can kill infected cells. Thus, a humoral immune response, primarily mediated by helper T cells that can activate B cells that result in antibody production, may also be induced. To analyze the type of immune response induced by a substance, various techniques well described in the art can be used, for example, as sufficiently described in Current Protocols in Immunology, Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001), John Wiley & Sons, NY, NY.

[0257] In T cell-mediated killing, CAR-ligand binding initiates CAR signaling to T cells, leading to the activation of various T cell signaling pathways that induce T cells to produce or release proteins that can induce target cell apoptosis through a variety of mechanisms. These T cell-mediated mechanisms include, but are not limited to, the transfer of intracellular cytotoxic granules from T cells to target cells, T cell secretion of pro-inflammatory cytokines that can directly induce target cell killing (or indirectly through the recruitment of other killer effector cells), and the upregulation of death receptor ligands (e.g., FasL) on the T cell surface that induce target cell apoptosis after binding to their genus death receptor (e.g., Fas) on target cells.

[0258] Embodiments described herein are methods for treating a subject diagnosed with cancer, comprising: isolating T cells from the subject; genetically modifying the T cells with a vector containing nucleic acid encoding the NKG2D CAR as intended herein, thereby producing a population of modified T cells; and administering the population of modified T cells to the same subject.

[0259] In certain embodiments, the disclosure also provides a method for stimulating an effector cell-mediated immunomodulatory response to a target cell population in a subject, comprising the step of administering to a population of immune effector cells expressing a nucleic acid construct encoding the NKG2D CAR molecule.

[0260] Methods for administering the cell compositions described herein include any method effective in bringing about the reintroduction of genetically modified immune effector cells ex vivo, either by directly expressing the manipulated NKG2D CAR in a subject or by reintroducing genetically modified precursor cells of immune effector cells that differentiate into mature immune effector cells expressing the NKG2D CAR molecule upon introduction into a subject. One method includes transducing peripheral blood T cells with the nucleic acid construct according to this disclosure ex vivo and returning the transduced cells to a subject.

[0261] While the foregoing disclosure is described in some detail as examples and illustrations to clarify understanding, it will be readily apparent to those skilled in the art, in light of the teachings of this disclosure, that certain modifications and alterations can be made without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only and are not limiting. Those skilled in the art will readily recognize a variety of non-essential parameters that can be changed or altered to produce essentially similar results. [Examples]

[0262] Example 1 NKG2D CAR construct design The engineered NKG2D chimeric antigen receptor construct was designed and synthesized in a retroviral vector, as used in the following examples. The first construct, referred to as NKG2D CAR1, contains a signaling domain from the N-terminus to the C-terminus, comprising a signal peptide, an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intercellular domain, and a CD3ζ signaling domain. This chimeric antigen receptor The amino acid sequence is shown below: MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 53).

[0263] The second construct, referred to as NKG2D CAR2, includes a signal peptide, an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intercellular domain, and a signaling domain containing CD3ε and CD3ζ signaling domains, from the N-terminus to the C-terminus. The amino acid sequence of this chimeric antigen receptor is shown below: MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWED GSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVT VAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 54).

[0264] The third construct, referred to as NKG2D CAR3, includes a signaling domain from the N-terminus to the C-terminus, comprising an NKG2D extracellular domain, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intercellular domain, and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor includes the following amino acid sequence: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 66). In one embodiment, CAR3 is encoded by the following nucleotide sequence: 1 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 61 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 121 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 181 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 241 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 301 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 361 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 421 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 481 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 541 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 601 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 661 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 721 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 781 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 841 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 901 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 961 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1021 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1081 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1141 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaaa actaccccta 1201 aaagccaaag cgccgccacc atggctcttc ctgtgacagc tcttctgctg cccctggccc 1261 tgcttctgca tgctgctaga cctgagcaaa agttgatttc tgaggaagac ctcgccggca 1321 gtttattcaa ccaagaagtc caaattccct tgaccgaaag ttactgtggc ccatgtccta 1381 agaactggat atgttacaaa aataactgtt accaattctt cgatgaatct aagaattggt 1441 atgagagcca ggcttcttgt atgtctcaaa atgccagcct tcttaaagta tacagcaaag 1501 aggaccagga tttacttaaa ctggtgaagt catatcattg gatgggacta gtacacattc 1561 caacaaatgg atcttggcag tgggaagaacg gctccattct ctcacccaac ctactaacaa 1621 taattgaaat gcagaaggga gactgtgcac tctatgcatc gagctttaaa ggctatatag 1681 aaaactgttc aactccaaat acatatattt gcatgcaaag gactgtgacc acgacgccag 1741 cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca acccctgtcc ctgaggcctg 1801 aagcgtgccg gccagcggcg ggcggcgcag tgcacacgag agggctggac ttcgcctgtg 1861 atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg tcactggtta 1921 tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa caaccattta 1981 tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt ccagaagaag 2041 aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc 2101 agcaagggca gaaccagctc tataacgagc tcaatctagg acgaagagag gagtacgatg 2161 ttttggacaa gaggcgtggc cgggaccctg agatgggggg aaagccgaga aggaagaacc 2221 ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc tacagtgaga 2281 ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac cagggtctca 2341 gtacagccac caaggacacc tacgacgccc ttcacatgca agctctgccc cctcgctga (SEQ ID NO: 67). CAR3 may be encoded by a nucleic acid having at least 75% sequence identity to (SEQ ID NO: 67) (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 67) may be modified to remove the Myc (human c-Myc proto-oncogene) epitope tag and the sequence encoding the linker between the Myc tag and the NKG2D extracellular domain. The nucleic acid sequence of (SEQ ID NO: 67) encodes a CD8a signal peptide at positions 1221-1283, and this signal peptide may be substituted with a different signal peptide.

[0265] The fourth construct, referred to as NKG2D CAR4, includes a signaling domain from the N-terminus to the C-terminus, comprising an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intercellular domain, and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor includes the following amino acid sequence: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 68). In one embodiment, CAR4 is encoded by the following nucleotide sequence: 1 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 61 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 121 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 181 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 241 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 301 gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 361 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 421 gaggcctggc ttgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 481 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 541 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 601 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 661 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 721 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 781 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 841 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt caccacaca aggaaaagg 901 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 961 cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1021 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1081 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1141 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaaa actaccccta 1201 aaagccaaag cgccgccacc atggctcttc ctgtgacagc tcttctgctg cccctggccc 1261 tgcttctgca tgctgctaga cctgagcaaa agttgatttc tgaggaagac ctcgccggca 1321 gtttattcaa ccaagaagtc caaattccct tgaccgaaag ttactgtggc ccatgtccta 1381 agaactggat atgttacaaa aataactgtt accaattctt cgatgaatct aagaattggt 1441 atgagagcca ggcttcttgt atgtctcaaa atgccagcct tcttaaagta tacagcaaag 1501 aggaccagga tttacttaaa ctggtgaagt catatcattg gatgggacta gtacacattc 1561 1621 1681 aaaactgttc aactccaaat acatatattt gcatgcaaag gactgtgacc acgacgccag 1741 cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca acccctgtcc ctgaggcctg 1801 aagcgtgccg gccagcggcg ggcggcgcag tgcacacgag agggctggac ttcgcctgtg 1861 atttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg ctagtaacag 1921 tggcctttat tattttctgg gtcaaacggg gcaagaagaa actcctgtat atattcaaac 1981 aaccatttat gagaccagta caaactactc aagagaaga tggctgtagc tgccgatttc 2041 2101 ccgcgtacca gcaagggcag aaccagctct ataacgagct caatctagga cgaagagagg 2161 agtacgatgt tttggacaag aggcgtggcc gggaccctga gatggggga aagccgagaa 2221 ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct 2281 acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc 2341 agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcaa gctctgcccc 2401ctcgctga (Sequence ID 69). CAR4 may be encoded by a nucleic acid having at least 75% sequence identity to (SEQ ID NO: 69) (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 69) may be modified to remove the Myc (human c-Myc proto-oncogene) epitope tag and the sequence encoding the linker between the Myc tag and the NKG2D extracellular domain. The nucleic acid sequence of (SEQ ID NO: 69) encodes a CD8a signal peptide at positions 1221-1283, and this signal peptide may be substituted with a different signal peptide.

[0266] The fifth construct, referred to as NKG2D CAR5, includes, from the N-terminus to the C-terminus, an NKG2D extracellular domain, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intercellular domain, and a signaling domain comprising a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor includes the following amino acid sequence: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDY EPIRKGQRDLYSGLNQRRILRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 70). In one embodiment, CAR5 is encoded by the following nucleotide sequence: 1 cgcggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc 61 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga 121 gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 181 gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag agaaccatca 241 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa 301 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc 361 acaacccctc actcggcgcg ccagtccttc gaagtagatc tttgtcgatc ctaccatcca 421 ctcgacacac ccgccagcgg ccgctgccaa gcttccgagc tctcgaatta attcacgccg 481 ccaccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt ctgcatgctg 541 ctagacctga gcaaaagttg atttctgagg aagacctcgc cggcagttta ttcaaccaag 601 aagtccaaat tcccttgacc gaaagttact gtggcccatg tcctaagaac tggatatgtt 661 acaaaaataa ctgttaccaa ttcttcgatg aatctaagaa ttggtatgag agccaggctt 721 cttgtatgtc tcaaaatgcc agccttcta aagtatacag caaagaggac caggatttac 781 ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca aatggatctt 841 ggcagtggga agacggctcc attctctcac ccaacctact aacaataatt gaaatgcaga 901 agggagactg tgcactctat gcatcgagct ttaaaggcta tatagaaaac tgttcaactc 961 1021 cccccgctcc tacaatcgcc agccaacctc tgagcctgag accggaggca tgcagacctg 1081 cggcaggggg agcagttcac acaagaggct tggacttcgc ttgcgacatc tacatctggg 1141 cccctctggc cggcacatgc ggagttcttc ttcttagcct ggtgatcacc ctgtactgca 1201 agaggccg gaagaagctg ctgtacatct tcaagcagcc cttcatgaga cctgtgcaga 1261 ccacacagga ggagaacggc tgcagctgta gattccccga ggagaggag ggcggctgtg 1321 agctgaaa ccgcaaagca aaggcaaaac c cgtcacacg aggagcgggc gcaggggac 1381 gacaacgcgg tcagaataag gaacgcccgc ctccagtacc aaatccagat tatgaaccaa 1441 ttcggaaggg aaacgcgat ctctactccg gtctcaatca gaggcgaatt ctgagagtta 1501 agttcagcag gagcgccgac gcccctgcct accagcaagg acagaatcaa ctgtacaacg 1561 agctgaacct gggcagacgg gaggaatacg atgtgctgga caagagga ggcagagacc 1621 1681 agaaggacaa gatggccgag gcctacagcg agatcggcat gaagggcgaa agaagaagag 1741 gcaagggcca cgacggcctc taccagggct taagcacagc tacaaaggac acctacgacg 1801 ccctgcacat gcaggccctg ccccctagat ga (Sequence ID 71) CAR5 may be encoded by a nucleic acid having at least 75% sequence identity to (SEQ ID NO: 71) (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 71) may be modified to remove the Myc (human c-Myc proto-oncogene) epitope tag and the sequence encoding the linker between the Myc tag and the NKG2D extracellular domain. The nucleic acid sequence of (SEQ ID NO: 71) encodes a CD8a signal peptide at positions 486-548, and this signal peptide may be substituted with a different signal peptide.

[0267] The sixth construct, referred to as NKG2D CAR6, contains, from the N-terminus to the C-terminus, an NKG2D extracellular domain, a CD8 alpha hinge, a CD8 transmembrane domain, a 4-1BB intercellular domain, and a signaling domain including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor includes the following: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 72). In one embodiment, CAR6 is encoded by the following nucleotide sequence: 1 cgcggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc 61 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga 121 gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 181 gccaagaaca gatggtcccc agatgcggtc cc gccctcag cagtttctag agaaccatca 241 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa 301 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc 361 acaacccctc actcggcgcg ccagtccttc gaagtagatc tttgtcgatc ctaccatcca 421 ctcgacacac ccgccagcgg ccgctgccaa gcttccgagc tctcgaatta attcacgccg 481 ccaccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt ctgcatgctg 541 ctagacctga gcaaaagttg atttctgagg aagacctcgc cggcagttta ttcaaccaag 601 aagtccaaat tcccttgacc gaaagttact gtggcccatg tcctaagaac tggatatgtt 661 acaaaaataa ctgttaccaa ttcttcgatg aatctaagaa ttggtatgag agccaggctt 721 cttgtatgtc tcaaaatgcc agccttcta aagtatacag caaagaggac caggatttac 781 ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca aatggatctt 841 ggcagtggga agacggctcc attctctcac ccaacctact aacaataatt gaaatgcaga 901 agggagactg tgcactctat gcatcgagct ttaaaggcta tatagaaaac tgttcaactc 961 caatacata tattgcatg haaaggactg tgaccacgac gccagcgccg cgaccacca 1021 caccggcgcc caccatcgcg tcgcacccc tgtccctgag gcctgaagcg tgccggcg 1081 cggcggggcgg cgcagtgcac acgagagggc tggactcgc ctgtgatatc tacatctggg 1141 cgcccttggc cgggacttgt gggtccttc tctgtcact gttatcacc ctttactgca 1201 aacggggcag aaagaaactc ctgtatatat tcaacaacc atttatgaga ccagtacaa 1261 ctactcaaga ggagatggc tgtagctgcc gatttccaga agagagaa ggaggatgtg 1321 aaagaaccg aaaagcaaa gccaagcctg ttacaagagg agcaggggca ggaggccgac 1381 aggaggggca aaaaaaagaaaggccccgcccgtcccaaa cccggattat gagccaatta 1441 ggaagggtca gagagacctg tattctgggc tcctgagagt gaagttcagc aggagcgcag 1501 acggcccccgc gtaccagcaa gggcagaacc agcttatta cgagctcaat ctaggacgaa 1561 gagaggatta cgatgttttg ganaagaggc gtggccggga ccctgagatg ggggaaagc 1621 cgagagagat aagatggcgg 1681 aggcctacag tgagattggg atgaaaggcg a gcgccggag gggcaagggg cacgatggcc 1741 tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaagctc 1801 tgccccctcg ctga (Sequence ID 73) CAR6 may be encoded by a nucleic acid having at least 75% sequence identity to (SEQ ID NO: 73) (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 73) may be modified to remove the Myc (human c-Myc proto-oncogene) epitope tag and the sequence encoding the linker between the Myc tag and the NKG2D extracellular domain. The nucleic acid sequence of (SEQ ID NO: 73) encodes a CD8a signal peptide at positions 486-548, and this signal peptide may be substituted with a different signal peptide.

[0268] The seventh construct, referred to as NKG2D CAR7, contains, from the N-terminus to the C-terminus, an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intercellular domain, and a signaling domain including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor includes the following: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACY SLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 74). In one embodiment, CAR7 is encoded by the following nucleotide sequence: 1 cgcggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc 61 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga 121 gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 181 gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag agaaccatca 241 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa 301 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc 361 acaacccctc actcggcgcg ccagtccttc gaagtagatc tttgtcgatc ctaccatcca 421 ctcgacacac ccgccagcgg ccgctgccaa gcttccgagc tctcgaatta attcacgccg 481 ccaccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt ctgcatgctg 541 ctagacctga gcaaaagttg atttctgagg aagacctcgc cggcagttta ttcaaccaag 601 aagtccaaat tcccttgacc gaaagttact gtggcccatg tcctaagaac tggatatgtt 661 acaaaaataa ctgttaccaa ttcttcgatg aatctaagaa ttggtatgag agccaggctt 721 cttgtatgtc tcaaaatgcc agccttcta aagtatacag caaagaggac caggatttac 781 ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca aatggatctt 841 ggcagtggga agacggctcc attctctcac ccaacctact aacaataatt gaaatgcaga 901 agggagactg tgcactctat gcatcgagct ttaaaggcta tatagaaaac tgttcaactc 961 1021 caccggcgcc caccatcgcg tcgcaacccc tgtccctgag gcctgaagcg tgccggccag 1081 cggcgggcgg cgcagtgcac acgagaggc tggacttcgc ctgtgatttt tgggtgctgg 1141 tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc tttattattt 1201 tctgggtcaa acggggcaga aagaaactcc tgtatatatt caaacaacca tttatgagac 1261 caatcaaac tactcaagag gaagatggct gtagctgccg atttccagaa gaagagaag 1321 gaggatgtga aaagaaccga aaagcaaaag ccaagcctgt tacaagga gcaggggcag 1381 gaggccgaca gagagggcaa aacaaagaaa ggccccccgcc cgtcccaaac ccggattatg 1441 agccaattag gaagggtcag agagacctgt attctgggct cctgagagtg aagttcagca 1501 ggagcgcaga cgccccgcg taccagcaag ggcagaacca gctctataac gagctacatc 1561 taggacgaag agaggagtac gatgttttgg acaagaggcg tggccgggac cctgagatgg 1621 ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg cagaaagata 1681 agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc 1741 acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca 1801 tgcaagctct gccccctcgc tga (Sequence ID 75) CAR7 may be encoded by a nucleic acid having at least 75% sequence identity to (SEQ ID NO: 75) (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 75) may be modified to remove the Myc (human c-Myc proto-oncogene) epitope tag and the sequence encoding the linker between the Myc tag and the NKG2D extracellular domain. The nucleic acid sequence of (SEQ ID NO: 75) encodes the CD8a signal peptide at positions 486-548, and this signal peptide may be substituted with a different signal peptide.

[0269] The eighth construct, referred to as NKG2D CAR8, contains, from the N-terminus to the C-terminus, an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intercellular domain, and a signaling domain including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor includes the following: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSL LVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRILRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 76). In one embodiment, CAR8 is encoded by the following nucleotide sequence: 1 cgcggaatga aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc 61 aaggcatgga aaatacataa ctgagaatag agaagttcag atcaaggtta ggaacagaga 121 gacagcagaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 181 gccaagaaca gatggtcccc agatgcggtc ccgccctcag cagtttctag agaaccatca 241 gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa 301 tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc 361 acaacccctc actcggcgcg ccagtccttc gaagtagatc tttgtcgatc ctaccatcca 421 ctcgacacac ccgccagcgg ccgctgccaa gcttccgagc tctcgaatta attcacgccg 481 ccaccatggc tcttcctgtg acagctcttc tgctgcccct ggccctgctt ctgcatgctg 541 ctagacctga gcaaaagttg atttctgagg aagacctcgc cggcagttta ttcaaccaag 601 aagtccaaat tcccttgacc gaaagttact gtggcccatg tcctaagaac tggatatgtt 661 acaaaaataa ctgttaccaa ttcttcgatg aatctaagaa ttggtatgag agccaggctt 721 cttgtatgtc tcaaaatgcc agccttcta aagtatacag caaagaggac caggatttac 781 ttaaactggt gaagtcatat cattggatgg gactagtaca cattccaaca aatggatctt 841 ggcagtggga agacggctcc attctctcac ccaacctact aacaataatt gaaatgcaga 901 agggagactg tgcactctat gcatcgagct ttaaaggcta tatagaaaac tgttcaactc 961 accaggac gccagcgccg cgaccaccaa 1021 caccggcgcc caccatcgcg tcgcaacccc tgtccctgag gcctgaagcg tgccggccag 1081 cggcgggcgg cgcagtgcac acgagaggc tggacttcgc ctgtgatttt tgggtgctgg 1141 tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc tttattattt 1201 tctgggtcaa acggggcaga aagaaactcc tgtatatatt caaacaacca tttatgagac 1261 caatcaaac tactcaagag gaagatggct gtagctgccg atttccagaa gaagagaag 1321 gaggatgtga aaagaaccgc aaagcaaagg caaaacccgt cacacgagga gcgggcgcag 1381 1441 aaccaattcg gaagggacaa cgcgatctct actccggtct caatcagagg cgaattctga 1501 1561 1621 gggaccctga gatggggggga aagccgagaa ggagaaccc tcaggaaggc ctgtacaatg 1681 aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc 1741 ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc aaggacacct 1801 acgacgccct tcacatgcaa gctctgcccc ctcgctga (Sequence ID 77) CAR8 can be encoded by a nucleic acid having at least 75% sequence identity to (SEQ ID NO: 77) (e.g., at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity, such as 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 77) can be modified to remove the Myc (human c-Myc proto-oncogene) epitope tag and the sequence encoding the linker between the Myc tag and the NKG2D extracellular domain. The nucleic acid sequence of (SEQ ID NO: 77) encodes the CD8a signal peptide at positions 486-548, and this signal peptide can be replaced with a different signal peptide.

[0270] Example 2 Transduction efficiency Retroviral vectors were used for all T cell transduction. HPV16+E7 11-19 Engineered TCRs targeting epitopes were used for single or simultaneous transduction with NKG2D CAR constructs (Jin BY et al., 2018 JCI Insight). Pre-frozen PBMCs obtained from healthy donors were thawed and activated for 48 hours with anti-CD3 in Optimizer T cell medium supplemented with 300 IU of IL-2. HPV16+E7 11-19 Epitope-targeted manipulated TCR (referred to as HPV-TCR in this example and the following examples), NKG2D A retrovirus containing CAR, or both, was transduced. T cells were grown for 10 days in Optimizer T cell medium supplemented with IL-7 (10 ng / mL), IL-15 (10 ng / mL), and Akt VIII inhibitor (AKTi, 1 μM). Transduction efficiency was measured by flow cytometry on days 8 and 15 after T cell activation. Antibodies used to detect TCR transduction efficiency included anti-mouse TCRβ clone H57-597 (BioLegend), and anti-human CD314 clone 1D11 (BioLegend) was used to detect NKG2D transduction efficiency. All flow cytometry data were collected using BD FACSDiva® software with LSR-Fortessa (BD LSR Fortessa®), and the data were analyzed using FlowJo (all BD Sciences). All antibody staining was performed at 4°C in PBS containing 1% BSA.

[0271] Table 4 shows the expression levels of transduced and non-transduced T cells (measured as mean fluorescence intensity (MFI) and positive cell percentage) after 8 and 15 days of culture by flow cytometry. High transduction efficiency was observed in all constructs. Small populations of only NKG2D+ cells were present in the HPV-TCR+NKG2D CAR1 and HPV-TCR+NKG2D CAR2 groups.

[0272] TIFF2026082962000004.tif81170

[0273] Table 19 shows the expression levels (as mean fluorescence intensity (MFI) and positive cell percentage) of transduced and non-transduced T cells after 7 / 8 days of culture, based on expression measurements by Myc staining and flow cytometry. High transduction efficiency was observed in all constructs.

[0274] TIFF2026082962000005.tif52170

[0275] Example 3 Proliferation and survival rate Cell proliferation was measured using a Vi-Cell XR cell counter (Beckman Coulter), and cell viability was measured by staining cells with Live / Dead Blue (ThermoFisher) in PBS on ice for 20 minutes. These viability was analyzed by flow cytometry on days 8 and 15 after T cell activation (Table 5). Transformed T cells were activated as described in Example 2. Table 5 shows the proliferation and cell viability of transduced and non-transduced T cells cultured in Optimizer medium supplemented with IL-7, IL-15, and AKTi. NKG2D CAR1 only, NKG2D CAR2 cells alone, HPV-TCR+NKG2D CAR1 cells, and HPV-TCR+NKG2D CAR2 cells showed higher levels of proliferation than HPV-TCR cells alone or non-transduced T cells.

[0276] TIFF2026082962000006.tif48170

[0277] Example 4 Determining Product Attributes Memory phenotype and CD4 / CD8 ratio were measured by flow cytometry on day 8 after T cell activation (Table 6). Antibodies used to evaluate memory phenotype included anti-human CD45RA clone HI100 (BioLegend), anti-human CD45RO clone UCHL1 (BioLegend), anti-human CCR7 clone G043H7 (BioLegend), and anti-human CD62L clone DREG-56 (BioLegend). Antibodies used to evaluate CD4 / CD8 ratio included anti-human CD3 clone SK7 (BioLegend), anti-human CD4 clone RPA-T4 (BioLegend), and anti-human CD8 clone SK1 (BioLegend). Transformed T cells were activated as described in Example 2. Memory phenotypes were evaluated as follows: naive T cells (CD45RA+CD45RO-CCR7+CD62L+); central memory T cells (CD45RA-CD45RO+CCR7+CD62L+); effector memory T cells (CD45RA-CD45RO+CCR7-CD62L-); terminal effector T cells (CD45RA+CD45RO-CCR7-CD62L-). All flow cytometry data were collected using BD FACSDiva® software on an LSR-Fortessa (BD LSR Fortessa®) and analyzed using FlowJo (all BD Sciences). All antibody staining was performed in PBS containing 1% BSA at 4°C.

[0278] Table 6 shows the CD4 / CD8 ratio and memory phenotype of transduced CD3+ T cells and non-transduced CD3+ T cells cultured in IL-2-supplemented TC medium after 8 days. There was no significant change in the CD4 / CD8 ratio between transduced and non-transduced T cells. Non-transduced T cells and T cells transduced with HPV-TCR only exhibited a larger naive T cell compartment than T cells transduced with NKG2D CAR1 only, NKG2D CAR2 only, HPV-TCR+NKG2D CAR1, or HPV-TCR+NKG2D CAR2. In addition, non-transduced T cells and T cells transduced with HPV-TCR only exhibited a larger naive T cell compartment than NKG2D These cells exhibited smaller memory T cell compartments than T cells transduced with CAR1 alone, NKG2D CAR2 alone, HPV-TCR+NKG2D CAR1, or HPV-TCR+NKG2D CAR2.

[0279] TIFF2026082962000007.tif48170

[0280] Example 5 Cytotoxicity HPV-TCR transducible T cells have been previously shown to effectively eliminate HPV16+ tumor cell lines and produce high levels of IFNγ. Sequencing of patient tumors from a Phase I clinical trial using HPV-TCR (Nagarsheth NB et al., 2021 Nat Med) suggests that tumors are highly heterogeneous and can exhibit mutations in the MHC-I antigen processing and presentation pathways, thereby making them resistant to HPV-TCR-manipulated adoptive TCR-T therapy. To model patient tumor data, HPV16+ tumor cell lines (CaSki) were transduced by knocking out β2M (β2MKO) using the CRISPR-Cas9 system (Vakulskas CA et al., 2018 Nature). * The 02:01 segment was omitted. Furthermore, the HLA-A segment is normally omitted. * SiHa HPV16+ tumor cell lines lacking 02:01 were converted using a retroviral vector. * The cells were manipulated to overexpress 02:01. CaSki and SiHa tumor cell lines were selected based on high and low E7 expression, respectively. Undamaged HLA-A was found in non-transduced and transduced PBMCs. * Regardless of the presence or absence of 02:01, the killing of these tumor cell lines was evaluated using the xCELLigence RTCA MP (Agilent Technologies) platform, and various effector-to-target (E:T) ratios are shown in Tables 7 and 8.

[0281] Table 7 shows the results for transduced T cells (see Example 2) that were sorted on day 8 and then co-cultured on day 10 with wild-type cells or β2MKO CaSki cells in various E:T ratios in 96-well xCELLigence plates. Impedance values ​​(IV) were measured after 72 hours. Control IV represents the impedance value of untransduced T cells, while experimental IV represents the impedance value of either HPV-TCR only, NKG2D CAR1 only, NKG2D CAR2 only, HPV-TCR + NKG2D CAR1, or HPV-TCR + NKG2D CAR2. Cytotoxicity percentage was calculated using the following formula: % Cytotoxicity=1-(Control IV-Experiment al IV) * 100

[0282] TIFF2026082962000008.tif57170

[0283] Table 8, sorting on day 8, then on day 10, A expressing wild-type cells or SiHa cells. * The results for transduced T cells (see Example 2) co-cultured in 96-well xCELLigence plates with various E:T ratios, including 02:01, are shown. The impedance value (IV) was measured after 72 hours, and the cytotoxicity percentage was calculated as described above.

[0284] TIFF2026082962000009.tif42170 HPV-TCR transduced T cells are wild-type CaSki cell lines and A * 02 SiHa cell lines were successfully eliminated, but β2M KO CaSki cell lines or wild-type SiHa cell lines could not be eliminated. Importantly, NKG2D CAR transducible T cells and NKG2D CARs co-transduced with HPV-TCR T cells were found to have HLA-A * Regardless of the 02:01 state, we were able to successfully eliminate both the CaSki and SiHa cell lines.

[0285] Example 6 Cytokine production After 24 hours of incubation, cytokine production was measured by collecting the supernatant from the xCELLigence 96-well plate used in Example 5. Cytokine levels were measured using the Meso Sector S 600 platform (Institute for Biopharmaceutical Research, Inc.). IFNγ production from HPV-TCR+NKG2D CAR T cells was compared with that of wild-type cells or A * 02 The results were similar to those of HPV-TCR transdermated cells when co-cultured with SiHa cells. However, IFNγ production from HPV-TCR+NKG2D CAR T cells was increased compared to HPV-TCR transdermated T cells when co-cultured with β2MKO CaSki cells or wild-type SiHa cells, and this correlated with cytotoxic data (see Tables 9 and 10). These data showed that NKG2D CAR T cells secreted small levels of IFNγ when HPV-TCR transdermated T cells eliminated target cells, but produced sufficient IFNγ when HPV-TCR transdermated T cells did not eliminate target cells. Interestingly, T cells transduced to NKG2D CAR1 and HPV-TCR+NKG2D CAR1 produced higher levels of IFNγ than T cells transduced to NKG2D CAR2 or HPV-TCR+NKG2D CAR2. .

[0286] Table 9 shows the results for transduced T cells, sorted on day 8 and then co-cultured on day 10 with wild-type or β2MKO CaSki cells in various E:T ratios in 96-well xCELLigence plates. After 24 hours, the supernatant was extracted and processed using a Meso Sector S 600 platform according to the manufacturer's instructions to determine the IFNγ concentration (pg / mL).

[0287] TIFF2026082962000010.tif44170

[0288] Table 10 shows the cells selected on day 8, and then on day 10, wild-type cells or A *The results of transduced T cells co-cultured with SiHa cells in 96-well xCELLigence plates at various E:T ratios are shown. After 24 hours, the supernatant was extracted and the Meso Sector was analyzed. The S 600 platform was used according to the manufacturer's instructions to process the samples and determine the IFNγ concentration (pg / mL).

[0289] TIFF2026082962000011.tif44170

[0290] Example 7 Long-term proliferation and depletion of transduced T cells Repeated antigen stimulation in long-term cell death assays is thought to functionally deplete T cells and correlate with in vivo efficacy. To complete the serial antigen stimulation assay, target cells (WT CaSki, β2MKO CaSki, wild-type SiHa, or A) are used. * 02 SiHa) was added to transduced T cells (see Example 2) every 2-3 days. At the 5th stimulation, wild-type CaSki, β2MKO CaSki, wild-type SiHa, or A * The cytotoxicity of any of the SiHa compounds was evaluated using the xCELLigence RTCA MP platform with various E:T ratios shown in Tables 11 and 12. Despite the addition of NKG2D CAR, HPV-TCR+NKG2D CAR transduced T cells successfully eliminated their targets after sequential stimulation and exhibited higher levels of proliferation compared to T cells transduced with HPV-TCR alone (see Tables 11 and 12).

[0291] Table 11 shows the results for transduced T cells, which were selected on day 8 and then co-cultured with wild-type CaSki cells in a 3:1 E:T ratio on day 10. Wild-type CaSki cells were then added every 2-3 days. After the 5th stimulation, the T cells were transferred to a 96-well xCELLigence plate. In the culture medium, wild-type cells or β2MKO CaSki cells were co-cultured with various E:T ratios. Impedance values ​​(IV) were measured after 72 hours, and cytotoxicity was calculated as described above.

[0292] TIFF2026082962000012.tif44170

[0293] Table 12 shows the results for transduced T cells that were selected on day 8 and then co-cultured with wild-type CaSki cells in a 3:1 E:T ratio on day 10. Wild-type CaSki cells were then added every 2-3 days. After the 5th stimulation, the T cells were compared with wild-type cells or A in a 96-well xCELLigence plate. * SiHa cells were co-cultured with various E:T ratios. Impedance values ​​(IV) were measured after 72 hours, and cytotoxicity was calculated as described above.

[0294] TIFF2026082962000013.tif44170

[0295] Example 8 Cytotoxicity and cytokine production of NKG2D CAR and NKG2D CAR co-expressed with HPV-TCR co-cultured with HPV16+ tumor cell lines To address the safety of NKG2D-based CARs, T cells transduced with NKG2D CAR1 alone, NKG2D CAR2 alone, HPV-TCR+NKG2D CAR1, and HPV-TCR+NKG2D CAR2 were co-cultured with C33A cell lines, which are primarily NKG2D ligand-negative, and primary normal cervical epithelial cells with various E:T ratios, as shown in Tables 13 and 14. Primary normal cervical epithelial cells were selected due to their higher NKG2D ligand expression. Cytotoxic data from the xCELLigence RTCA MP platform and IFNγ production from the Meso Sector S 600 platform showed low responsiveness to both C33A and primary normal cervical epithelial cells. These data suggest that NKG2D CAR2 has a strong safety profile and is unlikely to exhibit off-target effects.

[0296] Table 13 shows the results for transduced T cells, sorted on day 8 and then co-cultured on day 10 with C33A cells or primary normal cervical epithelial cells in various E:T ratios in 96-well xCELLigence plates. After 24 hours, the supernatant was extracted and processed using a Meso Sector S 600 platform to determine the IFNγ concentration (pg / mL).

[0297] TIFF2026082962000014.tif48170

[0298] Table 14 shows the results for transduced T cells selected on day 8 and then co-cultured on day 10 with C33A cell lines or primary normal cervical epithelial cells in 96-well xCELLigence plates at various E:T ratios. After 24 hours, the supernatant was extracted and processed using a Meso Sector S 600 platform to determine the IFNγ concentration (pg / mL).

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[0300] Example 9 In vivo efficacy of NKG2D CAR against HPV16+ tumors in a mouse xenograft model. To determine the in vivo efficacy of HPV-TCR only, NKG2D CAR only, and HPV-TCR + NKG2D CAR in eliminating both wild-type and β2MKO HPV16+ solid tumors, 5E6 wild-type CaSki and 5E6 β2MKO CaSki cells were subcutaneously transplanted into the left and right flanks of 6-8 week old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ), respectively. Five study groups were included: vehicle control (PBS), non-transduced T cells (NTD), HPV-TCR only, NKG2D CAR2 only, and HPV-TCR + NKG2D CAR2. The tumor volume of wild-type CaSki cells was approximately 70 mm². 3 The β2MKO CaSki cells reached approximately 30 mm in size. 3 If it reaches 20 x 10 per mouse, 6Numerous T cells were adopted on day 6. IL-2 supplementation was not added. Tumors were measured every 3-4 days using digital calipers, and mouse body weight was recorded. Peripheral blood was collected 24 hours after T cell adoptive transfer, and then weekly thereafter. Peripheral blood T cell persistence was characterized by flow cytometry. Antibodies used to evaluate peripheral blood T cell persistence included anti-mouse CD45 clone 30-F11 (BioLegend), anti-human CD45 clone HI30 (BioLegend), anti-human CD4 clone RPA-T4 (BioLegend), anti-human CD8 clone SK1 (BioLegend), anti-human CD279 clone EH12.2H7 (BioLegend), anti-human HLA-DR clone L243 (BioLegend), anti-mouse TCRβ clone H57-597 (BioLegend), and anti-human CD314 clone 1D1. Examples include 1 (Biolegend) and L / D Blue (ThermoFisher). All flow cytometry data were collected using BD FACSDiva® software on LSR-Fortessa (BD LSR Fortessa®), and the data were analyzed using FlowJo (all BD Sciences). All antibody staining was performed in PBS containing 1% BSA at 4°C. The study period was 62 days. Overall, HPV-TCR was able to control the tumor growth of wild-type CaSki cells on the left flank, but showed no signs of tumor control of β2MKO CaSki cells transplanted on the right flank. In contrast, NKG2D CAR2 alone and HPV-TCR + NKG2D CAR2 successfully controlled the tumor growth of both wild-type and β2 MKO CaSki cells (see Tables 15 and 16). Furthermore, the body weight of tumor-bearing mice did not significantly change during the first few weeks after adoptive T cell transfer until the mice were removed from the study due to tumor size (see Table 17). Despite the absence of observable tumors in all mice receiving NKG2D CAR2 alone or HPV-TCR+NKG2D CAR2 T cells, most mice demonstrated recurrence of tumor growth, suggesting a lack of persistence of transduced T cells. This is further supported by the absence of transduced T cells in the peripheral blood three weeks after adoptive T cell transfer (see Table 18).

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[0314] In general, the terms used in the following claims should not be construed as limiting the claims to any specific embodiment disclosed herein and in the claims, but rather as encompassing all possible embodiments, along with the entire scope of equivalents to which such claims are entitled. Therefore, the claims are not limited by this disclosure.

Claims

1. It is a chimeric antigen receptor (CAR), NKG2D ectodomain and Transmembrane domain and 4-1BB co-stimulatory domain and A signaling domain including the CD3-zeta signaling domain, A chimeric antigen receptor, including one.

2. The CAR according to claim 1, further comprising a CD8-alpha-hinge domain.

3. The CAR according to claim 2, wherein the CD8-alpha-hinge domain comprises the amino acid sequence described in Sequence ID No.

15.

4. The CAR according to any one of claims 1 to 3, wherein the NKG2D ectodomain comprises the amino acid sequence described in SEQ ID NO:

3.

5. The CAR according to any one of claims 1 to 4, wherein the transmembrane domain further comprises a CD28 transmembrane domain.

6. The CAR according to claim 5, wherein the CD28 transmembrane domain comprises the amino acid sequence described in SEQ ID NO:

21.

7. The CAR according to any one of claims 1 to 6, wherein the 4-1BB costimulatory domain comprises the amino acid sequence described in SEQ ID NO: 33 or SEQ ID NO:

63.

8. The CAR according to any one of claims 1 to 7, wherein the CD3 zeta signaling domain comprises the amino acid sequence described in SEQ ID NO:

27.

9. The CAR according to any one of claims 1 to 8, wherein the signal transduction domain further comprises a CD3-epsilon signal transduction domain.

10. The CAR according to claim 9, wherein the CD3-epsilon signaling domain comprises the amino acid sequence described in SEQ ID NO: 31 or SEQ ID NO:

61.

11. A CAR according to any one of claims 1 to 10, comprising an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, and SEQ ID NO:

76.

12. A nucleic acid encoding a CAR according to any one of claims 1 to 11.

13. The nucleic acid according to claim 12, comprising a nucleotide sequence having at least 90% sequence identity with respect to a nucleotide sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, and SEQ ID NO:

77.

14. A recombinant vector comprising the nucleic acid described in claim 12 or 13.

15. The recombinant vector or nucleic acid according to claim 14, or the nucleus according to claim 12 or 13, further comprising a nucleic acid encoding an engineered T cell receptor (TCR). acid.

16. The recombinant vector according to claim 14 or the nucleic acid according to claim 12 or 13, wherein the recombinant vector or nucleic acid further comprises a nucleic acid encoding a second CAR specific to a tumor antigen.

17. The tumor antigens include HPV-16E6 and HPV-16E7, alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, and FR. a, a recombinant vector or nucleic acid according to claim 15 or claim 16, comprising Flt3, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-IIIRα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEM, or VEGFRII.

18. A host cell transformed with the nucleic acid or recombinant vector according to any one of claims 12 to 17.

19. A host cell transformed with the nucleic acid described in claim 12 or 13 or the recombinant vector described in claim 14, and a nucleic acid or recombinant vector encoding a tumor antigen-specifically modified T cell receptor (TCR) or a second CAR.

20. The tumor antigens include HPV-16E6 and HPV-16E7, alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, CS1, EGFR, EGFR family including ErbB2 (HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, and fetus. A host cell according to claim 19, comprising AchR, FRa, Flt3, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-IIIRα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesoserine, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEM, or VEGFRII.

21. The host cell according to any one of claims 18 to 20, wherein the host cell includes iPSCs, T cells, or NK cells.

22. A pharmaceutical composition comprising the host cell described in claim 21.

23. A method for treating a disease in a patient requiring treatment for the disease, comprising administering to the patient the host cells described in claim 21 or the pharmaceutical composition described in claim 22.

24. The method according to claim 23, wherein the host cells are allogeneic to the patient.

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