Chimeric Antigen Receptor

Abstract

Chimeric antigen receptors (CARs) comprising the NGK2D ectodomain are provided. Compositions, cells and cell therapies comprising same are provided. Methods of treatment are further provided.

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 May 24, 2021, and entitled "Chimeric Antigen Receptor," which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to the field of cell therapy, and more particularly to NKG2D chimeric antigen receptors. [Background technology]

[0003] Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic conversion to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate 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 them.

[0004] Current T cell therapies are based on human T cells that have been enriched or engineered to target and kill cancer cells in patients. 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 T cells to specific target cancer cells. Chimeric antigen receptors (CARs) containing binding domains capable of interacting with specific tumor antigens and engineered T cell receptors (TCRs) enable T cells to target and kill cancer cells expressing specific tumor antigens. However, some tumor types, particularly solid tumors, are resistant to T cell immunotherapy, necessitating the development of next-generation enhancement strategies to target tumor-specific resistance mechanisms in T cell immunotherapy. NKG2D ligands are expressed on most types of tumors, demonstrating the relative selectivity of ligand expression on tumor cells compared to healthy cells and representing targets for enhancing traditional T cell therapies. Summary of the Invention

[0005] Disclosed is 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. In embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 33. In embodiments, the CD3-zeta signaling domain comprises the amino acid sequence set forth 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 set forth in SEQ ID NO: 15. In embodiments, the NKG2D ectodomain comprises the amino acid sequence set forth 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 set forth 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 set forth in SEQ ID NO: 31.

[0006] Nucleic acids encoding the disclosed CARs and vectors comprising the same are disclosed. In embodiments, the recombinant vector or nucleic acid further comprises a nucleic acid encoding an engineered T cell receptor (TCR) specific for a tumor antigen. In embodiments, the recombinant vector or nucleic acid further comprises a nucleic acid encoding a second CAR specific for a tumor antigen. In embodiments, the tumor antigen is 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, or CS1. EGFR, EGFR family including ErbB2 (HERII), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-AI+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-AI+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TACI, TAG72, TEM, or VEGFRII.

[0007] Disclosed are host cells transformed with the disclosed nucleic acids or recombinant vectors. In embodiments, host cells are transformed with the disclosed nucleic acids or recombinant vectors and a nucleic acid or recombinant vector encoding an engineered T cell receptor (TCR) specific for a tumor antigen or a second CAR specific for a tumor antigen. In embodiments, the tumor antigen is 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, EpC The host cells include AM, FAP, fetal AchR, FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-AI+MAGEI, HLA-A2+MAGE1, HLA-AA3+MAGE1, HLA-AI+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TACI, TAG72, TEM, or VEGFRII. In embodiments, the host cells include induced pluripotent stem cells (iPSCs), T cells, or NK cells. Pharmaceutical compositions comprising the disclosed T cells and / or NK cells are disclosed. Disclosed are methods of treating a disease in a patient in need thereof, comprising administering to the patient the disclosed T cells and / or NK cells or pharmaceutical compositions. In embodiments, the host cells are allogeneic to the patient. DETAILED DESCRIPTION OF THE INVENTION

[0008] term

[0009] In order that this disclosure may be more readily understood, certain terms are first defined below. Additional definitions of the following terms, as well as other terms, are found throughout the specification.

[0010] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] As used herein, unless otherwise stated or clear from context, the term "or" is understood to be inclusive and encompasses both "or" and "and."

[0012] The term "and / or" as used herein should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" 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 encompass each of the following aspects: 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] As used herein, the term "for example" is used merely as an example, is not intended to be limiting, and should not be construed to refer only to the items explicitly listed herein.

[0014] Terms such as "greater than," "at least," and "greater than," e.g., "at least one," are used to mean, but are not limited to, 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, , 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 This 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, as well as any larger numbers or fractions therebetween.

[0015] Conversely, the term "less than or equal to" includes each value less than the recited 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, 55, 56, 57, 58, 59 ... Included are 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 smaller number or fraction in between is also included.

[0016] Terms such as "plurality," "at least two," "two or more," and "at least a second" are intended to mean, but are not limited to, 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, 104, 105 5, 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, 137, 1 This is understood to include 38, 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, as well as any larger numbers or fractions therebetween.

[0017] Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps. Whenever an embodiment is described herein with the term "comprising," it will be understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also presented.

[0018] Unless specifically stated or clear from the context, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one or more standard deviations as practiced in the art. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can 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 can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean a value that is up to an order of magnitude or up to 5 times greater. When a particular value or composition is presented in this disclosure, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0019] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof, as appropriate (such as integer tenths and hundredths), unless otherwise specified.

[0020] Units, prefixes and symbols used herein are presented using the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, e.g., Juo, "The Concise Dictionary of Biomedicine and Molecular Biology," 2 nd ed., (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 ed., (2006), Oxford University Press, provides one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0022] "Administering" refers to the physical introduction of an agent, such as an engineered T cell disclosed herein, into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion. The phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0023] The terms "activated" and "activation" refer to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. In one embodiment, activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to proliferating T cells. Signals generated through the TCR alone may be insufficient for full activation of T cells; one or more secondary or costimulatory signals may also be required. Thus, T cell activation includes a primary stimulatory signal via the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0024] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds to an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains, or antigen-binding molecules thereof, interconnected by disulfide bonds. 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 embedded in more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of Abs may mediate the binding of the immunoglobulin 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 tetrameric entities of approximately 150 kD, composed of two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD) that associate together in what is commonly referred to as a "Y-shaped" structure. 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 together, resulting in the dimers being connected to each other to form a tetramer. Naturally produced antibodies, e.g., C H It is glycosylated on two domains.

[0025] The term "human antibody" is intended to include antibodies having variable and constant domain sequences generated, assembled, or derived from human immunoglobulin sequences or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) can be considered "human" even if their amino acid sequences include residues or elements not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term "humanized" is intended to include antibodies having variable domains having sequences derived from the variable domain of a non-human species (e.g., murine) that have been modified to more closely resemble human germline coding sequences. In some embodiments, a "humanized" antibody comprises 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 comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant domain. In some embodiments, a humanized antibody comprises the C, C, D, E, E, F ... H 1. Hinge, C H 2. C H 3, and optionally, C H It may contain four regions.

[0026] Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising 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 herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (single-chain Fvs). Examples of antibodies include Fv, scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, antibodies as described herein refer to polyclonal antibody populations. Antibodies can also include, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof, and human heavy chain antibodies (UniAbs)), single chain or tandem diabodies (TandAb®), camelid antibodies, Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®.

[0027] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass encoded by the heavy chain constant region genes (e.g., IgM or IgG1). The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human or non-human antibodies, fully synthetic antibodies, and single-chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated or the context dictates otherwise, the term "antibody" includes antigen-binding fragments or portions of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain antibodies.

[0028] An "antigen-binding molecule," "antigen-binding portion," "antigen-binding fragment," or "antibody fragment," or "antigen-binding domain" refers to any molecule comprising an antigen-binding portion of a molecule. In one example, the antigen-binding molecule is an antibody or a portion thereof, such as an scFv. In one example, the antigen-binding molecule is a portion 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-binding molecule may be a portion of NKG2D that binds to an NKG2D ligand. The antigen-binding molecule may comprise an antigen complementarity-determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide-binding domain) are another example of a suitable antigen-binding molecule. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a hyperproliferative disease, or a viral or bacterial antigen. In 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 the antigen, comprising 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 comprises or consists of an avimer.

[0029] In some examples, the CDRs are substantially identical to those found in a reference antibody (e.g., an antibody of the present disclosure) and / or the sequence of the CDRs provided herein. In some embodiments, the CDRs are substantially identical to a reference CDR in that they are either identical in sequence or contain one, two, three, four, or five (e.g., one to five) amino acid substitutions compared to the reference CDR. In some embodiments, the CDRs are substantially identical to a reference CDR in that they exhibit 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%) to the reference CDR. In some embodiments, the CDR is substantially identical to a 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 a reference CDR in that one amino acid within the CDR has been deleted, added, or substituted compared to the reference CDR, while the CDR otherwise has an amino acid sequence identical to the amino acid sequence of the reference CDR. In some embodiments, the CDR is substantially identical to a reference CDR in that two, three, four, or five (e.g., two to five) amino acids within the CDR have been deleted, added, or substituted compared to the reference CDR, while the CDR otherwise has an amino acid sequence 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, e.g., binds to substantially the same 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 enzymatically or chemically produced by fragmentation of an intact antibody. In some embodiments, antigen-binding fragments can be recombinantly produced (such as by expression of an engineered nucleic acid sequence). In some embodiments, antigen-binding fragments can be wholly or partially synthetically produced. In some embodiments, antigen-binding fragments can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, and in some embodiments, 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 either the light or heavy chain, typically the amino-terminal 110-120 amino acids of a mature heavy chain and approximately 90-115 amino acids of a mature light chain, which vary significantly in sequence among antibodies and are used to determine the binding and specificity of a particular antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions within the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FR).

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

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

[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 an analysis of available complex crystal structures.

[0035] TIFF0007815283000001.tif58163

[0036] Terms such as "Kabat numbering" are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding molecule thereof. In certain embodiments, the CDRs 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, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acid positions 31-35 (which can optionally include one or two additional amino acids following 35, designated 35A and 35B in the Kabat numbering scheme) (CDR1), 50-65 (CDR2), and 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acid positions 24-34 (CDR1), 50-56 (CDR2), and 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 CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the position of the immunoglobulin structural loops (see, e.g., Chothia C & 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, using the Kabat numbering convention, the Chothia CDR-H1 loop is located at amino acids 26-32, 33, or 34 in the heavy chain, the Chothia CDR-H2 loop is located at amino acids 52-56 in the heavy chain, and the Chothia CDR-H3 loop is located at amino acids 95-102 in the heavy chain, while the Chothia CDR-L1 loop is located at amino acids 24-34 in the light chain, the Chothia CDR-L2 loop is located at amino acids 50-56 in the light chain, and the Chothia CDR-L3 loop is located at amino acids 89-97 in the light chain. The end of the Chothia CDR-HI loop, when numbered using the Kabat numbering convention, 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 are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). In certain embodiments, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme.

[0038] The terms "constant region" and "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interaction with Fc receptors. 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 an antibody, the term "heavy chain" may refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which types give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0040] When used in reference to an antibody, the term "light chain" can refer to any of the different types, e.g., 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 T-cell responses in humans or animals, including compositions (such as those containing tumor-specific proteins) injected or absorbed into humans or animals. Antigens react with specific humoral or cellular immune products, including those elicited by heterologous antigens such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is substantially not found on the surface of other normal (desired) cells and to which the binding domain of a TCR or CAR contemplated herein is designed to bind. Those skilled in the art will readily understand that virtually any macromolecule, including all proteins or peptides, can function as an antigen. Antigens can be endogenously expressed, i.e., expressed by genomic DNA, or recombinantly expressed. Antigens can be specific to a particular tissue, such as cancer cells, or can be broadly expressed. Furthermore, fragments of larger molecules can act as antigens. A "target" is any molecule that is bound by a binding motif, CAR, TCR, or antigen-binding agent, e.g., an antibody.

[0042] An "antigen-specific targeting region" (ASTR) refers to the region of a CAR or TCR that targets a specific antigen. The targeting region on a CAR or TCR is extracellular. In some embodiments, the antigen-specific targeting region comprises an antibody or its functional equivalent or a fragment or derivative thereof, with each targeting region targeting a different antigen. The targeting region may comprise 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 for a target antigen. However, numerous alternatives exist, such as linked cytokines (resulting in recognition of cells bearing cytokine receptors), affibodies, ligand-binding domains from naturally occurring receptors 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 the present disclosure. Indeed, as will be appreciated by those skilled in the art, almost any molecule that binds with high affinity to a given antigen may 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 costimulation.

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

[0045] Two events or entities are "associated" with one another 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 with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). For example, two or more entities are physically "associated" with one another if they directly or indirectly interact with one another, such that they come into and / or remain in physical proximity (e.g., bound to) one another. In a further example, two or more entities that are physically associated with one another may be covalently linked or connected to one another or non-covalently associated, for example, by hydrogen bonding, 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 later reintroduced. For example, the engineered autologous cell therapy (eACT™) method described herein involves the collection of lymphocytes from a patient, which are then engineered to express, for example, a CAR construct, and then administered to the same patient.

[0047] "Binding affinity" generally refers to the strength of the sum 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, which 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 is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium association constant (K A K D is k off / k onis calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k off 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 particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. D There is an inverse relationship between the binding affinity and the K D The smaller the value, the higher or stronger the affinity. Thus, the terms "higher affinity" or "stronger affinity" refer to a higher ability to form an interaction and therefore a smaller K D Conversely, the terms "lower affinity" or "weaker affinity" refer to a lower ability to form an interaction and therefore a larger K D In some situations, a higher binding affinity (or K ) of a particular molecule (e.g., an antibody) to its interaction partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., an antibody) to another interaction partner molecule (e.g., antigen Y) may be involved. D ) is larger than K D (lower or weaker affinity) with a smaller K D The binding affinity may be expressed as a binding ratio determined by dividing by the binding affinity (higher or stronger affinity), for example, as a 5-fold or 10-fold greater binding affinity in some cases.

[0049] "k d The term "(sec-1 or 1 / s)" refers to the dissociation rate constant of a particular 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. 0i Also called the r value.

[0050] "k a The term "(M-1 x 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 particular 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 k d It is obtained by dividing by

[0052] The term "binding" generally refers to a non-covalent association between two or more entities. Direct binding involves physical contact between the entities or moieties. "Indirect" binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can be assessed in any of a variety of contexts, such as when the interacting entities or moieties are studied alone or in a more complex system (e.g., while covalently or otherwise associated with a carrier entity and / or within a biological system such as a cell).

[0053] The terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by those of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined, for example, by immunoassays, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K A At least 2 log, 2.5 log, 3 log, 4 log, or more than KA and binds to its antigen with a binding affinity of about 10. Binding can include preferential association of a 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 an entity that is not the target (i.e., a non-target). In some embodiments, a binding domain, antibody, or antigen-binding system selectively binds to a target if the binding of the binding domain, antibody, or antigen-binding system with the target is more than 2-fold, more than 5-fold, more than 10-fold, more than 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more than 100-fold greater than binding of the binding domain, antibody, or antigen-binding system with the non-target. In some embodiments, a binding domain, antibody, or antigen-binding system has a binding affinity of about 10 -5 Less than M, about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M or about 10 -9 If it is less than M, it will selectively bind to the target.

[0054] In another embodiment, the molecules that specifically bind to an antigen are about 1 x 10 -7 Dissociation constant of M (K d In some embodiments, the antigen-binding molecule binds at K d is about 1×10 -9 M ~ approx. 5×10 -9 In some embodiments, an antigen-binding molecule specifically binds an antigen with a "high affinity" when the K d is 1×10 -10 M ~ approx. 5×10 -10 In one embodiment, the antigen-binding molecule specifically binds to an antigen with "very high affinity" when the -9 K of M d In one embodiment, the dissociation rate is about 1×10 -5 is less than.

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

[0056] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation leads to the formation of malignant tumors that may invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors. In some embodiments, the methods of the present disclosure are used to treat cancers such as, for example, prostate cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma, and the like. lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, 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 of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal pelvis, cancer of the central nervous system The compounds may reduce tumor size in tumors resulting from neoplasms of the central nervous system (CNS), primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including those induced by asbestos, other B-cell malignancies, multiple myeloma, and combinations of such cancers. Certain cancers may be responsive to chemotherapy or radiation therapy, or certain cancers may be refractory.Refractory cancer refers to cancer that is not amenable to surgical intervention; refractory cancers either do not respond to chemotherapy or radiation therapy initially, or the cancer becomes unresponsive over time.

[0057] A "chemokine" is 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 chemokine (MDC or CCL22), monocyte chemoattractant protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1α, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0058] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to contain a binding domain and a means to activate immune cells (e.g., T cells such as naive T cells, central memory T cells, effector memory T cells, NK cells, or a combination 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 engineered to express a chimeric antigen receptor may be referred to as CAR T cells. Similarly, NK cells engineered to express a chimeric antigen receptor may be referred to as CAR NK cells.

[0059] "Decrease" or "lower" or "alleviate" or "reduce" or "attenuate" generally refers to the ability of a composition contemplated herein to produce, induce, or cause a lower physiological response (i.e., downstream effect) compared to the response caused by either the vehicle alone (i.e., active moiety) or a control molecule / composition. A "decrease" or "reduced" amount is typically a "statistically significant" amount and can include a 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, or more (e.g., 500-fold, 1000-fold) (e.g., 1.5, 1.6, 1.7, 1.8, etc., above 1, including all integers and decimals in between) decrease in the response produced by the vehicle, the control composition (reference response).

[0060] "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that is understood to reside outside the cell membrane, in the extracellular space, when the polypeptide resides in the cell membrane. Ectodomain may be used interchangeably herein with extracellular domain.

[0061] The term "extracellular ligand-binding domain," as used herein, refers to a ligand, e.g., an oligo- or polypeptide, capable of binding to a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state (e.g., cancer). Examples of cell surface markers that can act as ligands include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

[0062] The binding domain of a CAR may be followed by a "spacer" or "hinge," which refers to a region that moves the antigen-binding domain away from the effector cell surface to allow for proper cell-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, and may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region, such as an 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 the wild-type hinge regions of these molecules or may be modified.

[0063] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding moiety to the plasma membrane of an immune effector cell and facilitates binding of the binding domain to a target antigen. The transmembrane domain can be a CD3 zeta transmembrane domain; however, other transmembrane domains that can be used include those obtained 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 contains primarily 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 transmitting the message of effective CAR binding to a target antigen to the interior of immune effector cells and 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 elicited by antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, aid or activity, including cytolytic activity or cytokine secretion. Thus, the terms "intracellular signaling domain" or "signaling domain," used interchangeably herein, refer to the portion of a protein that transmits an effector function signal and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, as long as it transduces the effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal. The intracellular signaling domain, also known as the "signaling domain," is typically derived from a portion of the human CD3 or FcRy chain.

[0065] It is known that signals generated through the T cell receptor alone are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the T cell receptor (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling domains known as immunoreceptor tyrosine-based activation domains or ITAMs.

[0066] Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present 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 term "costimulatory signaling domain" or "costimulatory domain" refers to a portion of a CAR that comprises the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or an Fc receptor, that provides a second signal required for efficient activation and function of T lymphocytes upon binding to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, 2B4, CD137, DAP12B7-H2, and ligands that specifically bind to CD83. Thus, although the present disclosure provides exemplary costimulatory domains derived from CD28, CD3-epsilon, and 4-1BB, other costimulatory domains are contemplated for use with the CARs described herein. Inclusion of one or more costimulatory signaling domains can enhance the efficacy and proliferation of T cells and NK cells expressing the CAR receptor. The intracellular signaling and costimulatory signaling domains can be linked in tandem, in any order, to the carboxyl terminus of the transmembrane domain.

[0068] CARs engineered to contain signaling domains 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 concomitant costimulatory signals. Other CARs containing binding domains, hinges, transmembrane and signaling domains derived from CD3 zeta or FcR gamma, along with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from 4-1BB, CD28, CD137, CD134, and CD278), can more effectively direct anti-tumor 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] A "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, results in a T cell response, including, but not limited to, proliferation and / or up-regulation or down-regulation of key molecules.

[0070] "Costimulatory ligands" include molecules on antigen-presenting cells that specifically bind to cognate costimulatory molecules on T cells. Binding of a costimulatory ligand provides a signal that mediates T cell responses, including, but not limited to, proliferation, activation, and differentiation. Costimulatory ligands induce signals, for example, by binding of the T cell receptor (TCR) / CD3 complex to peptide-loaded major histocompatibility complex (MHC) molecules, in addition to the primary signal provided by the stimulatory molecule. Costimulatory ligands include, but are not limited to, 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), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MHC class I chain-related protein B), and the like. B, MICB), OX40 ligand, PD-L2, or programmed death (PD) L1.Examples of costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, 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 cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, "costimulatory molecules" that are cognate binding partners on a T cell that specifically bind to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.Costimulatory 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), CD1-la, CD1-lb, CD1-lc, CD1-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, ITGB1, 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(CD1) la / CD18), MHC class I molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule, SLAM (SLAMF1, CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, 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 have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar 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 framework region of the antibody or antigen-binding molecule thereof can be replaced with an amino acid residue having a similar side chain. In general, two sequences are generally considered "substantially similar" if they contain conservative amino acid substitutions at corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substitution of another amino acid of the same type may be considered a conservative substitution. Exemplary amino acid classifications are summarized in Tables 2 and 3 below.

[0073] TIFF0007815283000002.tif102151

[0074] TIFF0007815283000003.tif30142

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

[0076] "Corresponding to" may be used to indicate the location / identity of a structural element in a molecule or composition through comparison to an appropriate 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 "corresponding to" a residue in an appropriate reference polymer. For example, for purposes of simplicity, residues in a polypeptide may be designated using a standard numbering system based on the reference related polypeptide, so that, for example, an amino acid "corresponding to" a residue at position 100 need not actually be the 100th amino acid in the amino acid chain, as long 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 can be utilized to identify "corresponding" residues in polypeptides and / or nucleic acids according to the disclosure.

[0077] An antigen-binding molecule, such as an antibody, its antigen-binding fragment, CAR, or TCR, "cross-competes" with a reference binding molecule, such as an antibody or its antigen-binding fragment, if the interaction between the antigen and a first antigen-binding molecule blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule to interact with the antigen. Cross-competition can be complete, e.g., 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, e.g., 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, an antigen-binding molecule that cross-competes with a reference antigen-binding molecule binds to the same or overlapping epitope as the reference antigen-binding molecule. In other embodiments, an antigen-binding molecule that cross-competes with a 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 antigen-binding molecule: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (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-labeled assay, solid-phase direct-labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), solid-phase direct-labeled RIA using 1-125 label (Morel et al. al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

[0078] A "cytokine" refers to a non-antibody protein released by one cell in response to contact with a specific antigen; the cytokine interacts with a second cell and mediates a response in the second cell. Cytokines can be endogenously expressed by a cell or administered to a subject. Cytokines can be released by immune cells, such as macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effector cytokines, and acute phase proteins. For example, homeostatic cytokines, such as interleukin (IL) 7 and IL-15, can promote the survival and proliferation of immune cells, while proinflammatory cytokines can promote an inflammatory response. 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 proinflammatory cytokines include, but are not limited to, 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), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF).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, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0079] The term "domain" refers to a portion of an entity. In some embodiments, a "domain" is associated with a structural and / or functional characteristic of an entity, e.g., such that when the domain is physically separated from the rest of its parent entity, the domain substantially or completely retains the structural and / or functional characteristic. In some embodiments, a domain can comprise a portion of an entity that, when separated from its (parent) entity and linked or connected to a different (recipient) entity, substantially retains and / or confers on the recipient entity one or more structural and / or functional characteristics characterized in the parent entity, for example. In some embodiments, a domain is a portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a portion of a polypeptide. In some such embodiments, a domain is characterized by a structural element (e.g., amino acid sequence or sequence domain, α-helical character, β-sheet character, coiled-coil character, random coil character, etc.) and / or by a functional characteristic (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0080] The term "dosage form" can be used to refer to a physically discrete unit of active agent (e.g., an antigen-binding system or antibody) for administration to a subject. Generally, each such unit contains a predetermined amount of active agent. In some embodiments, such amount is a unit dose (whole fraction thereof) appropriate for administration according to a dosing regimen determined to correlate with a desired or beneficial outcome when administered to a relevant population. The total amount of a therapeutic composition or agent to be administered to a subject is determined by one or more physicians and may involve administration of more than one dosage form.

[0081] The term "dosing regimen" can 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 dosing regimen that can include one or more doses. In some embodiments, the dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the dosing regimen includes multiple doses, where consecutive doses are separated from each other by equal periods of time; in some embodiments, the dosing regimen includes multiple doses, where consecutive doses are separated from each other by at least two different periods of time. In some embodiments, all doses within the dosing regimen are the same unit dose amount. In some embodiments, different doses within the dosing regimen are different amounts. In some embodiments, the dosing regimen includes a first dose at a first dose amount, followed by one or more additional doses at a second dose amount that is different from the first dose amount. In some embodiments, the dosing regimen is periodically adjusted to achieve a desired or beneficial result.

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

[0083] "Effector function" refers to the biological outcome of the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector functions can be antigen binding-dependent, antigen binding-independent, or both. ADCC refers to the lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve effector cells containing Fc receptors (FcRs) recognizing and subsequently killing antibody-coated target cells (e.g., cells expressing on their surface the antigen to which the antibody binds). Effector cells that mediate ADCC can include immune cells, including, but not limited to, one or more of natural killer (NK) cells, macrophages, neutrophils, and eosinophils.

[0084] The term "engineered autologous cell therapy," also known as adoptive cell transfer and abbreviated as "eACT™," refers to the process of harvesting a patient's own T cells and subsequently genetically modifying them to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells or NK cells can be engineered to express, for example, a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR). In some instances, CAR-positive (+) T cells or NK cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. In some instances, CAR-positive (+) T cells or NK cells are engineered to express the extracellular domain of NKG2D specific for the NKG2D antigen linked to an intracellular signaling moiety comprising at least one costimulatory domain and at least one activation domain. The costimulatory domain can be derived from a naturally occurring costimulatory domain or a variant thereof, e.g., a variant having a truncated hinge domain ("THD"), and the activation domain can be derived from, e.g., CD3-zeta and / or CD3-epsilon. In certain embodiments, CARs are designed to have two, three, four, or more costimulatory domains.

[0085] In some embodiments, the CAR is engineered such that the costimulatory domain is expressed as a separate polypeptide chain. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties. "Adoptive cell therapy" or "ACT" involves the transfer of immune cells with anti-tumor activity into a subject, e.g., a cancer patient. In some embodiments, ACT is a therapeutic approach involving the use of lymphocytes (e.g., engineered lymphocytes) with anti-tumor activity.

[0086] "Epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, from a polypeptide or two or more non-contiguous regions of a polypeptide (a conformational, non-linear, discontinuous, or discontinuous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any of the methods known in the art (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 and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al., U.S. Patent Application Publication No. 2004 / 0014194) and BUSTER (see, Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW, Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art, e.g., see Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0087] "Endogenous" with respect to a gene, protein, and / or nucleic acid refers to the natural occurrence of that gene, protein, and / or nucleic acid in a cell, such as an immune cell.

[0088] "Exogenous" refers to an agent, such as a nucleic acid, gene, or protein, being introduced into a cell, e.g., from an external source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein that is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase expression of the protein above levels that would naturally be found in the cell under similar conditions, e.g., without the introduction of the exogenous nucleic acid.

[0089] The term "excipient" refers to an agent that may be included in a composition to, for example, 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, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc.

[0090] A "fragment" or "portion" of a material or entity described herein has a structure that comprises a distinct portion of a whole, e.g., a physical or abstract entity. In some embodiments, a fragment lacks one or more portions found in the whole. In some embodiments, a fragment consists of or comprises a characteristic structural element, domain, or portion found in the whole. In some embodiments, a polymer fragment comprises 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 monomeric units (e.g., residues) found throughout the polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polymer (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). The whole material or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0091] The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. Generally, a polypeptide containing at least two such segments is considered to be a fusion polypeptide if the two segments are (1) portions that are not inherently contained in the same peptide, and / or (2) portions that are not previously linked or connected to each other in a single polypeptide, and / or (3) portions that are linked or connected to each other through the action of the human hand. In embodiments, a CAR is a fusion protein. In embodiments, a TCR is a fusion protein.

[0092] The term "gene product" or "expression product" generally refers to the RNA (before and / or after processing) transcribed from a gene or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0093] The terms "genetically engineered" or "engineered" refer to methods of modifying a cell's genome, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell or an NK cell, and can be obtained from either a patient or a donor. In some embodiments, the modified cell is an induced pluripotent stem cell (iPSC) that can differentiate into a lymphocyte, such as a T cell or an NK cell. The cell may also be engineered to express an exogenous construct, e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the cell's genome. Other gene editing can also be performed, for example, to reduce rejection and / or enhance cytocompatibility. Engineering generally includes manipulation by the hand of man. For example, a polynucleotide is considered "engineered" if it is manipulated by the hand of man so that two or more sequences that are not naturally linked or connected in that order are directly linked or connected to each other in the engineered polynucleotide. In the context of cell manipulation by molecular biology techniques, a cell or organism is considered "engineered" if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutation, or by other protocols). In some embodiments, the binding agent is an engineered lymphocyte, e.g., a T cell or NK cell, and can be obtained from either a patient or a donor. Engineered cells can be engineered to express an exogenous construct, e.g., a chimeric antigen receptor (CAR) or T cell receptor (TCR), that is integrated into the genome of the cell. Progeny of an engineered polynucleotide or binding agent are generally referred to as "engineered," even if the actual manipulation was performed on the previous entity. In some embodiments, "engineered" refers to a designed and manufactured entity.The term "designed" refers to an agent that (i) has a structure selected by or through the human hand, (ii) is produced by a process requiring the human hand, and / or (iii) is distinct from natural substances and other known agents.

[0094] "T cell receptor" or "TCR" refers to an antigen-recognition molecule present on the surface of a T cell. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to result in a wide variety of TCR proteins. Examples of TCR-based T cell therapy are disclosed in International Application Nos. PCT / US2013 / 059608 and PCT / US2015 / 033129, which are incorporated by reference in their entireties.

[0095] The term "heterologous" refers to a sequence derived from any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein is an amino acid that does not naturally occur as a wild-type human costimulatory protein, i.e., does not align with the wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human costimulatory protein coding sequence.

[0096] The term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for calculating the percent identity between two provided polypeptide sequences are known. For example, calculating the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When 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, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, optionally 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. Comparison or alignment of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0097] To calculate percent identity, the sequences to be compared are typically aligned in a way that maximizes the correspondence between the sequences. An example of a computer program that can be used to determine percent 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 whose percent sequence identity is to be determined. The sequences are aligned for optimal matching of each amino acid or nucleotide (the "matched span" determined by the algorithm). In certain embodiments, standard comparison matrices (for the PAM250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used in the algorithm. Other algorithms are available for comparing amino acid 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.Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, 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, the above programs generally provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be 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 (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) over a relevant stretch of residues. In some embodiments, the relevant stretch is the entire sequence.In some embodiments, the related stretch is 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, at least 500 or more residues. Sequences having substantial sequence similarity may be homologs of each other.

[0098] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate 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 of the nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0099] The terms "substantial similarity" or "substantially similar," as applied to polypeptides, mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

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

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

[0102] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from or relapsing from a disease by methods that involve inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, NK cell and T cell therapy. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one skilled in the art will understand that the conditioning methods disclosed herein will enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and WO 2008 / 081035. Examples of TCR-based T cell therapies are disclosed in International Application Nos. PCT / US2013 / 059608 and PCT / US2015 / 033129, which are incorporated by reference in their entireties.

[0103] T cells or NK cells for immunotherapy can 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 can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood drawn from a subject using various techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

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

[0105] The term "isolated" refers to (1) a substance that is separated from at least some components with which it was previously associated or would otherwise be associated, and / or (2) a substance that is present in a composition that includes limited or defined amounts or concentrations of one or more known or unknown contaminants. An isolated substance may, in some embodiments, be separated from 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., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of other components that are not the substance with which the substance was previously associated, e.g., other components or contaminants with which the substance was previously or otherwise associated. In certain instances, a substance is isolated when it is present in a composition comprising a limited or reduced amount or concentration of the same or similar type of molecule. For example, in certain instances, a nucleic acid, DNA, or RNA substance is isolated when it is present in a composition containing a limited or reduced amount or concentration of the nucleic acid, DNA, or RNA molecule that is not the substance. For example, in certain instances, a polypeptide substance is isolated when it is present in a composition containing a limited or reduced amount or concentration of the polypeptide molecule that is not the substance. In certain embodiments, the amount can be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, a limited amount can be an amount that is 100% or less of the amount of the substance in the composition, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or less (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of the amount of the substance in the composition. In certain instances, the composition is pure or substantially pure with respect to the selected substance.In some embodiments, an isolated material is 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 greater than about 99% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) pure. A material is "pure" if it is substantially free of other components or contaminants. In some embodiments, a material may still be considered "isolated" or "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the material is calculated without including such carriers or excipients.

[0106] "Linker" (L) or "linker domain" or "linker region" refers to an oligo- or polypeptide region, about 1-100 amino acids in length, that links together, for example, any of the domains / regions of a CAR, TCR, and / or scFv, or at least one of many of these polypeptides. Linkers can be composed of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to one another. Longer linkers may be used if it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, a porcine teschovirus (P2A), a CHYSEL sequence (SEQ ID NO: 1) of a virus (T2A), or combinations, variants, and functional equivalents thereof. In other embodiments, the linker sequence is Asp-Val / Ile-Glu-X-Asn-Pro-Gly, which results in cleavage between 2A glycine and 2B proline. (2A) -Pro (2B)The linker may comprise the domain (SEQ ID NO: 2). Other linkers will be apparent to those skilled in the art and may be used in connection with the present disclosure. A linker may be part of a multi-element agent that connects different elements to one another. For example, a polypeptide comprising two or more functional or structural domains may comprise a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, where S1 and S2 may be the same or different and represent two domains associated with one another by the linker. The linker may connect or link together either the CAR or TCR domains / regions. In some embodiments, the polypeptide linker comprises 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, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 11 The linker may be 0, 95, 100, or more amino acids in length (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 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 amino acids in length). In some embodiments, the linker is characterized by not tending to adopt a rigid three-dimensional structure, but instead providing flexibility to the polypeptide. In another example, it may be used to connect multiple polypeptides to be expressed, such as a CAR and / or a TCR.

[0107] Other linkers include non-cleavable linkers. To realize the present invention, a variety of linkers, including "flexible linkers," are used. The latter are glycine-rich. 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, e.g., 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, e.g., 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, e.g., 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 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 (cell-toxic) lymphocyte that represent a component 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 were 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 specialized 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, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells), memory T cells (i.e., stem memory T cells), and cytotoxic T cells (TCs). SCM(ii) central memory T cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, similar to naive cells, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting multiple functional attributes unique to memory cells; CM (iii) effector memory T cells express L-selectin and CCR7 and secrete IL-2, but they do not secrete IFNγ or IL-4. EM There are various types of T cells: CD4+CD25+ regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. B cells, on the other hand, play a role in humoral immunity (involving antibodies). B cells produce antibodies and antigens, act as antigen-presenting cells (APCs), and transform into memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence the name.

[0111] The term "neutralizing" 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 otherwise alters the ability of the ligand to bind through indirect means (such as a structural or energetic change in 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 polymeric chain of nucleotides. Nucleic acids can be DNA, RNA, or a combination thereof. In some embodiments, nucleic acids comprise one or more naturally occurring nucleic acid residues. In some embodiments, nucleic acids are comprised of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from natural sources, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, reproduction in recombinant cells or systems, and chemical synthesis. 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 nucleic acid may be 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length (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; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide.

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

[0114] A "patient" includes any human 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, with no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins, of which there are many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural 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 the recipient or the 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 ingredients of the composition and not harmful to the recipient, or any harmful effects must be outweighed by the benefits to the recipient. The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a drug from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or the benefits to the recipient must outweigh any harmful effects. Some examples of materials that can serve 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; powdered tragacanth; 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, 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, compatible 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 treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, the pharmaceutical compositions may be formulated for administration in solid or liquid form, including, but not limited to, forms adapted for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, as a sterile solution or suspension, or as a sustained release formulation; topical administration, e.g., as a cream, ointment, or sustained release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingually; ophthalmically; transdermally; or intranasally, to the lungs, and other mucosal surfaces.

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

[0119] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially contemporaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, a reference or control is determined or characterized under conditions or circumstances comparable to those under evaluation, where similarity is sufficient to justify reliance on and / or comparison to the selected reference or control.

[0120] "Regulatory T cells" ("Tregs," "Treg cells," or "Tregs") refer to a lineage of CD4+ T lymphocytes involved in controlling certain 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 specific innate immune system cell types. Tregs can be identified by expression of the biomarkers CD4, CD25, and Foxp3, as well as low expression of CD127. Naturally occurring Treg cells typically comprise approximately 5-10% of peripheral CD4+ T lymphocytes. However, Treg cells within the tumor microenvironment (i.e., tumor-infiltrating Treg cells) can comprise as much as 20-30% of the total CD4+ T lymphocyte population.

[0121] The term "sample" generally refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, the source of interest is a biological source or an environmental source. In some embodiments, the source of interest may include a cell or organism, such as a cell population, a tissue, or an animal (e.g., a human). In some embodiments, the source of interest comprises a biological tissue or fluid. In some embodiments, the biological tissue or fluid may include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural effusion, pus, catarrhal secretions, saliva, sebum, semen, semen, semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, biological fluids may include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cellular fluid. In some embodiments, biological fluids may include plant exudates. In some embodiments, biological tissues or samples 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, ductal, nasal, ocular, oral, uterine, vaginal, or other lavage). In some embodiments, biological samples include cells obtained from an individual. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as is clear from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample). Such a "processed sample" 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, etc.

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

[0123] The term "cancer stage" refers to a qualitative or quantitative assessment of the level of progression of cancer. In some embodiments, criteria used to determine the stage of cancer 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, whether the cancer has spread to one or more different parts of the body, etc. In some embodiments, cancer may be staged using the so-called TNM system, where 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 that have cancer, and M refers to whether the cancer has metastasized. In some embodiments, cancer may be referred to as Stage 0 (abnormal cells are present without spreading to nearby tissues, also known as carcinoma in situ or CIS; CIS is not cancer but may become cancer), Stage I-III (cancer is present, and the larger the number, the larger the tumor and the more it has spread to nearby tissues), or Stage IV (cancer has spread to distant parts of the body). In some embodiments, the cancer may be assigned a stage selected from the group consisting of: in situ, localized (cancer is limited to where it began and there is no sign it has spread), regional (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] "Stimulation" refers to a primary response induced by binding of a stimulatory molecule to its cognate ligand, which mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an APC, a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, such as, but not limited to, activation, initiation of an immune response, or proliferation. Stimulatory ligands include, but are not limited to, anti-CD3 antibodies (e.g., OKT3), peptide-loaded MHC class I molecules, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0125] The phrase "therapeutic agent" may refer to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be therapeutic if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a model organism or a population of human subjects. In some embodiments, the appropriate population may be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical conditions, the presence or absence of biomarkers, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, reduce, inhibit, prevent, delay the onset of, reduce the severity of, 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 a drug that has been or needs to be approved by a government agency before it can be commercially available for administration to humans. In some embodiments, a therapeutic agent is a drug that requires a medical prescription for administration to humans.

[0126] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective administration amount" of a therapeutic agent, e.g., 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 developing disease or promotes regression of disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of disability or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or 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, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0128] "Transformation" refers to any process by which exogenous DNA is introduced into a host cell. Transformation can occur under natural or artificial conditions using a variety of methods. Transformation can be achieved using any known method for the insertion of foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. In some embodiments, some transformation methodologies are selected based on the host cell to be transformed and / or the nucleic acid to be inserted. Methods of transformation may include, but are not limited to, viral infection, electroporation, and lipofection. In some embodiments, a "transformed" cell is stably transformed in that the inserted DNA is capable of replicating as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cell can express the introduced nucleic acid.

[0129] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical manifestations associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment can be treatment of a subject who does not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment can be treatment of a subject who exhibits one or more definitive signs of the associated disease, disorder, and / or condition. In some embodiments, treatment can be treatment of a subject who has been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment can be treatment of a subject who is known to have one or more susceptibility factors statistically correlated with an increased risk of developing the associated 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 are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Furthermore, certain vectors contain sequences that direct the expression of inserted genes to which they are operably linked. Such vectors may be referred to herein as "expression vectors." Standard techniques can be used to manipulate vectors, for example, as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d 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 either single-stranded or double-stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. The donor sequence may be of any length, for example, 2 to 10,000 nucleotides in length (or any integer value therebetween or greater), preferably about 100 to 1,000 nucleotides in length (or any integer value therebetween), and more preferably about 200 to 500 nucleotides in length.

[0132] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product (see below) and all DNA regions that regulate the production of that gene product, whether or not such regulatory sequences flank the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0133] A "transmembrane domain" is a domain of a polypeptide that comprises at least one contiguous amino acid sequence that spans a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. For example, a transmembrane domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acid sequences that each span a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. A transmembrane domain may comprise, for example, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) contiguous amino acid sequence that has an α-helical secondary structure in a lipid bilayer (which spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). In some embodiments, a transmembrane domain may comprise two or more contiguous amino acid sequences that form a β-barrel secondary structure in a lipid bilayer (which each spans the lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell). Non-limiting examples of transmembrane domains are described herein. Further examples of transmembrane domains are known in the art.

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

[0135] The present disclosure may employ, unless specifically indicated to the contrary, methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art, many of which are described below by way of example, and such techniques are explained more fully 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 Genomes, (Academic Press, New York, 1992), Transcription and Translation (B. Hames & S. Higgins, Eds., 1984), Perbal, A Practical Guide to Molecular See monographs in journals such as Cloning (1984), Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998), Current Protocols in Immunology QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991), Annual Review of Immunology, and Advances in Immunology.

[0136] The present disclosure provides antigen receptors (CARs), referred to herein as NKG2D CARs, that comprise a portion of the extracellular domain of NKG2D capable of binding to one or more NKG2D ligands. Among other things, the present disclosure provides methods and compositions useful for treating cancer and / or initiating or modulating an immune response. In some embodiments, the NKG2D CAR is expressed with a TCR specific for one or more tumor antigens and / or one or more additional CARs specific for one or more tumor antigens.

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

[0138] Various embodiments of the present disclosure provide cells encoding or expressing an NKG2D CAR, e.g., induced pluripotent stem cells (iPSCs), T cells, or NK cells engineered to encode or express an NKG2D CAR. Various embodiments of the present disclosure provide cells encoding or expressing an NKG2D CAR and a TCR or one or more additional CARs, e.g., T cells or NK cells engineered to encode or express an NKG2D CAR and a TCR or one or more additional CARs. The present disclosure provides immune cells genetically modified with an integrated gene, e.g., a nucleotide sequence of interest (e.g., a constitutive expression construct and / or an inducible expression construct comprising such a nucleotide sequence). In embodiments, the immune cells are further engineered to express a TCR or one or more additional CARs. In some embodiments, the present disclosure provides a method of treating a subject having a tumor, comprising administering to the subject an NKG2D CAR therapy described herein. In some embodiments, the methods further comprise administration of one or more additional therapies (e.g., a second binding agent (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-engaging bispecific antibody, an engineered antibody, and / or a polypeptide described herein).

[0139] Natural killer cells preferentially express several calcium-dependent (C-type) lectins, which are 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 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 protein. Surface expression of these ligands is important for the recognition of stressed cells by the immune system; therefore, this protein and its ligands are therapeutic targets for the treatment of immune diseases and cancer.

[0140] NKG2D ligands are inducible self-proteins that are absent or present at low levels on the surface of normal cells but are overexpressed by infected, transformed, senescent, and stressed cells. Their expression is regulated at different stages (transcription, mRNA and protein stabilization, and 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 the disclosed NKG2D CARs, an NKG2D ligand) and, upon binding to that target, activate immune cells to attack and destroy cells bearing that target. If these targets are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells. CARs generally include an extracellular binding domain (e.g., an NKG2D ectodomain) that mediates antigen binding, a transmembrane domain that is understood to span or translocate the cell membrane if the CAR is present on the cell surface or membrane, and an intracellular (or cytoplasmic) signaling domain.

[0142] According to at least one non-limiting aspect, there are at least three "generations" of CAR compositions. In first-generation CARs, a binding domain (e.g., a single-chain fragment variable binding domain) is linked or connected to a signaling domain (e.g., CD3ζ) via a transmembrane domain, optionally including a hinge domain and one or more spacers. In second-generation CARs, a costimulatory domain (CM1, such as CD28, 4-1BB, or OX-40) is introduced along with the signaling domain (e.g., CD3ζ). In third-generation CARs, a second costimulatory domain (CM2) is included.

[0143] TCR is a heterodimer composed of an α chain and a β chain. TCR signaling requires the recruitment of signaling proteins to generate an immune synapse. In addition, the localization of the TCR at the plasma membrane depends on the CD3 complex expressed in T cells. Engineered single-chain 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 TCR β chain with CD28 TM and CD28 and CD3ζ signaling modules).

[0144] The NKG2D CARs of the present disclosure may comprise an extracellular NKG2D domain that binds to an NKG2D ligand. In some embodiments, the antigen binding system further comprises 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, a CD3-zeta, and / or a CD3-epsilon activation domain.

[0145] In certain embodiments, an NKG2D CAR comprises an NKG2D ectodomain (extracellular domain) polypeptide, which refers to a polypeptide 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 SEQ ID NO: 3: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (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 a nucleic acid having the sequence set forth 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, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth 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 a nucleic acid having the sequence set forth below: TTATTCAACCAAGAAGTCCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAGAACTGGATATGTTACAAAAATAACTGTTACCAATTCTTCGATGAATCTAAGAA TTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTTAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGG CAGTGGGAAGACGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCATCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACATATATTTGCATGCAAAG GACTGTG (SEQ ID NO: 55).

[0146] In some embodiments, an NKG2D CAR of the present disclosure may comprise an antigen binding system comprising one or more or all of 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 activation domain (A). In some examples, an NKG2D CAR is configured according to the following BETA: In certain examples, the activation domain comprises one or more activation domains. In certain aspects, the activation domain comprises CD3ζ, CD3ε, or both CD3ζ and CD3ε. In some examples, an NKG2D CAR is configured according to the following PBETA: In some examples, an NKG2D CAR is configured according to the following BETCA: In some examples, an NKG2D CAR is configured according to the following PETCA: In some examples, an NKG2D CAR is configured according to the following BETC C' A: In some examples, an NKG2D CAR is configured according to the following PBETC C' A:

[0147] In certain embodiments, CARs contemplated herein may include linker residues added between the various domains for proper spacing and conformation of the molecule. 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. In some embodiments, the linker is 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 amino acids in length.

[0148] Illustrative examples of linkers include glycine polymers (G), glycine-serine polymers (G 1~5 S 1~5)n (where 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 the CARs described herein. Glycine also has better access to phi-psi space than alanine and is much less restricted 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 CAR designs in some embodiments can include a fully or partially flexible linker, whereby the linker can include a flexible linker as well as one or more moieties that confer less flexibility to provide a desired CAR structure. In one embodiment, any of the constructs described herein can include a "GS" linker. In another embodiment, any of the constructs described herein includes a "GSG" linker. In one example, the glycine-serine linker comprises or consists of the amino acid sequence GS (SEQ ID NO: 6), which can be encoded by a nucleic acid sequence according to ggatcc (SEQ ID NO: 7) or gggtcc (SEQ ID NO: 8). In one example, the glycine-serine linker comprises or consists of the amino acid sequence GGGSGGGS (SEQ ID NO: 9), which can be encoded by a nucleic acid sequence according to ggcggtggaagcggaggaggttcc (SEQ ID NO: 10). In another embodiment, a CAR described herein comprises an amino acid sequence 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 that of SEQ ID NO: 11 (GSTSGSGKPGSGEGSTKG (SEQ ID NO: 11)).In one embodiment, the linker is encoded by a nucleic acid sequence having at least 75% sequence identity (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%) to the following nucleic acid sequences: gggagcactagcggctctggcaaacctggatctggcgagggatctaccaagggc (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 serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. CARs generally include one or more hinge domains between the binding domain and the transmembrane domain. The hinge domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0150] In some embodiments, CARs contemplated herein may include a hinge that is, is from, or is derived from an immunoglobulin-like hinge domain (e.g., comprising all or a fragment of an immunoglobulin-like hinge domain). In some embodiments, the hinge domain is from an immunoglobulin. In some embodiments, the hinge domain is selected from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM hinge, or a fragment thereof. The hinge may be derived from a natural or synthetic source. 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 the wild-type hinge regions of these molecules or may be modified. The hinge may be derived from a natural or synthetic source. In some embodiments, the antigen binding system of the present disclosure is selected from the group consisting of CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, 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(BAF FR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD3 35(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, activating NK cell receptor, or Toll ligand receptor, or a fragment or combination thereof, or may comprise a hinge that is, is, or is derived from (e.g., includes all or a fragment thereof).

[0151] The polynucleotide and polypeptide sequences of these hinge domains are known. In some embodiments, a polynucleotide encoding a hinge domain comprises a nucleotide sequence that is 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 to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the hinge domain comprises a polypeptide sequence that is 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 to a known polypeptide sequence.

[0152] In embodiments, the hinge domain comprises a CD8α hinge region. In embodiments, a CAR described herein comprises a hinge domain derived from CD8α having an amino acid sequence with 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 SEQ ID NO: 15TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 15). In embodiments, the hinge domain 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 a nucleic acid having the sequence set forth below: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 16). In embodiments, the hinge domain 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 a nucleic acid having the sequence set forth below: ACAACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGAGGCCTGAAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCTTGTGAC (SEQ ID NO: 17).In embodiments, the hinge domain 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 a nucleic acid having the sequence set forth below: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGCGCCCCGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 18). In embodiments, the hinge domain 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 a nucleic acid having the sequence set forth below: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAACCCCTGTCCCTGAGGCCTGAAGCGTGCCGGCCAGCGGCGGGCGGCGCAGTGCACACGAGAGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 56).

[0153] In embodiments, the hinge domain comprises a truncated CD28 hinge region (CD28T) hinge region, as disclosed in International Application No. PCT / US2017 / 025351, filed March 31, 2017, which is incorporated herein by reference in its entirety. In embodiments, a CAR described herein comprises a CD28T hinge domain having an amino acid sequence with 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 SEQ ID NO: 19 (LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 19)) to SEQ ID NO: 230. In embodiments, 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 a sequence according to the following: CTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC (SEQ ID NO: 20).

[0154] Generally, a "transmembrane domain" (e.g., of an antigen-binding system) refers to a domain that, when present in a molecule on the cell surface or in the cell membrane, has the property of being intramembrane (e.g., spanning part or all of the cell membrane). The costimulatory domain of the antigen-binding system of the present disclosure may further comprise a transmembrane domain and / or an intracellular signaling domain. Not all amino acids in a transmembrane domain need be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized by a specified stretch or portion of a protein being located substantially within the membrane. Amino acid or nucleic acid sequences can be analyzed using various algorithms to predict the subcellular localization (e.g., transmembrane localization) of proteins. 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 is naturally associated with the binding domain and / or the intracellular domain is selected. In some instances, the transmembrane domain contains one or more amino acid modifications (e.g., deletions, insertions, and / or substitutions) to, for example, prevent binding of such domains to transmembrane domains of the same or different surface membrane proteins and minimize interaction with other members of the receptor complex.

[0156] The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains include the alpha, beta, or zeta chains of the T cell receptor, 2B4, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, 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, activating NK cell receptor, immune Globulin proteins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRTAM, cytokine receptors, 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, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, CD83-binding ligand, LIG HT, 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), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244;2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof (e.g., may include at least the transmembrane domain). In some embodiments, the transmembrane domain may be synthetic (and may comprise primarily hydrophobic residues, such as, for example, leucine and valine). In some embodiments, a phenylalanine, tryptophan, and valine triplet is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked or connected to the cytoplasmic domain. In some embodiments, a short oligo- or polypeptide linker (e.g., 2-10 amino acids in length) may form the 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 the transmembrane domains provided herein are known. In some embodiments, a polynucleotide encoding a transmembrane domain comprises a nucleotide sequence that is 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 to a known nucleotide sequence. In some embodiments, the polypeptide sequence of the transmembrane domain comprises a polypeptide sequence that is 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 to a known polypeptide sequence. Optionally, a short spacer can form a link between any or some of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0158] In embodiments, the NKG2D CAR described herein comprises a TM domain derived from CD28 having an amino acid sequence with 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 that of SEQ ID NO: 21 (FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 21)). In embodiments, the TM domain 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 a nucleic acid having the sequence set forth below: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 22). In embodiments, the TM domain 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 a nucleic acid having the sequence set forth below: TTTTGGGTATTGGTAGTAGTGGGCGGAGTCCTGGCTTGCTATAGTCTGCTAGTAACAGTGGCTTTTATTATATTTTGGGTG (SEQ ID NO: 23).In embodiments, the TM domain 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 a nucleic acid having the sequence set forth below: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 24).

[0159] In embodiments, a CAR described herein comprises a TM domain from CD8α having an amino acid sequence with 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 that of SEQ ID NO: 25 (IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 25)). In embodiments, the TM domain 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 a nucleic acid having the sequence set forth below: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTATTGC (SEQ ID NO: 26). In embodiments, the TM domain 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 a nucleic acid having the sequence set forth below: ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 57).

[0160] Intracellular signaling domains that can transmit signals upon antigen binding to immune cells are known, and any of these can be included in the antigen-binding system of the present disclosure. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction after TCR binding to an antigen (e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0161] In some embodiments, a CAR contemplated herein comprises an intracellular signaling domain. An "intracellular signaling domain" refers to the portion of a CAR that is responsible for transmitting the message of an effective CAR binding to a target antigen to the interior of an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into the CAR-bound target cell or other cellular responses elicited by antigen binding to the extracellular CAR domain. In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor 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 (see, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2:a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27:591-619 (2009)).In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin proteins, 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, 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, ligand binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108), 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), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0162] The term "effector function" refers to a specialized function of a cell. Effector functions of T cells can be, for example, aid or activities including cytolytic activity or cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be used, but in many cases, it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the entire domain, so long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

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

[0164] Illustrative examples of ITAMs containing primary signaling domains useful in the present disclosure include those derived from TCRζ, FcRγ, FcRβ, DAP12, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In some embodiments, a CAR comprises a CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order. In one embodiment, a CAR comprises a CD3ζ domain having an amino acid sequence 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 SEQ ID NO: 27. LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 27).In embodiments, the CD3 zeta 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 sequence set forth below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGGCAGAACCAACTCTATAACGAGCTCAATCTAGGAAGGAGA GAAGAGTACGATGTTCTAGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCACGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTACAAAAAGATAAAATGGCGGAGGCCTACAGT GAGATTGGCATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGCCTCAGTACAGCCACCAAGGACACCTATGACGCCCTTCACATGCAAGCTCTGCCCCCTCGC (SEQ ID NO: 28).In embodiments, the CD3 zeta 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 sequence set forth below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAACGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGCATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGACGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 29).

[0165] In embodiments, the CD3 zeta 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 sequence set forth below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 30).In embodiments, the CD3 zeta 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 sequence set forth below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAAGCTCTGCCCCCTCGCTGA (SEQ ID NO: 58).In embodiments, the CD3 zeta 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 sequence set forth below: CTGAGAGTTAAGTTCAGCAGGAGCGCCGACGCCCCTGCCTACCAGCAAGGACAGAATCAACTGTACAACGAGCTGAACCTGGGCAGACGGGA GGAATACGATGTGCTGGACAAGAGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCTAGAAGAAAGAACCCCCAGGAGGGCCTGTATAACGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGA GATCGGCATGAAGGGCGAAAGAAGAAGAGGCAAGGGCCACGACGGCCTCTACCAGGGCTTAAGCACAGCTACAAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGATGA (SEQ ID NO: 59).In embodiments, the CD3 zeta 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 sequence set forth below: CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAAGGGCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA GGAGTACGATGTTTTGGACAAGAGGCGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAAGCTCTGCCCCCTCGCTGA (SEQ ID NO: 60).

[0166] In some embodiments, the CAR comprises a CD3ζ signaling domain, a CD3ε signaling domain, and one or more costimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains can be linked in tandem to the carboxyl terminus of the transmembrane domain in any order. In embodiments, the CAR has a CD3ε domain having an amino acid sequence with 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 that of SEQ ID NO: 31. KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGL (SEQ ID NO: 31). In embodiments, the CAR has a CD3ε domain having an amino acid sequence with 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 that of SEQ ID NO: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 61).

[0167] In embodiments, the CD3 epsilon 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 sequence set forth below: AAGAACCGAAAAGCAAAAGCCAAGCCTGTTACAAGAGGAGCAGGGGCAGGAGGCCGACAGAGAGGGCAAAACAAAGAAAGGCCCCCGCCCGTCCCAAACCCGGATTATGAGCCAATTAGGAAGGGTCAGAGAGACCTGTATTCTGGGCTC (SEQ ID NO: 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, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth below: AAGAACCGCAAAGCAAAGGCAAAACCCGTCACACGAGGAGCGGGCGCAGGGGGACGACAACGCGGTCAGAATAAGGAACGCCCGCCTCCAGTACCAAATCCAGATTATGAACCAATTCGGAAGGGACAACGCGATCTCTACTCCGGTCTCAATCAGAGGCGAATT (SEQ ID NO: 62).

[0168] The CARs contemplated herein comprise one or more costimulatory signaling domains to enhance the efficacy and proliferation of T cells expressing a CAR receptor. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. In some embodiments, costimulatory molecules may include 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-1, CD11a, Lck, TNFR-I, TNFR-II, FasR, NKG2C, B7-H3, and CD83.

[0169] In embodiments, the CAR comprises a 4-1BB costimulatory domain having an amino acid sequence with 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 SEQ ID NO: 33: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE (SEQ ID NO: 33). In embodiments, the CAR has a 4-1BB costimulatory domain having an amino acid sequence with 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 SEQ ID NO:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:63). In embodiments, 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 a nucleic acid having the sequence set forth below: AAACGAGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACAACTCAGGAGGAGGATGGCTGTAGCTGCCGATTCCCGGAAGAAGAAGAAGGTGGCTGTGAA (SEQ ID NO: 34).In embodiments, 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 a nucleic acid having the sequence set forth below: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA (SEQ ID NO: 35). In embodiments, 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 a nucleic acid having the sequence set forth below: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAA (SEQ ID NO: 36). In embodiments, 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 a nucleic acid having the sequence set forth below: AAGAGAGGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCTGTGCAGACCACACAGGAGGAAGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGTGAGCTG (SEQ ID NO: 64).

[0170] In embodiments, the CAR comprises a CD28 costimulatory domain comprising an amino acid sequence 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 SEQ ID NO: 37: 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 a nucleic acid having the sequence set forth below: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 38).

[0171] The engineered NKG2D CAR described herein may also comprise an N-terminal signal peptide or tag at the N-terminus of the NKG2D ectodomain. In one embodiment, a heterologous signal peptide can be used. The antigen-binding domain can be fused to a leader or signal peptide that directs the nascent protein to the endoplasmic reticulum and subsequently translocated to the cell surface. It is understood that when a polypeptide containing a signal peptide is expressed on the cell surface, the signal peptide is generally proteolytically removed during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, polypeptides such as the CAR constructs described herein are generally expressed on the cell surface as mature proteins lacking a signal peptide, although precursor forms of the polypeptide contain a signal peptide. Any suitable signal sequence known in the art can 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 comprises a CD8α signal sequence having an amino acid sequence with 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 SEQ ID NO: 39; MALPVTALLLPLALLLHAARP (SEQ ID NO: 39). In embodiments, 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, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth below: ATGGCTCTTCCTGTGACTGCACTACTGCTGCCCCTGGCCTTACTTCTTCATGCTGCGCGTCCT (SEQ ID NO: 40). In embodiments, 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, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth below: ATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCT (SEQ ID NO: 65).

[0173] In one embodiment, the signal sequence is a CSF2RA signal sequence. In embodiments, an NKG2D CAR described herein comprises a CSF2RA signal sequence having an amino acid sequence with 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 MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 41), MEWTWVFLFLLSVTAGVHS (SEQ ID NO: 42), or MALPVTALLLPLALLLHAARP (SEQ ID NO: 43). In embodiments, 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, e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to a nucleic acid having the sequence set forth below: ATGGCTCTTCCTGTGACAGCTCTTCTGCTGCCCCTGGCCCTGCTTCTGCATGCTGCTAGACCT (SEQ ID NO: 44).

[0174] Components of a CAR can be exchanged or "swapped" for equivalent components using routine techniques of biotechnology. In some non-limiting examples, a CAR of the present disclosure can include a binding domain provided herein in combination with a hinge provided herein and a costimulatory domain provided herein. In certain examples, a CAR of the present disclosure can include a leader sequence provided herein in combination with a hinge provided herein and a costimulatory domain provided herein, together with a binding domain provided herein.

[0175]

[0176]

[0177]

[0178]

[0179] The present disclosure contemplates the use of the NKG2D CARs described herein in conjunction with engineered T cell receptors (TCRs) used in T cell immunotherapy. Libraries of TCRs can be screened for their selectivity for target antigens. In this manner, natural TCRs with high avidity and reactivity to target antigens can be selected, cloned, and subsequently introduced into T cell populations used in adoptive immunotherapy. T cells or NK cells bearing engineered TCRs that also express the NKG2D CARs described herein can not only target specific antigens due to TCR specificity, but also target cells expressing NKG2D ligands. Thus, combining the NKG2D CARs described herein with TCRs may provide a method for maintaining or enhancing the therapeutic efficacy of adoptive T cell or NK cell immunotherapy. In embodiments, the NKG2D CARs described herein are coexpressed with a TCR.

[0180] In one embodiment described herein, T cells or NK cells are modified by introducing a polynucleotide encoding a subunit of a TCR capable of forming a TCR that confers specificity to the T cell or NK cell for tumor cells expressing a target antigen and / or an NKG2D ligand. In some embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, so long as the subunit retains the ability to form a TCR conferred on the transfected T cell or NK cell, the ability to home to target cells, and participate in immunologically relevant cytokine signaling. The engineered TCR can also bind to target cells that display relevant tumor-associated peptides with high avidity and, optionally, mediate efficient killing of target cells presenting the relevant peptide in vivo.

[0181] Nucleic acids encoding engineered TCRs can be isolated from their natural context in 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 can be transferred into cells, which may be T cells. The modified T cells can then express one or more chains (and in some aspects, two chains) of the TCR encoded by the transduced nucleic acid or nucleic acids. In some embodiments, the engineered TCR is an exogenous TCR because it is introduced into T cells that do not normally express the introduced TCR. An essential aspect of an engineered TCR is that it has high avidity for tumor antigens presented by the major histocompatibility complex (MHC) or similar immunological components. In contrast to engineered TCRs, CARs are engineered to bind target antigens in an MHC-independent manner.

[0182] The proteins encoded by the nucleic acids described herein can be expressed with additional polypeptides attached to the amino- or carboxyl-terminal portions of the α or β chains of the TCR, so long as the attached additional polypeptides do not interfere with the ability of the α or β chains to form a functional T cell receptor and MHC-dependent antigen recognition.

[0183] Antigens recognized by the engineered TCRs contemplated herein include, but are not limited to, cancer antigens, including antigens of both blood cancers and solid tumors and virus-induced cancers. TCR therapy for the treatment of HPV-induced cervical cancer is a promising area of ​​interest. Thus, the oncolytic proteins HPV-16 E6 and HPV-16 E7 may be potential target antigens for use with TCRs (see, for example, International Application No. PCT / US2015 / 033129). Other exemplary antigens include HPV-16 E6 and HPV-16 HPV oncoproteins including 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 These include, but are not limited to, HLA-3, glypican-3 (GPC3), HLA-AI+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, MAGA-A3, HLA-AI+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NY-ES0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TAG72, TACI, TEM, and VEGFRII.

[0184] Combining any of the TCR constructs described herein with an NKG2D CAR of the present disclosure may restore, maintain, or enhance the therapeutic effect of TCR therapy. Thus, in one embodiment described herein, an NKG2D CAR is co-expressed in a T cell or NK cell with a TCR directed against HPV. In another embodiment described herein, an NKG2D CAR is co-expressed in a T cell or NK cell with a TCR directed against the HPV-16 E6 protein. In another embodiment described herein, an NKG2D CAR is co-expressed in a T cell or NK cell with a TCR directed against the HPV-16 E7 protein.

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

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

[0187] In some embodiments, one or more additional CARs comprise an extracellular binding domain, including but not limited to, an extracellular domain of an antibody or antigen-binding fragment thereof, a tethered ligand, or a co-receptor, that specifically binds to a target antigen. By way of non-limiting example, target antigens may include: HPV-16 E6 and HPV-16 HPV oncoproteins including 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, GD3, glypican-3 (GPC3), HLA-AI+MAGEI, HLA-A2 +MAGE1, HLAA3 +MAGE1, HLA-AI +NY-ES0-1, HLA-A2 +NY-ES0-1, HLA-A3 +NY-ES0-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TACI, TAG72, TEM, and VEGFRII; in embodiments described herein, the CAR binds to a tumor antigen 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 comprise an extracellular binding domain that specifically binds to a target polypeptide, e.g., a target antigen, expressed on a tumor cell. 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 used interchangeably to provide a CAR capable of specifically binding to a target antigen of interest. A binding domain can include any protein, polypeptide, oligopeptide, or peptide capable of specifically recognizing and binding to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule thereof, or component thereof). A binding domain includes natural, synthetic, semi-synthetic, or recombinantly produced binding partners for a biological molecule of interest.

[0189] In some embodiments, the extracellular binding domain of one or more additional CARs comprises an antibody or antigen-binding fragment thereof. "Antibody" refers to a binding agent that is a polypeptide comprising at least a light 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 one recognized by immune cells. Antibody includes antigen-binding fragments thereof. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0190] In some embodiments, the target antigen is an epitope of: 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-AI+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-AI+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TAG72, TEM, TACI, and VEGFRII polypeptides. In one embodiment described herein, the CAR binds to a tumor antigen epitope including 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 and VL domains, for proper spacing conformation of the molecule. A CAR 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. In some embodiments, the linker is 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 amino acids in length.

[0192] Illustrative examples of linkers include glycine polymers (G), glycine-serine polymers (G, S) (where 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 the CARs described herein. Glycine also has better access to phi-psi space than alanine and is much less restricted 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 CAR designs in some embodiments can include a fully or partially flexible linker, whereby the linker can include a flexible linker as well as one or more moieties that confer less flexibility to provide a desired CAR structure. In one embodiment, any of the constructs described herein can include a "GS" linker. In another embodiment, any of the constructs described herein includes a "GSG" linker. In another embodiment, a CAR described herein includes an amino acid sequence 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 that of SEQ ID NO: 40. GSTSGSGKPGSGEGSTKG (SEQ ID NO: 40).

[0193] In other embodiments, the CAR comprises an scFv that further comprises a variable region linking sequence. A "variable region linking sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of the two sub-binding domains such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. In one embodiment, the variable region linking sequence is 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 amino acids in length.

[0194] In other embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refer to regions that move the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999;6:412-419). Spacer domains can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

[0195] The binding domain of a CAR can generally be followed by one or more "hinge domains," which serve to position the antigen-binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. CARs generally include one or more hinge domains between the binding domain and the transmembrane domain. The hinge domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region.

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

[0197] "Polypeptide," "polypeptide fragment," "peptide," and "protein," unless specified to the contrary, are used in accordance with their conventional meaning, i.e., as a sequence of amino acids. A polypeptide is not limited to a particular length and can include, for example, a full-length protein sequence or a fragment of a full-length protein, and can include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications, both naturally occurring and non-naturally occurring, known in the art. In various embodiments, a polypeptide contemplated herein includes a signal (or leader) sequence at the N-terminal end of the protein, which directs translocation of the protein co- or post-translationally.

[0198] Polypeptides include "polypeptide variants." Polypeptide variants can differ from naturally occurring polypeptides in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or synthetically produced, for example, by modifying one or more of the above polypeptide sequences. For example, in some embodiments, it may be desirable to improve the binding affinity and / or other biological properties of an engineered NKG2D CAR by introducing one or more substitutions, deletions, additions, and / or insertions. Preferably, polypeptides of the present disclosure include polypeptides having at least about 50%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity thereto. Polypeptides of the present disclosure include variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., the Sequence Listing); typically, the variants retain at least one biological activity of the reference sequence. Polypeptides include "polypeptide fragments." Polypeptide fragments refer to polypeptides that may be monomeric or multimeric, having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions in naturally occurring or recombinantly produced polypeptides. In certain embodiments, polypeptide fragments may comprise an amino acid chain of at least 5 to about 500 amino acids in length. It will be appreciated that in certain embodiments, 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 in length.

[0199] The polypeptides can also be fused or conjugated in frame to linkers or other sequences to facilitate synthesis, purification, or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. As described above, the polypeptides of the present disclosure can be modified in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations 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 nucleotide sequence changes are well known in the art. See, e.g., Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154:367-382), U.S. Pat. No. 4,873,192, Watson, J.D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance for appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0200] In certain embodiments, variants comprise conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid with similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic properties of the polypeptide to remain substantially unchanged. Modifications can be made to the polynucleotide and polypeptide structures of the present disclosure and still obtain functional molecules that encode variant or derivative polypeptides with desirable properties.

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

[0202] Polypeptides of the present disclosure include fusion polypeptides. In some embodiments, fusion polypeptides and polynucleotides encoding the 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 linked C-terminus to N-terminus, but they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein can be in any order or a specific order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired transcriptional activity of the fusion polypeptide is preserved. Fusion polypeptides can be produced by chemical synthesis, by chemical conjugation between two moieties, or generally prepared using other common techniques. The linked DNA sequences comprising the fusion polypeptide are operably linked to suitable transcriptional or translational control elements, as discussed elsewhere herein.

[0203] In one embodiment, the fusion partner contains a sequence that aids in expressing the protein at a higher yield than the native recombinant protein (an expression enhancer). Other fusion partners may be selected to increase the solubility of the protein, or to allow targeting of the protein to a desired intracellular compartment, or to facilitate transport of the fusion protein across the cell membrane.

[0204] The fusion polypeptide may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein. Furthermore, the polypeptide domain may be inserted into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as 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, e.g., Ryan et al., 1997. J. Gener. Viral. 78:699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus Nia protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus Nia protease, biovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picoma 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, the TEV (tobacco etch virus) protease cleavage site can be used. In other embodiments, the self-cleaving peptide can include polypeptide sequences obtained from potyvirus and cardiovirus 2A peptides, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teschovirus.In another embodiment, the self-cleaving polypeptide site comprises 2A or an analogous site, sequence or domain of 2A (Donnelly et al., 2001. J Gen. Viral. 82:1027-1041).

[0205] Generally, it is understood that any suitable viral vector or vectors can be used to transduce the engineered constructs described herein. In one embodiment described herein, cells (e.g., T cells, NK cells, or iPSCs) are transduced with a retroviral vector, e.g., 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, which then covalently integrates the 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 sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine 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 2), visna-maedi virus (VMV), caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0207] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may contain sequences that direct autonomous replication within a cell, or may contain sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0208] As will be apparent to those skilled in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that contains viral-derived nucleic acid elements that facilitate the transfer or integration of the nucleic acid molecule into a cellular genome, or a viral particle that mediates nucleic acid transfer. In addition to the nucleic acid, the viral particle typically also contains various viral and sometimes host cell components.

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

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

[0211] Both ends of the provirus contain structures called "long terminal repeats" or "LTRs." The term "long terminal repeat (LTR)" refers to base-pair domains 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 essential functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. Viral LTRs are divided into three regions, designated U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is located between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat) region is adjacent to the U3 and U5 regions. LTRs are composed of U3, R, and U5 regions and appear at both the 5' and 3' ends of the viral genome. Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site).

[0212] As used herein, the term "packaging signal" or "packaging sequence" refers to a sequence located within the retroviral genome that is necessary for insertion of viral RNA into the viral capsid or particle; see, e.g., Clever et al., 1995. J of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors use a minimal packaging signal (also referred to as a psi ['P] sequence) required for encapsidation of the viral genome. Thus, as used herein, the terms "packaging sequence," "packaging signal," "psi," and the symbol "'P" are used in reference to non-coding sequences required for encapsidation of retroviral RNA strands 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. Modification of the 3'LTR is often performed to improve the safety of lentiviral or retroviral systems by rendering the virus replication-defective. As used herein, the term "replication-defective" refers to a virus that is unable to replicate completely and efficiently so that infectious virions are not produced (e.g., replication-defective lentiviral progeny). The term "replication-competent" refers to a wild-type or mutant virus that is capable of replication such that viral replication of the virus can produce infectious virions (e.g., replication-competent lentiviral progeny).

[0214] A "self-inactivating" (SIN) vector refers to a replication-defective vector, e.g., a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because during viral replication, the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region, and therefore, no viral transcripts can be made without the U3 enhancer-promoter. In a further embodiment of the present disclosure, the 3' LTR is modified such that the U5 region is replaced, for example, with an ideal poly(A) sequence. Note that modifications to the LTRs, such as modifications to the 3' LTR, the 5' LTR, or both the 3' and 5' LTRs, are also contemplated herein.

[0215] Further safety enhancement can be achieved by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters are capable of driving high levels of transcription in a Tat-independent manner. Because the virus production system lacks a complete U3 sequence, this replacement reduces the possibility of generating replication-competent virus through recombination. In certain embodiments, heterologous promoters have the added advantage of controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such 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 temperature or pH, under which the host cells are cultured.

[0216] In some embodiments, the viral vector comprises a TAR element. The term "trans-activation response (TAR)" refers to a "trans-activation response" genetic element located in the R region of a lentivirus (e.g., HIV) LTR. This element interacts with the lentivirus transactivator (tat) genetic element to enhance viral replication.

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

[0218] As used herein, the term "FLAP element" refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Pat. No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, the central initiation of plus-strand DNA at the central polypurine tract (cPPT) and the central termination at the central termination sequence (CTS) result in the formation of a triple-stranded DNA structure: the HIV-1 central DNA flap. Without wishing to be bound by any theory, the DNA flap may act as a cis-acting determinant for lentiviral genome nuclear import and / or increase viral titer.

[0219] In one embodiment, the 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 regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, 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 the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, RNA export elements are located within the 3'UTR of a gene and can be inserted in one or multiple copies.

[0220] In other embodiments, 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 protein expression of heterologous nucleic acids, 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) present 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, a vector may contain a regulatory oligonucleotide with transcriptional or translational regulatory activity. Such oligonucleotides can be used in various gene expression configurations to regulate expression control. Transcriptional regulatory oligonucleotides can increase (enhance) or decrease (silence) the expression level of a recombinant expression construct. Regulatory oligonucleotides can selectively regulate expression in a context-specific manner, including, for example, to confer tissue-specific, developmental stage-specific, or similar expression of a polynucleotide, including constitutive or inducible expression. The regulatory oligonucleotides of the present disclosure can also be components of an expression vector or a recombinant nucleic acid molecule comprising a regulatory oligonucleotide operably linked to an expressible polynucleotide. Regulatory elements can vary in length from a few nucleotides to hundreds of nucleotides.

[0222] Elements that direct 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, vectors contain a polyadenylation sequence 3' of a polynucleotide encoding an expressed polypeptide. As used herein, the term "polyA site" or "polyA sequence" refers to a DNA sequence that directs both the termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thus contributing to increased translation efficiency. Efficient polyadenylation of recombinant transcripts is desirable because transcripts lacking a polyA tail are unstable and rapidly degraded. Illustrative examples of poly A signals that can be used in the vectors of the present disclosure include ideal poly A sequences (e.g., AATAAA, ATTAAA, AGTAAA), bovine growth hormone poly A sequence (BGHpA), rabbit β-globin poly A sequence (rβgpA), or another suitable heterologous or endogenous poly A sequence known in the art.

[0223] Also described herein are "codon-optimized" nucleic acids. A "codon-optimized" nucleic acid refers to a nucleic acid sequence in which the codons have been altered to be optimal for expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence can 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 the codons of the native sequence with codons that are more frequently or most frequently used in the genes of that species. Codon optimization does not change the amino acid sequence of the encoded protein.

[0224] Codon-optimized nucleotide sequences can exhibit improved properties related to expression efficiency. In some embodiments, transcribed DNA sequences can be optimized to promote more efficient transcription and / or translation. In some embodiments, DNA sequences can 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, cryptic CpG islands, GC content, polymerase slippage sites, and / or other elements related to transcription. DNA sequences can be optimized with respect to cryptic splice sites, mRNA secondary structure, stable free energy of mRNA, repetitive sequences, RNA instability domains, and / or other elements related to mRNA processing and stability. The DNA sequence may be optimized with respect to codon usage bias, codon compatibility, internal Chi sites, ribosome binding sites (e.g., IRES), premature poly(A) sites, Shine-Dalgarno (SD) sequences, and / or other elements associated with translation, and / or the DNA sequence may be optimized with respect to codon context, codon-anticodon interactions, translational pause sites, and / or other elements associated with protein folding.

[0225] A vector may have one or more LTRs, any of which may contain one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector may further contain one or more accessory elements to increase transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., Psi (Ψ) packaging signal, RRE), and / or other elements that increase therapeutic gene expression (e.g., poly(A) sequences), and may optionally include a WPRE or HPRE. Those skilled in the art will understand that many other different embodiments can be formed from the existing embodiments of the present disclosure.

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

[0227] In many cases, large-scale production of viral particles is required to achieve reasonable viral titers. Viral particles are produced by transfecting transfer vectors into packaging cell lines containing viral structural and / or accessory genes, such as the 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 a polynucleotide encoding one, two, three, four, or more viral structural 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 the present disclosure can be introduced into a packaging cell line via transfection, transduction, or infection to generate producer cells or cell lines. The packaging vectors of the present disclosure can 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, blastocidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be physically linked to the gene encoded by the packaging vector, for example, by an IRES or a self-cleaving viral peptide.

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

[0230] Illustrative examples of env genes from retroviruses that may be used in the embodiments described herein include, but are not limited to: MLV envelope, IOAI envelope, BAEV, FeLV-B, RDI 14, SSAV, Ebola, Sendai, FPV (avian plague virus), and influenza virus envelope. Similarly, genes encoding envelopes from RNA viruses (e.g., RNA virus families Picomaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Bimaviridae, Retroviridae) and DNA viruses (Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae families) can be used. Representative 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, envelope proteins for pseudotyping viruses of the present disclosure include, but are not limited to, any of the following viruses: influenza A viruses, such as H1N1, H1N2, H3N2, and H5N1 (avian influenza), influenza B viruses, influenza C viruses, hepatitis A viruses, hepatitis B viruses, hepatitis C viruses, hepatitis D viruses, hepatitis E viruses, rotaviruses, any virus of the Norwalk virus group, enteric adenoviruses, parvoviruses, dengue virus, monkeypox, Mononegavirales, lyssaviruses, such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat viruses 1 and 2, Australian bat virus, ephemeroviruses, vesiculoviruses, vesicular stomatitis virus, varicella ... Virus (VSV), herpes viruses such as herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, murine gammaherpesvirus, arenaviruses 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 virusvirus (LCMV), Bunyaviridae (Filoviruses) including Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever viruses that cause renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever, and Marburg hemorrhagic fever, Flaviviridae (Flaviviridae) including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, and viruses that cause tick-borne encephalitis, Paramyxoviridae (Paramyxoviridae) including Hendra virus, Nipah virus, variola major and variola minor (smallpox), Alphaviruses (Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, or any virus that causes encephalitis.

[0232] As used herein, the term "pseudotype" or "pseudotyping" refers to a virus in which a viral envelope protein has been replaced with that of another virus with other characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein, allowing HIV to infect a wider 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 in reference to a cell line that does not contain a packaging signal but stably or transiently expresses viral structural proteins and replicative enzymes (e.g., gag, pol, and env) necessary for correct 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 packaging cell lines include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, P A317 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 packaging cell line and a cell line capable of producing recombinant retroviral particles containing a transcription vector construct including a packaging signal. The production of infectious viral particles and viral stocks can be carried out using conventional techniques. Methods for preparing viral 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 viral particles can be harvested from packaging cells using conventional techniques. For example, infectious particles can be harvested by cell lysis or by harvesting the cell culture supernatant, as known in the art. Optionally, the harvested viral particles can 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 transduces a cell through infection and proviral integration. In certain embodiments, a target cell, e.g., a T cell or NK cell, is "transduced" when it contains a gene or other polynucleotide sequence delivered to the cell by infection with a viral or retroviral vector. In some embodiments, the transduced cell contains one or more genes or other polynucleotide sequences delivered by the retroviral or lentiviral vector in its cellular genome.

[0236] Disclosed are host cells expressing one or more of the constructs of the present disclosure. The host cells can be transduced with one or more viral vectors comprising nucleic acid sequences encoding one or more polypeptides that express the engineered TCR and / or CAR. Other methods relating to the use of viral vectors in gene therapy that may be utilized in accordance with certain embodiments of the present disclosure are described, for example, in Kay, MA (1997) Chest 111(6Supp.):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, SL and Crystal, RG (1994) Ann. NY Acad. Sci. 716:90-101, Strayer, DS (1999) Expert Opin. Investig. Drugs 8:2159-2172, Smith-Arica, JR and 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 containing them, T cell compositions, and NK compositions, as contemplated herein. One embodiment described herein is a composition comprising engineered T cells expressing an NKG2D CAR. Another embodiment described herein is a composition comprising engineered NK cells expressing an NKG2D CAR. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated into a pharmaceutically or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapies. It will also be understood that, if desired, the compositions of the present disclosure may be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceutical active agents. There is virtually no limit to the other components that may be included in the composition, so long as the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0238] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0239] As used herein, "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for human or veterinary use. Exemplary pharmaceutically acceptable carriers include, but are not limited to, 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; 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; phosphate buffer; and any other compatible substance used in pharmaceutical formulations.

[0240] In one embodiment described herein, the compositions of the present disclosure comprise an amount of modified T cells or NK cells as contemplated herein. Pharmaceutical compositions comprising T cells or NK cells as contemplated herein may be administered in amounts of 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 8It can be generally stated that the compositions may be administered at a dose of 1000 cells / kg body weight (including all integer values ​​within these ranges). The number of cells will depend on the end use for which the composition is intended, as will the type of cells contained therein. T cells or NK cells modified to express an engineered NKG2D CAR may be administered multiple times at doses within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing treatment. If desired, the treatment may also include administration of a mitogen (e.g., PHA) or lymphokine, cytokine, and / or chemokine (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 engraftment and function of the infused T cells.

[0241] Generally, compositions comprising cells activated and expanded as described herein can be used to treat and prevent diseases occurring in immunocompromised or immunosuppressed individuals. Some compositions comprising the modified T cells or NK cells contemplated herein are used to treat cancer. The modified T cells or NK cells described herein can be administered as pharmaceutical compositions 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. In some embodiments, pharmaceutical compositions contemplated herein comprise a quantity 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 the engineered T cells or NK cells contemplated herein may further comprise a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol, etc.; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The compositions of the present disclosure can be formulated for parenteral administration, for example, intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0243] Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain one or more of the following: a sterile diluent such as water for injection; saline solutions such as saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; 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 enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Sterile injectable pharmaceutical compositions are also included.

[0244] In some embodiments, compositions contemplated herein comprise an effective amount of a proliferation-modified T cell or NK cell composition, alone or in combination with one or more therapeutic agents. Thus, the T cell or NK cell composition can be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, or photodynamic therapy. The compositions can also be administered in combination with antibiotics and antiviral agents. Such therapeutic agents would be accepted in the art for the treatment of disease states described herein, such as cancer. In one embodiment, compositions contemplated herein can also be administered with an inhibitor of TGF-β, e.g., the small molecule inhibitor LY55299. Exemplary contemplated therapeutic agents include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapeutic agents, therapeutic antibodies, or other active and adjunctive agents.

[0245] In certain embodiments, compositions comprising T cells or NK cells contemplated 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™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metoledopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphamide, ethyleneimines and methylameramines, including trimethylmelamine regimes; nitinamides. erogen mustards, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine , ranimustine; antibiotics, such as aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detrevicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivivir; Bomycin, peplomycin, pofilomycin, puromycin, queramycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, such as calsterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine, bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformitine; elliptinium acetate; etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; topoisomerase inhibitor RPS 2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin);Included within this definition are ONTAK™ (denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Also included within this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0246] A variety of additional therapeutic agents can be used in conjunction with the compositions or agents / treatments described herein. In one embodiment, a composition comprising T cells is administered with an anti-inflammatory agent. 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, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, 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 sialylate. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary disease-modifying anti-rheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0248] In other embodiments, therapeutic antibodies suitable for combination with the CAR or TCR modified T cells or NK cells contemplated herein include abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drotumumab, and drotumumab. Zizumab, durigotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, framvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imatuzumab, indatuximab, ino Tuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minletumomab, mitumomab, moxetumomab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratuzumab, onatuzumab, oportuzumab, oregovomab, panitumumab, palsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab These include, but are not limited to, tzatzumab, pritumumab, racotumomab, radletuzumab, rilotumumab, rituximab, lobatumumab, satumomab, sibrotuzumab, siltuximab, simtuzumab, solitumab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, borsetuzumab, votumumab, zalutumumab, CC49, and 3F8.

[0249] In some embodiments, the compositions described herein are administered with cytokines. As used herein, "cytokine" refers to a general term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, chemokines, and traditional polypeptide hormones.Cytokines include growth hormones, e.g., human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, e.g., follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH); hepatic growth factors; fibroblast growth factors; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; Müllerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factors, e.g., NGF-beta; platelet growth factors; transforming growth factors; TGFs, such as TGF-alpha and TGF-beta; insulin-like growth factor-I and growth factor-II; erythropoietin (EPO); bone morphogenetic factors; interferons, such as interferon-alpha, beta, and gamma; colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs), such as 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, such as TNF-alpha or TNF-beta; and LIF and kit ligand (kit As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0250] Any cell can be used as a host cell for the polynucleotides, vectors, or polypeptides of the present disclosure. In some embodiments, the cell can 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., Enterobacteriaceae, 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. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cells are selected from the group consisting of T cells, B cells, tumor infiltrating lymphocytes (TILs), TCR-expressing cells, natural killer (NK) cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, platelets, thymocytes, and myeloid cells. In one embodiment, the immune cells are T cells. In another embodiment, the immune cells are NK cells. In certain embodiments, the T cells are tumor infiltrating lymphocytes (TILs), autologous T cells, engineered autologous T cells (eACT™), allogeneic T cells, xenogeneic T cells, or any combination thereof. The T cells (or any of the above) are distinct from antibody therapy or stand-alone NKG2D CAR-modified T cells.

[0251] Another embodiment described herein is a method of treating cancer in a subject in need thereof, comprising administering an effective amount, e.g., 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 will be determined by factors such as the patient's condition, the type and severity of the patient's disease, and appropriate dosages may be determined by clinical trials.

[0252] In other embodiments, methods are provided that include administering a therapeutically effective amount of the engineered T cells contemplated herein or a composition comprising same, alone or in combination with one or more therapeutic agents, to a patient in need thereof. In certain embodiments, the cells of the present disclosure are used to treat a patient 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 engineered T cells of the present disclosure.

[0253] Those skilled in the art will recognize that multiple administrations of the disclosed compositions may be required to affect the desired treatment. For example, the compositions may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over the course of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0254] In certain embodiments, it may be desirable to administer activated T cells to a subject, then re-draw blood (or perform apheresis), activate T cells therefrom according to the present disclosure, and re-infuse these activated and expanded T cells into the patient. This process may be performed multiple times, every few weeks. In certain embodiments, T cells may be activated from a blood draw of 10 cc to 400 cc. Without being bound by theory, this multiple blood draw / multiple re-infusion protocol may be used to help select for certain populations of T cells.

[0255] Administration of the compositions contemplated herein can be carried out in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, transplantation, or implantation. In some embodiments, the compositions are administered parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and substernal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0256] In one embodiment, a subject in need thereof is administered an effective amount of a composition to increase the cellular immune response against cancer in the subject. The immune response may include cellular immune responses mediated by cytotoxic T cells, regulatory T cells, and helper T cell responses, which can kill infected cells. Thus, a humoral immune response, primarily mediated by helper T cells, which can activate B cells leading to antibody production, may also be induced. Various techniques well-described in the art can be used to analyze the type of immune response elicited by the compositions of the present disclosure, for example, 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 the case of T cell-mediated killing, CAR-ligand binding initiates CAR signaling to the T cell, resulting in activation of various T cell signaling pathways that induce the T cell to produce or release proteins that can induce target cell apoptosis by various mechanisms. These T cell-mediated mechanisms include (but are not limited to) the transfer of intracellular cytotoxic granules from the T cell to the target cell, T cell secretion of proinflammatory cytokines that can directly induce target cell killing (or indirectly via recruitment of other killer effector cells), and upregulation of death receptor ligands (e.g., FasL) on the T cell surface that induce target cell apoptosis after binding to their cognate death receptor (e.g., Fas) on the target cell.

[0258] Described herein are embodiments of a method of treating a subject diagnosed with cancer, comprising removing T cells from the subject, genetically modifying the T cells with a vector comprising a nucleic acid encoding an NKG2D CAR as contemplated 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 present disclosure also provides a method for stimulating an effector cell-mediated immunomodulatory response against a target cell population in a subject, the method comprising administering to the subject a population of immune effector cells that express a nucleic acid construct encoding an NKG2D CAR molecule.

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

[0261] Although the foregoing disclosure has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present disclosure that certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Section 1] A chimeric antigen receptor (CAR), NKG2D ectodomain, a transmembrane domain; a 4-1BB costimulatory domain; a signaling domain comprising a CD3-zeta signaling domain; A chimeric antigen receptor comprising: [Section 2] The CAR of paragraph 1, further comprising a CD8-alpha hinge domain. [Section 3] The CAR of paragraph 2, wherein the CD8-alpha hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 15. [Section 4] Item 4. The CAR according to any one of Items 1 to 3, wherein the NKG2D ectodomain comprises the amino acid sequence set forth in SEQ ID NO:3. [Section 5] The CAR of any one of items 1 to 4, wherein the transmembrane domain further comprises a CD28 transmembrane domain. [Section 6] The CAR of paragraph 5, wherein the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 21. [Section 7] Item 7. The CAR according to any one of Items 1 to 6, wherein the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 63. [Section 8] 8. The CAR of any one of items 1 to 7, wherein the CD3 zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 27. [Section 9] Item 9. The CAR of any one of items 1 to 8, wherein the signaling domain further comprises a CD3-epsilon signaling domain. [Section 10] The CAR of paragraph 9, wherein the CD3-epsilon signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO: 61. [Section 11] Item 11. The CAR of any one of Items 1 to 10, comprising an amino acid sequence having at least 90% sequence identity to 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. [Section 12] A nucleic acid encoding the CAR according to any one of items 1 to 11. [Section 13] 13. The nucleic acid of claim 12, comprising a nucleotide sequence having at least 90% sequence identity 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. [Section 14] Item 14. A recombinant vector comprising the nucleic acid of Item 12 or 13. [Section 15] 15. The recombinant vector of paragraph 14 or the nucleic acid of paragraph 12 or 13, wherein the recombinant vector or nucleic acid further comprises a nucleic acid encoding an engineered T cell receptor (TCR). [Section 16] 15. The recombinant vector of paragraph 14 or the nucleic acid of paragraph 12 or 13, wherein the recombinant vector or nucleic acid further comprises a nucleic acid encoding a second CAR specific for a tumor antigen. [Section 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, The recombinant vector or nucleic acid of paragraph 15 or 16, comprising FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-AI+MAGEI, HLA-A2+MAGE1, HLAA3+MAGE1, HLA-AI+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TACI, TAG72, TEM, or VEGFRII. [Section 18] A host cell transformed with the nucleic acid or recombinant vector according to any one of items 12 to 17. [Section 19] A host cell transformed with the nucleic acid of paragraph 12 or 13 or the recombinant vector of paragraph 14, and a nucleic acid or recombinant vector encoding an engineered T cell receptor (TCR) or second CAR specific for a tumor antigen. [Section 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, fetal 20. The host cell of paragraph 19, comprising: HLA-AchR, FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-AI+MAGEI, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-AI+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TACI, TAG72, TEM, or VEGFRII. [Section 21] 21. The host cell according to any one of items 18 to 20, wherein the host cell comprises an iPSC, a T cell, or a NK cell. [Section 22] A pharmaceutical composition comprising the host cell of Item 21. [Section 23] 23. A method for treating a disease in a patient in need thereof, the method comprising administering to the patient the host cell of paragraph 21 or the pharmaceutical composition of paragraph 22. [Section 24] 24. The method of paragraph 23, wherein the host cells are allogeneic to the patient. [Example]

[0262] Example 1 NKG2D CAR construct design As used in the following examples, engineered NKG2D chimeric antigen receptor constructs were designed and synthesized in retroviral vectors. The first construct, designated NKG2D CAR1, contains signaling domains comprising, from N- to C-terminus, a signal peptide, the NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intracellular domain, and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor is shown below: MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (sequence number 53).

[0263] The second construct, designated NKG2D CAR2, contains, from N- to C-terminus, a signal peptide, the NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intracellular domain, and signaling domains including the CD3ε signaling domain and the CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor is shown below: MALPVTALLLPLALLLHAARPLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWED GSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVT VAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 54).

[0264] The third construct, designated NKG2D CAR3, comprises signaling domains including, from N- to C-terminus, the NKG2D extracellular domain, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intracellular domain, and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor comprises 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 can be 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 (SEQ ID NO: 67). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 67) 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: 67) encodes a CD8a signal peptide at positions 1221-1283, which can be replaced with a different signal peptide.

[0265] The fourth construct, designated NKG2D CAR4, comprises signaling domains comprising, from N- to C-terminus, the NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intracellular domain, and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor comprises 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 (SEQ ID NO: 69). CAR4 can be 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 (SEQ ID NO: 69). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 69) 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: 69) encodes a CD8a signal peptide at positions 1221-1283, which can be replaced with a different signal peptide.

[0266] The fifth construct, designated NKG2D CAR5, contains, from N- to C-terminus, an NKG2D extracellular domain, a CD8 hinge, a CD8 transmembrane domain, a 4-1BB intracellular domain, and signaling domains including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor contains the following amino acid sequence: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAIYIWAPLAGTCGVLLL SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRILRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (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 agctgaagaa ccgcaaagca aaggcaaaac ccgtcacacg 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 (SEQ ID NO: 71) CAR5 can be 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 (SEQ ID NO: 71). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 71) 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: 71) encodes a CD8a signal peptide at positions 486-548, which can be replaced with a different signal peptide.

[0267] The sixth construct, designated NKG2D CAR6, contains, from N- to C-terminus, an NKG2D extracellular domain, a CD8 alpha hinge, a CD8 transmembrane domain, a 4-1BB intracellular domain, and signaling domains including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor contains the following amino acid sequence: 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 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 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 agcgccggag gggcaagggg cacgatggcc 1741 tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaagctc 1801 tgccccctcg ctga (SEQ ID NO: 73) CAR6 can be 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 (SEQ ID NO: 73). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 73) 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: 73) encodes a CD8a signal peptide at positions 486-548, which can be replaced with a different signal peptide.

[0268] The seventh construct, designated NKG2D CAR7, contains, from N- to C-terminus, an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intracellular domain, and signaling domains including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor contains the following amino acid sequence: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACY SLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 74). In one aspect, 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 (SEQ ID NO: 75) CAR7 can be 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 (SEQ ID NO: 75). In certain embodiments, the nucleic acid sequence of (SEQ ID NO: 75) 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: 75) encodes a CD8a signal peptide at positions 486-548, which can be replaced with a different signal peptide.

[0269] The eighth construct, designated NKG2D CAR8, contains, from N- to C-terminus, an NKG2D extracellular domain, a CD8 alpha hinge, a CD28 transmembrane domain, a 4-1BB intracellular domain, and signaling domains including a CD3ε signaling domain and a CD3ζ signaling domain. The amino acid sequence of this chimeric antigen receptor contains the following amino acid sequence: 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 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 (SEQ ID NO: 77) CAR8 can be 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 (SEQ ID NO: 77). 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 a CD8a signal peptide at positions 486-548, which 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 the epitope were used for single or co-transduction with the NKG2D CAR construct (Jin BY et al., 2018 JCI Insight). Pre-cryopreserved PBMCs obtained from healthy donors were thawed and activated with anti-CD3 for 48 hours in Optimizer T cell medium supplemented with 300 IU of IL-2. PBMCs were transfected with HPV16+E7. 11-19T cells were transduced with retroviruses containing an engineered TCR targeting the epitope (referred to as HPV-TCR in this and following examples), an NKG2D CAR, or both constructs. 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 an Akt VIII inhibitor (AKTi, 1 μM). Transduction efficiency was measured by flow cytometry on days 8 and 15 after T cell activation. Antibodies for detecting TCR transduction efficiency include anti-mouse TCR β clone H57-597 (BioLegend), and antibodies for detecting NKG2D transduction efficiency include anti-human CD314 clone 1D11 (BioLegend). All flow cytometry data were collected on an LSR-Fortessa (BD LSR Fortessa™) using BD FACSDiva™ software, and 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 (measured as mean fluorescence intensity (MFI) and % positive cells) of transduced and non-transduced T cells by flow cytometry after 8 and 15 days of culture. High transduction efficiency was observed for all constructs. Small NKG2D+-only populations are present in the HPV-TCR+NKG2D CAR1 and HPV-TCR+NKG2D CAR2 groups.

[0272] TIFF0007815283000004.tif81170

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

[0274] TIFF0007815283000005.tif52170

[0275] Example 3 Growth and viability 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 and analyzed by flow cytometry on days 8 and 15 after T cell activation (Table 5). Transduced T cells were activated as described in Example 2. Table 5 shows the proliferation and cell viability of transduced and untransduced T cells cultured in Optimizer medium supplemented with IL-7, IL-15, and AKTi. NKG2D CAR1 alone, NKG2D CAR2 alone, HPV-TCR + NKG2D CAR1, and HPV-TCR + NKG2D CAR2 showed higher levels of proliferation than HPV-TCR alone or untransduced T cells.

[0276] TIFF0007815283000006.tif48170

[0277] Example 4 Determining product attributes The memory phenotype and CD4 / CD8 ratio were measured by flow cytometry 8 days after T cell activation (Table 6). Antibodies used to assess the 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 assess the CD4 / CD8 ratio included anti-human CD3 clone SK7 (BioLegend), anti-human CD4 clone RPA-T4 (BioLegend), and anti-human CD8 clone SK1 (BioLegend). Transduced T cells were activated as described in Example 2. Memory phenotypes were assessed 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-); and peripheral effector T cells (CD45RA+CD45RO-CCR7-CD62L-). All flow cytometry data were collected on an LSR-Fortessa (BD LSR Fortessa™) using BD FACSDiva™ software, and data were analyzed using FlowJo (all BD Sciences). All antibody staining was performed at 4°C in PBS containing 1% BSA.

[0278] The CD4 / CD8 ratios and memory phenotypes of transduced and untransduced CD3+ T cells cultured in TC medium supplemented with IL-2 after 8 days are shown in Table 6. There was no significant change in the CD4 / CD8 ratio between transduced and untransduced T cells. Untransduced T cells and T cells transduced with HPV-TCR only displayed 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, untransduced T cells and T cells transduced with HPV-TCR only displayed a smaller memory T cell compartment than T cells transduced with NKG2D CAR1 only, NKG2D CAR2 only, HPV-TCR+NKG2D CAR1, or HPV-TCR+NKG2D CAR2.

[0279] TIFF0007815283000007.tif48170

[0280] Example 5 Cytotoxicity HPV-TCR-transduced T cells have previously been 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 pathway, thereby rendering them resistant to HPV-TCR-engineered adoptive TCR-T therapy. To model the patient tumor data, we transformed an HPV16+ tumor cell line (CaSki) into an Aβ2M-targeted tumor by knocking out β2M (β2MKO) using the CRISPR-Cas9 system (Vakulskas CA et al., 2018 Nature). * It was engineered to lack 02:01. Furthermore, it is usually HLA-A * SiHa HPV16+ tumor cell lines lacking 02:01 were transfected with retroviral vectors. *The CaSki and SiHa tumor cell lines were engineered to overexpress E7 and 02:01, respectively. The tumor cell lines were selected due to their high and low E7 expression. The expression of intact HLA-A in untransduced and transduced PBMCs was significantly increased. * Killing of these tumor cell lines with or without 02:01 was assessed 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 of transduced T cells (see Example 2) sorted on day 8 and then co-cultured with wild-type or β2MKO CaSki cells at various E:T ratios in 96-well xCELLigence plates on day 10. Impedance values ​​(IV) were measured after 72 hours. Control IV represents the impedance value of untransduced T cells, and experimental IV represents the impedance value of either HPV-TCR alone, NKG2D CAR1 alone, NKG2D CAR2 alone, HPV-TCR + NKG2D CAR1, or HPV-TCR + NKG2D CAR2. Percent cytotoxicity was calculated by the following formula: % Cytotoxicity=1-(Control IV-Experimental IV) * 100

[0282] TIFF0007815283000008.tif57170

[0283] Table 8. Sorting on day 8 followed by wild-type or SiHa expressing cells on day 10. * Figure 1 shows the results of transduced T cells (see Example 2) co-cultured in 96-well xCELLigence plates at various E:T ratios with 02:01. Impedance values ​​(IV) were measured after 72 hours and percent cytotoxicity was calculated as above.

[0284] TIFF0007815283000009.tif42170HPV-TCR transduced T cells were expressed in wild-type CaSki cell lines and A *02 SiHa cell line, but failed to eliminate the β2M KO CaSki cell line or the wild-type SiHa cell line. Importantly, NKG2D CAR-transduced T cells and NKG2D CAR co-transduced with HPV-TCR T cells were able to express their HLA-A * CaSki and SiHa cell lines were successfully killed regardless of 02:01 status.

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

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

[0287] TIFF0007815283000010.tif44170

[0288] Table 10 shows the results of sorting on day 8 and then on day 10 on wild-type or A * Figure 1 shows the results of transduced T cells co-cultured with 02 SiHa cells at various E:T ratios in 96-well xCELLigence plates. At 24 hours, media supernatants were extracted and processed to determine IFNγ concentrations (pg / mL) using a Meso Sector S 600 platform according to the manufacturer's instructions.

[0289] TIFF0007815283000011.tif44170

[0290] Example 7 Long-term expansion and depletion of transduced T cells Repeated antigen stimulation in the long-term killing assay is believed to functionally deplete T cells and correlate with in vivo efficacy. To complete the continuous antigen stimulation assay, target cells (WT CaSki, β2MKO CaSki, wild-type SiHa, or A * β2MKO CaSki, wild-type SiHa, or Aβ2MKO CaSki were added to the transduced T cells (see Example 2) every 2-3 days. *The cytotoxicity of either 02 SiHa was evaluated using the xCELLigence RTCA MP platform at 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 continuous stimulation and exhibited high 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 sorted on day 8 and then co-cultured with wild-type CaSki cells at a 3:1 E:T ratio on day 10. Wild-type CaSki cells were then added every 2-3 days. At the fifth stimulation, T cells were co-cultured with wild-type or β2MKO CaSki cells at various E:T ratios in 96-well xCELLigence plates. Impedance values ​​(IV) were measured after 72 hours, and cytotoxicity was calculated as described above.

[0292] TIFF0007815283000012.tif44170

[0293] Table 12 shows the results for transduced T cells sorted on day 8 and then co-cultured with wild-type CaSki cells at a 3:1 E:T ratio on day 10. Wild-type CaSki cells were then added every 2-3 days. At the fifth stimulation, T cells were cultured in 96-well xCELLigence plates with either wild-type or A cells. * The cells were co-cultured with 02 SiHa cells at various E:T ratios. Impedance values ​​(IV) were measured after 72 hours, and cytotoxicity was calculated as described above.

[0294] TIFF0007815283000013.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 cocultured with either the C33A cell line, which is primarily NKG2D ligand-negative, or primary normal cervical epithelial cells at 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. Cytotoxicity data from the xCELLigence RTCA MP platform and IFNγ production from the Meso Sector S 600 platform demonstrated low reactivity against 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 of transduced T cells sorted on day 8 and then co-cultured with C33A cells or primary normal cervical epithelial cells at various E:T ratios in 96-well xCELLigence plates on day 10. At 24 hours, media supernatants were extracted and processed to determine IFNγ concentrations (pg / mL) using a Meso Sector S 600 platform.

[0297] TIFF0007815283000014.tif48170

[0298] Table 14 shows the results of transduced T cells sorted on day 8 and then co-cultured with C33A cell line or primary normal cervical epithelial cells at various E:T ratios in 96-well xCELLigence plates on day 10. At 24 hours, media supernatants were extracted and processed to determine IFNγ concentrations (pg / mL) using a Meso Sector S 600 platform.

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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 alone, NKG2D CAR alone, 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 implanted into the left and right flanks, respectively, of 6- to 8-week-old female NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ). Five study groups were included: vehicle control (PBS), non-transduced T cells (NTD), HPV-TCR alone, NKG2D CAR2 alone, and HPV-TCR + NKG2D CAR2. Tumor volumes of wild-type CaSki cells were approximately 70 mm. 3 β2MKO CaSki cells reached approximately 30 mm 3 When the number of mice reaches 20 × 10 6T cells were adoptively transferred on day 6. No IL-2 supplementation was added. Tumors were measured every 3–4 days using digital calipers, and mouse weights were recorded. Peripheral blood was collected 24 h after T cell adoptive transfer and then weekly thereafter. Persistence of T cells in the peripheral blood was characterized by flow cytometry. Antibodies used to assess T cell persistence in peripheral blood 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), anti-human CD314 clone 1D11 (Biolegend), and L / D Blue (ThermoFisher). All flow cytometry data were collected on an LSR-Fortessa (BD LSR Fortessa™) using BD FACSDiva™ software, and data were analyzed using FlowJo (all BD Sciences). All antibody staining was performed at 4°C in PBS containing 1% BSA. The study period was 62 days. Overall, HPV-TCR was able to control tumor growth of wild-type CaSki cells on the left flank, but did not show any signs of tumor control of β2MKO CaSki cells implanted on the right flank. In contrast, NKG2D CAR2 alone and HPV-TCR + NKG2D CAR2 successfully controlled tumor growth of both wild-type and β2MKO CaSki cells (see Table 15, Table 16). Furthermore, the weight of tumor-bearing mice did not fluctuate significantly 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 recurrent tumor growth, suggesting a lack of persistence of the transduced T cells.This is further supported by the absence of transduced T cells in the peripheral blood 3 weeks after adoptive T cell transfer (see Table 18).

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[0314] Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. A chimeric antigen receptor (CAR) comprising, from the N-terminus to the C-terminus: NKG2D ectodomain; and a transmembrane domain; a 4-1BB costimulatory domain; and a signaling domain comprising a CD3-epsilon signaling domain and a CD3-zeta signaling domain; Including, the CD3-epsilon signaling domain consists solely of an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 31; Chimeric antigen receptor.

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

3. The CAR of claim 2, wherein the CD8-alpha hinge domain comprises the amino acid sequence set forth in SEQ ID NO:

15.

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

3.

5. The CAR according to any one of claims 1 to 3, wherein the transmembrane domain further comprises a CD28 transmembrane domain.

6. The CAR of claim 5, wherein the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:

21.

7. The CAR according to any one of claims 1 to 3, wherein the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO:

63.

8. The CAR of any one of claims 1 to 3, wherein the CD3-zeta signaling domain comprises the amino acid sequence set forth in SEQ ID NO:

27.

9. The CAR according to any one of claims 1 to 3, comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO:

74.

10. A nucleic acid encoding the CAR of claim 1.

11. 11. The nucleic acid of claim 10, comprising a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:73, and SEQ ID NO:

75.

12. A recombinant vector comprising the nucleic acid of claim 10.

13. 13. The recombinant vector of claim 12 or the nucleic acid of claim 10 or 11, wherein the recombinant vector or nucleic acid further comprises a nucleic acid encoding an engineered T cell receptor (TCR).

14. 13. The recombinant vector of claim 12 or the nucleic acid of claim 10 or 11, wherein the recombinant vector or nucleic acid further comprises a nucleic acid encoding a second CAR specific for a tumor antigen.

15. 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 Ach 15. The recombinant vector or nucleic acid of claim 14, comprising R, FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGEI, HLAA3+MAGEI, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TACI, TAG72, TEM, or VEGFRII.

16. A host cell transformed with a recombinant vector according to claim 12 or a nucleic acid according to claim 10 or 11.

17. 13. A host cell transformed with a nucleic acid according to claim 10 or 11 or a recombinant vector according to claim 12 and a nucleic acid or recombinant vector encoding a second CAR or an engineered T cell receptor (TCR) specific for a tumor antigen.

18. 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 18. The host cell of claim 17, comprising AchR, FRa, Flt3, GD2, GD3, glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGEI, HLAA3+MAGEI, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, kappa, mesothelin, Mucl, Mucl6, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, survivin, TACI, TAG72, TEM, or VEGFRII.

19. 17. The host cell of claim 16, wherein the host cell comprises an iPSC, a T cell, or an NK cell.

20. A pharmaceutical composition comprising the host cell of claim 19.

21. 21. The pharmaceutical composition of claim 20 for treating a disease in a patient in need thereof.

22. 22. The pharmaceutical composition of claim 21, wherein the host cells are allogeneic to the patient.

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