CD3-expressing natural killer cells with enhanced function for adoptive immunotherapy

Abstract

Embodiments of the present disclosure include methods and compositions in which NK cells are engineered by humans to express T cell receptors and CD3 co-receptors that are not naturally expressed on NK cells. Such engineered NK cells can effectively interact with bispecific or multispecific antibodies engineered to include anti-CD3 antibodies that bind to the engineered NK cells, thereby inducing signaling, activation, and cytotoxicity of target cells to which the antibodies also bind. Thus, the NK cells are specifically configured to effectively interact with bispecific NK cell engagers (BiKEs) and bispecific T cell engagers (BiTEs).

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 225,281, filed July 23, 2021, which also claims priority to U.S. Provisional Patent Application No. 63 / 310,526, filed February 15, 2022, and to U.S. Provisional Patent Application No. 63 / 344,931, filed May 23, 2022, each of which is incorporated by reference in its entirety.

[0002] I.Technical field The present disclosure relates to the fields of immunology, cell biology, molecular biology, and medicine, including at least cancer medicine. [Background technology]

[0003] II. Background Natural killer (NK) cells have been investigated as potential antitumor effectors, but several barriers, primarily related to a lack of antigen specificity, limit their therapeutic use. One approach to overcome this is to transduce NK cells with chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) to target desired antigens. For T cells, bispecific or multispecific antibodies, such as bispecific T cell engagers (BiTEs), can be used, which bind to CD3 on the T cell surface and also to antigens on cancer cells. CD3 is composed of four distinct chains; in mammals, the complex contains the CD3γ chain, CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and ζ chain (zeta chain), generating activation signals in T lymphocytes. However, NK cells do not naturally express the CD3 receptor complex or TCR. Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure fulfills a long-felt need in the art for improved immunotherapies, including immunotherapies that utilize NK cells. [Means for solving the problem]

[0005]

[0003] Embodiments of the present disclosure include methods and compositions for treating individuals with cancer using adoptive cell therapy. In specific embodiments, individuals are provided with a therapeutically effective amount of a two-part therapy comprising both engineered NK cells and an antibody capable of binding to the NK cells to initiate signaling, activation, and killing of target cells. The present disclosure relates to NK cells engineered to express multiple proteins not naturally expressed on NK cells that act in concert, including heterologous proteins on the surface of NK cells that are not naturally present on NK cells.

[0006] In specific embodiments, NK cells are engineered to express one or more proteins from the CD3 co-receptor complex and, optionally, the TCR receptor complex, each of which is normally present on the surface of T cells. Such engineering allows for increased versatility of the NK cells, which can be utilized in conjunction with various bispecific or multispecific antibodies, including anti-CD3 antibodies (e.g., anti-CD3 scFvs). In certain embodiments, the engineered NK cells are administered to an individual in need thereof in conjunction with one or more bispecific or multispecific antibodies, each of which has one antibody targeting CD3 and one antibody binding to a desired antigen, such as a cancer antigen. As a result, in certain cases, CD3-expressing NK cells can bind to the anti-CD3 antibody portion of the bispecific or multispecific antibody, and the antibody binding to the cancer antigen binds to the cancer antigen on the surface of the cancer cells. This cooperative binding between the NK cells and the antibodies results in the activation of cytotoxicity against the target cancer antigen.

[0007] In certain embodiments, the present disclosure relates to modified NK cells that express complete or partial CD3 complexes with or without TCRs, and in some cases, individual CD3 chains heterologously linked to NK-associated signaling domains, all of which allow the modified NK cells to be utilized with a variety of bispecific antibodies.

[0008] Embodiments of the present disclosure include compositions comprising NK cells modified to express a single chain or part or all of any combination of CD3δ, CD3ε, CD3γ, or CD3ζ. In some cases, the NK cells are modified to express the T cell receptor (TCR) αβ chain or the TCRγδ chain. The NK cells may be modified to express part or all of CD3ζ, or two of CD3ε, CD3δ, and CD3γ. In some cases, the NK cells are modified to express the full length of CD3ζ, CD3ε, CD3δ, and / or CD3γ. In certain cases, any one or more of CD3ζ, CD3ε, CD3δ, and CD3γ are heterologously linked to one or more intracellular signaling domains. The intracellular signaling domain may be selected from the group consisting of CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof. In some embodiments, the intracellular signaling domain is fused to CD3ζ. In some embodiments, the intracellular signaling domain is derived from DAP10. In some embodiments, the intracellular signaling domain is derived from CD28. In some embodiments, the intracellular signaling domain comprises a sequence derived from DAP10 and a sequence derived from CD28. In some embodiments, the intracellular signaling domain may also comprise other costimulatory signals associated with NK cell function, such as, but not limited to, 2B4, DNA, 4-1BB, DAP12, NKG2D, etc. In specific embodiments, the composition further comprises one or more bispecific or multispecific antibodies, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody. The NK cell may express and / or be complexed with an antibody. In some embodiments, the TCR is directed against a cancer antigen or a viral antigen. In specific embodiments, the NK cell is derived from cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a mixture thereof. In some embodiments, the TCR is directed against the NY-ESO antigen. In some embodiments, the TCR is directed against the PRAME antigen.The NK cells may be preactivated, e.g., with one or more cytokines, including IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. In some embodiments, the NK cells are expanded, e.g., in the presence of IL-2. In specific embodiments, the NK cells are modified to express one or more heterologous proteins, e.g., one or more engineered antigen receptors, one or more cytokines, one or more homing receptors, and / or one or more chemokine receptors. In certain cases, the engineered antigen receptor is a chimeric antigen receptor and / or an engineered T cell receptor. In some cases, the heterologous protein is a cytokine, e.g., selected from the group consisting of IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokine may be membrane-bound, and the membrane-bound cytokine may include a transmembrane domain from CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, or DAP12. In certain cases, the NK cell expresses a chimeric antigen receptor and a cytokine. In some cases, the bispecific antibody includes an antibody that targets a cancer antigen.

[0009] Embodiments of the present disclosure include compositions comprising a complex comprising: (1) NK cells engineered to express part or all of the CD3 receptor complex, and optionally engineered to express a T cell receptor (TCR) αβ chain or a TCR γδ chain; and (2) a bispecific or multispecific antibody comprising an anti-CD3 antibody that binds to CD3 on the NK cell. In specific embodiments, the complex is encapsulated in a pharmaceutically acceptable excipient. The complex may be contained in a delivery device.

[0010] In certain embodiments, there is a method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of any one of the compositions encompassed herein. In some embodiments, the NK cells and the antibody are administered to the individual simultaneously. The NK cells and the antibody may or may not be administered in the same formulation. The NK cells and the antibody may be pre-complexed before administration to the individual. In a specific embodiment, the NK cells and the antibody are administered to the individual at different times. The NK cells and the antibody may be administered by infusion. In a specific embodiment, the NK cells are autologous or allogeneic with respect to the individual.

[0011] Embodiments of the present disclosure include methods of redirecting NK cell specificity for a cancer antigen for treatment of an individual with a bispecific or multispecific anti-CD3 antibody, comprising administering to the individual the antibody and NK cells that express part or all of the CD3 receptor complex and, optionally, part or all of the TCRαβ chain or the TCRγδ chain. In specific embodiments, the method further comprises modifying the NK cells to express part or all of the CD3 receptor complex. In specific embodiments, the method further comprises modifying the NK cells to express the TCRαβ chain or the TCRγδ chain. In some cases, the method further comprises modifying the NK cells to express one or more heterologous proteins.

[0012] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0014] [Figure 1A] Various embodiments of NK cells engineered to express CD3 are shown, including their use with various heterologous proteins such as cytokines, bispecific NK cell engagers, and engineered antigen receptors (CAR and / or TCR). [Figure 1B] Demonstrate CD3 and TCR matched NK cells for optimal cancer immunotherapy. [Figure 1C] An example of a single chimeric CD3 construct is shown.

[0015] [Figure 2A] 1 shows an example of an expression construct for CD3 receptor complex components for transduction or transfection of NK cells. [Figure 2B] An example of a plasmid map of a representative expression construct is shown.

[0016] [Figure 3] A table of various TCR / CD3 expression construct designs for NK-TCR engineering is provided.

[0017] [Figure 4] An example of a CMV-directed TCR complex is shown, showing CD3 expression on engineered NK cells 4 days after transduction.

[0018] [Figure 5] Demonstrates TCR expression on engineered NK cells 4 days after transduction with CMV-directed TCR complexes.

[0019] [Figure 6]Figure 1 shows TCR / CD3 expression on engineered NK cells 6 days after intracellular transduction of CMV-directed TCR complexes.

[0020] [Figure 7] 1 demonstrates the binding of an example CD3-CD19 BiTE on NK cells at different concentrations via the CD3 / TCR complex on NK cells.

[0021] [Figure 8] NK-TCR cytokine production of TNFα and CD107a after stimulation with plate-bound CD3 antibody is shown.

[0022] [Figure 9] 1 demonstrates the phosphorylation of CD3z in NK TCR / CD3 cells after cross-linking of CD3.

[0023] [Figure 10A] Figures 10A-B show that pre-incubation of CD3-CD19 BiTE with TCR / CD3-expressing NK cells enhanced the killing activity against Raji cells. Figure 10A shows an effector:target ratio of 1:1. [Figure 10B] FIG. 10B depicts an effector:target ratio of 1:5.

[0024] [Figure 11] A schematic of multiple retroviral transduction to generate NK cells expressing CD3, IL-15, and the TCR complex is provided.

[0025] [Figure 12] 1 shows the expression of NY-ESO TCR on NK cells transduced with uTNK15. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; C refers to CD3-CD28-Dap10 with IL-15.

[0026] [Figure 13]The number of TCR molecules expressed per cell on NK cells is shown. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15. A Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences) was used to determine the number of NY-ESO TCR molecules on NK cells.

[0027] [Figure 14] Expression of NY-ESO TCR on T cells is shown.

[0028] [Figure 15] NK cells transduced with NY-ESO TCR kill target cells pulsed with NY-ESO peptide in a dose-dependent manner. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 molecules with IL-15; B refers to CD3-DAP10 molecules with IL-15; and C refers to CD3-CD28-Dap10 molecules with IL-15.

[0029] [Figure 16] We demonstrate endogenous NY-ESO expression in human tumor cell lines.

[0030] [Figure 17] We show that T cells transduced with the NY-ESO TCR kill tumor targets expressing NY-ESO.

[0031] [Figure 18] Results show that NY-ESO TCR-transduced NK cells can kill NY-ESO-expressing tumor targets even at low E:T ratios. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15.

[0032] [Figures 19A-19B] Figures 19A and 19B show that NY-ESO-transduced NK cells have a similar phenotype (19A) and expression pattern (19B) to NT NK cells. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15.

[0033] [Figure 20] A table showing the cellular composition of the expanded uTNK15 product is provided. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15.

[0034] [Figure 21] Figure 21A shows that NK cells can be successfully transduced with CD3 and TCR constant alpha-beta (TCRCab) (referred to as the TCR6 construct), and that the engineered NK cells can bind blinatumumab (Figure 21B) and selectively kill CD19+ lymphoma targets (Figure 21C).

[0035] [Figure 22A] Figures 22A-C show the in vivo activity of effector cells (e.g., NK cells, or T cells) containing NY-ESO-targeted TCRs. Figure 22A is a schematic diagram outlining the experimental procedures performed. [Figure 22B]Figure 22B shows bioluminescence imaging over time (days 1, 7, 14, and 21) of mice transplanted with FireFlyluciferase (FFluc)-transduced U266B.1 cells and treated with control, NY-ESO TCR NK cells, or NY-ESO TCR T cells (NK cells containing WT, #A, or #B UT-NK15-NY ESO TCR constructs, respectively; WT refers to wild-type CD3 molecules with IL-15; #A refers to CD3-CD28 with IL-15; #B refers to CD3-DAP10 with IL-15). [Figure 22C] Figure 22C is a graphical quantification of the mean bioluminescence brightness displayed in Figure 22B. These results demonstrated that effector cells containing the NY-ESO TCR construct described herein robustly inhibit tumor growth in vivo.

[0036] [Figure 23A] Figures 23A-B show the in vitro activity of effector cells (e.g., NK cells or T cells) containing the NY-ESO1-targeting TCR and the UT-NK15 construct. Figure 23A shows an image of a spheroid formed by the osteosarcoma tumor cell line Saos-2 stably transduced to express GFP, which was used to test the cytotoxic activity of NK cells and T cells expressing the NY-ESO1-specific TCR. [Figure 23B]Figure 23B is a graph showing the percentage of cytotoxicity (Y-axis) in representative images after 3 days of coculture. NK cells were cotransduced with the NY-ESO-TCR and the UT-NK15 signaling complex coexpressing different costimulatory molecules fused to the CD3ζ signaling chain or a TCR complex without IL-15. T cells were transduced with the NY-ESO TCR alone. Abbreviations in the graph: 28 = CD3ζ fused to the CD28 costimulatory domain; 10 = CD3ζ fused to the Dap10 costimulatory domain; 8 = CD8 alpha / beta coreceptor as part of the NY ESO TCR construct; wo IL-15 = construct containing only the CD3 zeta, epsilon, gamma, and delta TCR complex without costimulation or IL-15.

[0037] [Figure 24A] Figures 24A-D show the in vivo activity of effector cells (e.g., NK cells or T cells) containing NY-ESO-targeted TCRs and the UT-NK15 construct. Figure 24A shows the design of an in vivo study to test the activity of NK cells and T cells transduced with different NY-ESO TCRs. [Figure 24B] Figure 24B shows the results of BLI imaging of a study outlined and performed according to Figure 24A, in which mice were injected with U266 tumor cells and 3 days later received T cells transduced with NY-ESO-specific TCRs or NK cells co-transduced with NY-ESO TCR and UT-NK15 with CD3ζ fused to CD28 (labeled as NY-ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15 CD28 NK cells). A tumor-only group was used as a control. [Figure 24C] FIG. 24C shows the mean luminance intensity of the region of interest in animals tested according to FIG. 24A and imaged in FIG. 24B. [Figure 24D] FIG. 24D is a graph showing the cohort survival curves of the aforementioned animals.

[0038] [Figure 25]This figure shows the in vivo activity of effector cells (e.g., NK cells) engineered to express the NY-ESO TCR and CD3 complex with or without the IL-15 transgene contained in the construct. NSG mice were irradiated (300 cGy) and the following day injected with 500,000 U266 cells (an HLA-A2-positive, NY-ESO-expressing myeloma cell line) via the tail vein. Three days later, the mice received 5 million TCR-transduced T cells or NK cells. Tumor control in mice was monitored by BLI imaging. NK cells were transduced with NY-ESO-specific TCRs expressing or not expressing the CD8 alpha / beta coreceptor and cotransduced with a CD3 complex without an IL-15 transgene, or with the UT-NK15 costimulatory molecule expressing CD3ζ fused to CD28 (UT-NK15 CD28) or the UT-NK15 costimulatory molecule expressing CD3ζ fused to DAP10 (UT-NK15 DAP10).

[0039] [Figure 26A] Figures 26A-C show the in vitro expression of the Preferentially Expressed Antigen in Melanoma (PRAME) TCR in effector cells (e.g., NK cells or T cells) and the in vitro activity of the cells. Figure 26A shows the expression of both UT-NK15 (x-axis, CD3) and PRAME-specific TCR (y-axis, TCR) in NK cells (TCR clones 46, 54, or DSK3, respectively) or the expression of PRAME-specific TCR in T cells transduced with it (TCR clones 46 or 54). [Figure 26B]Figure 26B shows the in vitro cytotoxicity of NK cells expressing a PRAME-specific TCR against the U266 myeloma cell line. Using Incucyte live cell imaging, the cytotoxicity of PRAME-specific TCR-transduced T cells and NK cells transduced with UT-NK15 and PRAME-specific TCRs against U266 myeloma cells was measured. GFP-expressing U266 cells were cocultured with PRAME-specific TCR-expressing T cells or NK cells at a 1:1 effector:target ratio. A reduction in GFP expression indicated cell death. 26 hours later, 50,000 tumor cells (labeled "reload") were added to each well in duplicate for tumor reload assays. Open symbols represent T cells, and filled symbols represent NK cells. NT = no transduction. [Figure 26C] Figure 26C shows the in vitro cytotoxicity of NK cells expressing a PRAME-specific TCR against the UA375 melanoma cell line. Using Incucyte live-cell imaging, the cytotoxicity of PRAME-specific TCR-transduced T cells and NK cells transduced with UT-NK15 and a PRAME-specific TCR (PRAME-specific TCR clone 46 (TCR-46), PRAME-specific TCR clone 54 (TCR-54), or PRAME-specific TCR clone DSK3 (DSK)) against UA375 melanoma cells was measured. GFP-expressing UA375 cells were cocultured with PRAME-expressing T cells or NK cells at a 1:1 effector:target ratio. A reduction in GFP expression indicated cell death. After 26 hours, 50,000 tumor cells were added to each well in duplicate for tumor rechallenge assays. Open symbols represent T cells, and closed symbols represent NK cells. NT = no transduction. DETAILED DESCRIPTION OF THE INVENTION

[0040] Following long-standing patent law practice, the words "a" and "an," when used in conjunction with the word compris- ing, herein, including the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0041] Throughout this specification, unless the context dictates otherwise, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are necessary or essential, and that other elements may not be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0042] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0043] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0044] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0045] As used herein, the term "CD3 receptor complex" or "CD3 co-receptor complex" refers to the protein complex that acts as a T cell co-receptor in nature and is composed of the CD3ζ chain, the CD3γ chain, the CD3δ chain, and two CD3ε chains (although alternatively, only one CD3ε chain is used).

[0046] The term "engineered," as used herein, refers to an entity made by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, an engineered entity is synthetic and includes elements that do not occur in nature or are not constructed by the methods utilized in the present disclosure. In specific embodiments, the vector is engineered by recombinant nucleic acid techniques and the cell is engineered by transfection or transduction of the engineered vector. The cell can be engineered to express a heterologous protein that is not naturally expressed by the cell, either because the heterologous protein is a recombinant or synthetic protein or because the cell does not naturally express the protein.

[0047] The phrases "pharmaceutically or pharmacologically acceptable" refer, as appropriate, to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human. The preparation of pharmaceutical compositions containing an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, it will be understood that if administered to an animal (e.g., a human), preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0048] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., parenteral vehicles such as water, alcoholic / aqueous solutions, saline, sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient replenishers, such materials, and combinations thereof, as known to those skilled in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0049] The term "subject," as used herein, generally refers to an individual who has or is suspected of having cancer. A subject can be any organism or animal subject that is the subject of a method or material, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject can be, for example, a patient who has or is suspected of having a disease (sometimes referred to as a pathology), such as a benign or malignant neoplasm or cancer. A subject can be undergoing or have undergone treatment. A subject can be asymptomatic. A subject can also be an individual who is healthy but desires cancer prevention. The terms "individual" can be used interchangeably, at least in some cases. A "subject" or "individual," as used herein, may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the Internet.An individual may include humans or non-human animals of any age, and thus includes both adults and young (i.e., children) and infants, including individuals in utero.The term is not intended to imply the need for medical treatment, and an individual may voluntarily or involuntarily participate in an experiment, whether clinical or supporting basic scientific research.

[0050] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment may optionally include either a reduction or amelioration of one or more symptoms of the disease or condition, or a delay in the progression of the disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of the disease or condition, or the symptoms associated therewith. "Treating" may also mean the alleviation of at least one symptom of a disease or condition.

[0051] As used herein, "TCR / CD3 complex" refers to the protein complex that is naturally present on the surface of T cells and that contains the CD3ζ, CD3γ, CD3δ, and CD3ε chains, as well as the T cell receptor α and β chains and / or the T cell receptor γ and δ chains.

[0052] I. Embodiments of the Disclosure Natural killer (NK) cells are an emerging cellular immunotherapy for patients with hematologic malignancies as well as solid tumors. Specifically, the present disclosure relates to NK cells that have been modified to enhance their immunotherapeutic function compared to unmodified NK cells. This modification makes the NK cells more versatile when used in combination with other therapeutic agents and, at least in some embodiments, confer T-cell-like activity by utilizing the CD3 / TCR receptor complex. In specific embodiments, the NK cells are engineered to express either (i) a single CD3 chain (CD3-zeta, CD3-epsilon, CD3-delta, or CD3-gamma) or part or all of the human CD3 receptor complex (comprising any combination of CD3-delta, epsilon (one or two copies of epsilon), gamma, and zeta); or (ii) a single CD3 chain or human CD3 receptor complex (comprising any combination of CD3-delta, epsilon (one or two molecules), gamma, and zeta, either as full-length proteins or as partial proteins heterologously linked to one or more intracellular signaling domains); and (iii) the CD3 complex may or may not include a T-cell receptor (αβ or γδ). The present disclosure relates to the use of CD3-expressing NK cells in the diagnosis and treatment of disease, including the use of cells in combination with bispecific or multispecific antibodies in which one epitope of the antibody binds to CD3 on the CD3-expressing NK cells. CD3-expressing NK cells can be pre-complexed with bispecific / multispecific antibodies ex vivo and / or combined in vivo to redirect their specificity for target antigens. In diagnostic embodiments, labeled NK cells can be loaded with any type of bispecific or multispecific antibody, including at least an anti-CD3 antibody, and the loaded labeled NK cells can be monitored for trafficking to the site of the target antigen where another antibody on the bispecific or multispecific antibody binds.

[0053] II. Compositions of the Present Disclosure The present disclosure relates to compositions comprising at least modified NK cells that express at least a portion of a TCR / CD3 complex. In some cases, the compositions also include bispecific or multispecific antibodies, including in the same formulation, although in alternative embodiments, the NK cells and antibodies are utilized as physically separate compositions.

[0054] A. TCR / CD3 modification of NK cells In certain embodiments, provided herein are compositions comprising NK cells engineered to express part or all of the TCR receptor complex and part or all of the CD3 co-receptor complex. In specific embodiments, the NK cells are engineered to contain all components of the CD3 complex, including CD3ζ, CD3ε, CD3γ, and CD3δ. In certain cases, the full length of CD3ζ, CD3ε, CD3γ, and CD3δ are utilized, including their extracellular, transmembrane, and intracellular domains; however, in alternative embodiments, only a portion of one or more of CD3ζ, CD3ε, CD3γ, and CD3δ are utilized, each of which may or may not be combined with one or more intracellular signaling domains, such as CD16, NKG2D, DAP10, DAP12, CD28, 41BB, 2B4, CD27, OX40, or any combination thereof. NK cells can also be engineered to express a TCR receptor complex; however, in alternative embodiments, none of the TCR receptor complex components are utilized.

[0055] In certain embodiments, an amino acid sequence (eg, a polypeptide) can include amino acids represented by the single letter "X" or the three-letter code "Xaa." In some embodiments, the amino acid represented by "X" or "Xaa" is any naturally occurring amino acid, such as, but not limited to, arginine (Arg, R), histidine (His, H), lysine (Lys, K), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), glycine (Gly, G), proline (Pro, P), cysteine ​​(Cys, C), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), or tryptophan (Trp, W).

[0056] In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is arginine (Arg, R). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is histidine (His, H). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is lysine (Lys, K). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is aspartic acid (Asp, D). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is glutamic acid (Glu, E). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is serine (Ser, S). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is threonine (Thr, T). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is asparagine (Asn, N). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is glutamine (Gln, Q). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is glycine (Gly, G). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is proline (Pro, P). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is cysteine ​​(Cys, C). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is alanine (Ala, A). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is valine (Val, V).In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is isoleucine (Ile, I). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is leucine (Leu, L). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is methionine (Met, M). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is phenylalanine (Phe, F). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is tyrosine (Tyr, Y). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is tryptophan (Trp, W).

[0057] In certain embodiments, any particular sequence of the CD3 receptor components, including wild-type or mutant versions of the components, is utilized, so long as the mutant CD3 receptor allows signaling through the CD3 complex leading to target activation and killing. In some cases, the following example sequences of CD3ε, CD3δ, CD3γ, and CD3ζ are utilized to engineer NK cells:

[0058] CD3 Epsilon(UniProtKB-P07766(CD3E_HUMAN))

[0059] signal peptide MQSGTHWRVLGLCLLSVGVW (SEQ ID NO: 1)

[0060] Extracellular domain sp|P07766|23-126 DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 2)

[0061] Transmembrane domain sp|P07766|127-152 VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 3)

[0062] Intracellular domain sp|P07766|153-207 KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 4)

[0063] An example of the Homo sapiens CD3e molecule (CD3E), mRNA, is present in the NCBI Reference Sequence GENBANK® accession number NM_000733.4. (SEQ ID NO: 5)

[0064] An example of a complete CD3 epsilon sequence, in nucleic acid and amino acid form, is as follows (underlined indicates signal peptide sequence): ATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAG(SEQ ID NO: 37) MQSGTHWRVLGLCLLSVGVWGQ DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 38)

[0065] CD3 Delta(UniProtKB-P04234(CD3D_HUMAN))

[0066] signal peptide MEHSTFLSGLVLATLLSQVS (SEQ ID NO: 6)

[0067] Extracellular domain sp|P04234|22-105 FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA (SEQ ID NO: 7)

[0068] Transmembrane domain sp|P04234|106-126 GIIVTDVIATLLLALGVFCFA (SEQ ID NO: 8)

[0069] Intracellular domain sp|P04234|127-171 GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 9)

[0070] Homo sapiens CD3d molecule, delta (CD3-TCR complex), mRNA (cDNA clone MGC:88324 IMAGE:30412345), complete cds GENBANK®:BC070321.1 ATGGAACATAGCACGTTTCTCTCTGGCCTGGTACTGGCTACCCTTCTCTCGCAAGTGAGCCCCTTCAAGATACCTATAGAGGAACTTGAGGACAGAGTGTTTGTGAATTGCAATACCAGCATCACATGGGT AGAGGGAACGGTGGGAACACTGCTCTCAGACATTACAAGACTGGACCTGGGAAAACGCATCCTGGACCCACGAGGAATATATAGGTGTAATGGGACAGATATATACAAGGACAAAGAATCTACCGTGCAAG TTCATTATCGAATGTGCCAGAGCTGTGTGGAGCTGGATCCAGCCACCGTGGCTGGCATCATTGTCACTGATGTCATTGCCACTCTGCTCCTTGCTTTGGGAGTCTTCTGCTTTGCTGGACATGAGACTGGA AGGCTGTCTGGGGCTGCCGACACACAAGCTCTGTTGAGGAATGACCAGGTCTATCAGCCCCTCCGAGATCGAGATGATGCTCAGTACAGCCACCTTGGAGGAAACTGGGCTCGGAACAAGTGA (SEQ ID NO: 10)

[0071] An example of a complete CD3 delta sequence, in nucleic acid and amino acid form, is as follows (underlined indicates signal peptide sequence): ATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGG AGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAG (SEQ ID NO: 35) MEHSTFLSGLVLATLLSQVSP FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 36)

[0072] CD3 Gamma (T cell surface glycoprotein CD3 gamma chain gene CD3G P09693) signal peptide MEQGKGLAVL ILAIILLQGTLA (SEQ ID NO: 11)

[0073] Extracellular domain sp|P09693|23-116 QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO: 12)

[0074] Transmembrane domain sp|P09693|117-137 GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 13)

[0075] Intracellular domain sp|P09693|138-182 GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 14)

[0076] Homo sapiens CD3g molecule (CD3G), mRNA;NM_000073.3:81-629 Homo sapiens CD3g molecule (CD3G), mRNA (SEQ ID NO: 15)

[0077] An example of the entire CD3 gamma sequence, in nucleic acid and amino acid form, is as follows (underlined indicates signal peptide sequence): ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCC CAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGT GTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAAT (SEQ ID NO: 33) MEQGKGLAVLILAIILLQGTLA QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 34)

[0078] CD3 Zeta

[0079] signal peptide sp|P20963| SP MKWKALFTAAILQAQLPITEA (SEQ ID NO: 16)

[0080] Extracellular domain sp|P20963|22-30 ECD QSFGLLDPK (SEQ ID NO: 17)

[0081] Transmembrane domain sp|P20963|31-51 tmd LCYLLDGILFIYGVILTALFL (SEQ ID NO: 18)

[0082] Intracellular domain sp|P20963|52-164 ICD RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19)

[0083] An example of a complete CD3 zeta sequence, in nucleic acid and amino acid form, is as follows (underlined indicates signal peptide sequence): ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA CAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 31) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 32)

[0084] Homo sapiens CD247 molecule (CD247; also known as CD3 Zeta), transcript variant 1, mRNA NCBI reference sequence: NM_198053.3 NM_198053.3:65-559 Homo sapiens CD247 molecule (CD247), transcript variant 1, mRNA ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTA TGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 20)

[0085] In specific embodiments, NK cells are engineered to express one or more of the TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain, and any combination thereof can be utilized. In particular cases, NK cells are engineered to express the T cell receptor (TCR) αβ chain or TCR γδ chain. In particular cases, NK cells are engineered to express only part or all of the constant region of one or more of the TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. NK cells may also be engineered to express only part or all of the constant region of the T cell receptor (TCR) αβ chain or TCR γδ chain. When a portion of a constant region is utilized, the portion of the constant region may be at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids, including contiguous amino acids of any constant region. A portion of a constant region may constitute at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the amino acids of the constant region, including consecutive amino acids of the constant region.

[0086] In certain cases, any of the sequences encompassed herein are utilized to modify NK cells, while in other cases, sequences related in identity are utilized, for example, related sequences that are at least 80, 85, 90, 95, 96, 97, 98, 99% identical to any of the sequences encompassed herein may be utilized in the present disclosure.

[0087] Specific constructs for the expression of various TCR / CD3 proteins in NK cells can be utilized in a variety of configurations. In certain cases, NK cells can be transduced or transfected with one or more vectors to express any of the various proteins encompassed herein, including at least any one or more components of the TCR / CD3 complex. In certain cases, the vector or vectors themselves may or may not be multicistronic, by virtue of ultimately being capable of producing two or more separate polypeptides. When one or more multicistronic vectors are used, they may utilize one or more internal ribosome entry sites (IRES) and / or one or more 2A self-cleaving peptide sites. When one or more 2A sequences are utilized, the following can be used, with GSG as the optional linker:

[0088] T2A (GSG) EGRGSLLTCGDVEENPGP (SEQ ID NO: 21)

[0089] P2A (GSG) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 22)

[0090] E2A (GSG) QCTNYALLKLAGDVESNPGP (SEQ ID NO: 23)

[0091] F2A (GSG) VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24)

[0092] In situations where multiple protein components are expressed from a multicistronic vector, the order from 5' to 3' on the polynucleotide vector can be any order, although in alternative cases they are present on the vector in a specific order. A multicistronic vector may express multiple components of the CD3 receptor complex and no other heterologous proteins, or a multicistronic vector may express multiple components of the CD3 receptor complex and one or more other heterologous proteins. A multicistronic vector may express multiple components of the TCR receptor complex and no other heterologous proteins, or a multicistronic vector may express multiple components of the TCR receptor complex and one or more other heterologous proteins. A multicistronic vector may or may not express one or more components of the TCR receptor complex and one or more components of the CD3 complex. In specific embodiments, a multicistronic vector comprises one or more components of the CD3 receptor complex and one or more heterologous proteins, such as a cytokine and an engineered antigen receptor, such as a CAR.

[0093] Figure 2A shows an example of a multicistronic vector in which full-length CD3ε, CD3δ, CD3γ, and CD3ζ are present, separated by the same or different 2A self-cleaving peptide sites. As further illustrated in the plasmid map in Figure 2B, the multicistronic vector may contain the signal peptide, extracellular domain, transmembrane domain, and intracellular domain of each of CD3ε, CD3δ, CD3γ, and CD3ζ.

[0094] Figure 3 provides a table showing examples of various TCR expression constructs for engineering TCR-expressing NK cells. In certain embodiments of the present disclosure, the CD3 receptor component and the TCR receptor component are expressed in NK cells from different vectors. In each case, the vector can express a TCR directed against a specific antigen, such as a cancer antigen or a viral antigen. The TCR may or may not include at least a portion of CD3ζ, including the intracellular domain of CD3ζ, and NK cells also express CD3ζ as a molecule separate from the TCR and as part of the CD3 receptor complex. Similarly, the CAR may or may not include at least a portion of CD3ζ, including the intracellular domain of CD3ζ, and NK cells also express CD3ζ as a molecule separate from the TCR and as part of the CD3 receptor complex.

[0095] In a specific embodiment, the TCR of the modified NK cell is not necessarily used as a therapeutic aspect of the cell, but as a structural support or scaffold to promote the function or enhancement of the CD3 receptor complex. That is, the TCR can be any TCR, and it does not have to be used for its ability to target a specific antigen. In such a case, for example, a TCR that targets a viral antigen can be used in NK cells used against cancers that are not necessarily related to that particular virus. In other cases, the TCR is selected for its ability to target a specific cancer antigen. Examples of antigens that can be targeted by TCRs are described elsewhere herein.

[0096] In Figure 3 the following example constructs are described:

[0097] TCR1: refers to TCRpp65 (TCR for HLA-A2 restricted CMVpp65) linked to the intracellular CD3 zeta domain and full length CD3 gamma, full length CD3 delta, and full length CD3 epsilon; the construct may also be referred to as TCRpp65ZicdGDEFL and may include the following sequence:

[0098] In TCRpp65ZicdGDEFL, the sequences of the corresponding components are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0099] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 40) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 41)

[0100] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO: 42)(where the P2A sequence is present at the C-terminus) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO: 43) (where the lowercase sequence is the P2A sequence)

[0101] TCRa-extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 44) ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO: 45)

[0102] CD3 gamma delta epsilon (CD3GDE): (SEQ ID NO: 46) (wherein the E2A sequence is at the C-terminus)

[0103] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0104] TCR2: refers to TCRpp65 linked to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, full length CD3 epsilon, lacking IL-15. Representative sequences are as follows:

[0105] TCR3: refers to TCRpp65 bound to the intracellular CD3z domain and IL-15, sometimes referred to as TCRpp65Zicd15, with a representative sequence as follows: *(SEQ ID NO: 52)

[0106] For TCRpp65Zicd15, the sequences of the corresponding components are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0107] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 40) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 41)

[0108] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO: 42)(Here, the P2A sequence is present at the C-terminus) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO: 43) (where the lowercase sequence is the P2A sequence)

[0109] TCRa-extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 44) (SEQ ID NO: 45)

[0110] CD3 zeta intracellular domain (Z-ICD) (in specific embodiments, two or more Z-ICD sequences may be utilized): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP (SEQ ID NO: 53) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC (SEQ ID NO: 54)

[0111] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0112] TCR4: refers to TCRpp65, also known as TCRpp65 beta alpha, and the representative sequence is as follows: *(SEQ ID NO: 55)

[0113] For TCRpp65betaalpha, the sequences of the corresponding components are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0114] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 40) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 41)

[0115] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO: 42) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC (SEQ ID NO: 54)

[0116] TCRa-extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 44) ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO: 45)

[0117] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP(SEQ ID NO: 53) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 54)

[0118] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0119] Further representative sequences of TCRpp65betaalpha are as follows: *(SEQ ID NO: 57)

[0120] Z1: Refers to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, and full length CD3 epsilon linked to IL15 (see Figures 2A and 2B), and may also be referred to as CD3ZFLGDEFL15, and a representative sequence may be as follows: (SEQ ID NO: 58)

[0121] Z2: refers to full-length CD3 zeta, full-length CD3 gamma, full-length CD3 delta, and full-length CD3 epsilon linked to membrane-bound IL21 (membrane-bound IL21 has a CD8 transmembrane domain), also referred to as CD3ZGDEFLSP821CD28, with a representative sequence as follows:

[0122] In CD3ZGDEFLSP821CD28, the sequences of the corresponding components are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0123] CD3: (SEQ ID NO: 61)

[0124] SP CD8: MRICLTSDRLAPAAGLAAPRRQAV (SEQ ID NO: 63) atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg (SEQ ID NO: 64)

[0125] IL-21: HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 65) CATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAACGGA TAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT (SEQ ID NO: 66)

[0126] CD8 Hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 67) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 68)

[0127] CD28 transmembrane domain: FWVLVVVGGVLACYSLLVTVAFIIFWV* (SEQ ID NO: 69) TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 70)

[0128] Z3: refers to full-length CD3 zeta, full-length CD3 gamma, full-length CD3 delta, and full-length CD3 epsilon linked to membrane-bound IL21 (membrane-bound IL21 has the CD28 transmembrane domain), also referred to as CD3ZGDEFL8SP21CD8, with a representative sequence as follows:

[0129] In CD3ZGDEFL8SP21CD8, the sequences of the corresponding components are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0130] CD3: (SEQ ID NO: 61)

[0131] SP CD8: MRICLTSDRLAPAAGLAAPRRQAV (SEQ ID NO: 63) atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg (SEQ ID NO: 64)

[0132] IL-21: HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 65) cataaatcttcctctcaaggtcaggaccgccatatgattcgaatgcggcagctgattgacatagtcgatcaactgaagaactatgtgaatgatcttgtgcccgag tttttgccagcccctgaagacgtagaaactaattgtgagtggagtgccttttcctgctttcaaaaggcacagctgaaatccgccaacacgggcaataacgaacgga taattaacgtatccattaagaagctgaagcggaagccgccctcaaccaatgcgggacggcggcaaaagcatcgcttgacctgtccgtcatgcgacagctacgaga aaaagcccccgaaggagttcttggaacgcttcaagagtctccttcagaaaatgattcaccagcacctgtcctcacggacgcacggaagcgaggacagt (SEQ ID NO: 65)

[0133] CD8 Hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 67) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 68)

[0134] CD8 transmembrane domain: IYIWAPLAGTCGVLLLSLVIT* (SEQ ID NO: 72) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC (SEQ ID NO: 73)

[0135] In certain embodiments, provided herein are CD3 constructs comprising a fusion with an intracellular costimulatory domain derived from CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, or any combination thereof. In certain embodiments, the intracellular costimulatory domain is fused to CD3δ, CD3ε, CD3γ, and / or CD3ζ. In certain embodiments, such CD3 fusion constructs comprise CD3ζ fused to the DAP10 intracellular costimulatory domain. In certain embodiments, such CD3 fusion constructs comprise CD3ζ fused to the CD28 intracellular costimulatory domain. In certain embodiments, such CD3 fusion constructs comprise CD3ζ fused to the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain. In certain embodiments, CD3ζ fused to a DAP10 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:106. In certain embodiments, CD3ζ fused to a CD28 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:107. In certain embodiments, CD3ζ fused to a DAP10 intracellular costimulatory domain and a CD28 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:108.In certain embodiments, CD3ζ fused to a DAP10 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 109. In certain embodiments, CD3ζ fused to a CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:110. In certain embodiments, CD3ζ fused to the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 111. In certain embodiments, CD3ζ fused to the intracellular domain may not include a C-terminal 2A domain. In certain embodiments, CD3ζ fused to the intracellular domain may not include an N-terminal signal peptide domain. ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 106) ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 107) ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 108) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP(SEQ ID NO: 109) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (SEQ ID NO: 110) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (SEQ ID NO: 111)

[0136] In certain embodiments, the DAP10 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 112. In certain embodiments, the CD28 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 113. In certain embodiments, the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain are represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 114. In certain embodiments, the DAP10 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 115. In certain embodiments, the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 116. In certain embodiments, the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain are represented by amino acid sequences that are at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 117. CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 112) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCA (SEQ ID NO: 113) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 114) LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 115) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 116) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 117)

[0137] UTNK15-DAP10: refers to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, full length CD3 epsilon linked to IL15 comprising a fusion with the intracellular costimulatory domain from DAP10, which may be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 118. In certain embodiments, the UTNK15-DAP10 amino acid sequence may be represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 119. MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQ.DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 119).

[0138] UTNK15-28: refers to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, full length CD3 epsilon linked to IL15 comprising a fusion with an intracellular costimulatory domain from CD28, which may be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 120. In certain embodiments, the UTNK15-28 amino acid sequence may be represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 121. MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQ.DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 121).

[0139] UTNK15-28-DAP10: refers to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, full length CD3 epsilon linked to IL15 comprising a fusion of the intracellular costimulatory domain from DAP10 and the intracellular costimulatory domain from CD28, which may be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 122. In certain embodiments, the UTNK15-28-DAP10 amino acid sequence may be represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 123. MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQ.DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 123).

[0140] As shown in Figure 3 and described above, the term "linked" refers to the presence on the same polynucleotide vector and does not necessarily mean that the two polypeptides are expressed as a single polypeptide. For example, a cytokine produced from a vector of the present disclosure may ultimately be produced as a molecule separate from any one or more TCR / CD3 receptor complex components. In contrast, the term "fused" or "fusion" refers to two polypeptides that contain a peptide bond joining the two molecules, i.e., two polypeptides that are covalently linked by an amide bond and are not separated by a dividing element such as a 2A element.

[0141] One specific example of a TCR that can be utilized intracellularly is the NY-ESO TCR, and specific examples of sequences include at least the following:

[0142] TCRα: XQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 25)

[0143] TCRβ: GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 26)

[0144] In certain embodiments, the TCR may comprise a TCR alpha chain variable region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:85. aaacaggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaactgcagtttcact gatagcgctatttacaac ctccagtggtttaggcaggaccctgggaaaggtctcacatctctgttgctt attcagtcaagtcagagagagcaaacaagtggaagacttaatgcctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgactcagccacctacctc tgtgctgtgaggcccctttatggaggaagctacatacctacattt ggaagaggaaccagccttattgttcatccgtat (SEQ ID NO: 85)

[0145] In certain embodiments, the TCR may comprise a TCR alpha chain constant region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:86. atccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaag taaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcat gtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacg aacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc (SEQ ID NO: 86)

[0146] In certain embodiments, the TCR may comprise a TCR alpha chain encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:87. atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagcaaacaggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaactgcagtttcact gatagcgctatttacaac ctccagtggtttaggcaggaccctgggaaaggtctcacatctctgttgctt attcagtcaagtcagagagag caaacaagtggaagacttaatgcctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgactcagccacctacctc tgtgctgtgaggcccctttatggaggaagctacatacctacattt ggaagaggaaccagccttattgttcatccgtatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttg attctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaa caaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaa acagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc (SEQ ID NO: 87)

[0147] In certain embodiments, the TCR may comprise an amino acid sequence of a TCR alpha chain variable region that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:88. XQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVRPLYGGSYIPTF GRGTSLIVHPY (SEQ ID NO: 88)

[0148] In certain embodiments, the TCR may comprise an amino acid sequence of a TCR alpha chain constant region that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:89. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 89)

[0149] In certain embodiments, the TCR may comprise an alpha chain CDR1 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:90. DSAIYN (SEQ ID NO: 90)

[0150] In certain embodiments, the TCR may comprise an alpha chain CDR2 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:91. IQSSQRE (SEQ ID NO: 91)

[0151] In certain embodiments, the TCR may comprise an alpha chain CDR3 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:92. CAVRPLYGGSYIPTF (SEQ ID NO: 92)

[0152] In certain embodiments, the TCR may comprise a TCR beta chain variable encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:93. ggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactgcagtgtgcccaggat atgaaccatgaatac atgtcctggtatcgacaagacccaggcatggggctgaggctgattcattac tcagttggtgctggtatc actgaccaaggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctcccagacatctgtgtacttc tgtgccagcagttacgtcgggaacaccggggagctgtttttt ggagaaggctctaggctgaccgtactggag (SEQ ID NO: 93)

[0153] In certain embodiments, the TCR may comprise a TCR beta chain constant region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:94. (SEQ ID NO: 94)

[0154] In certain embodiments, the TCR may comprise a TCR beta chain encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:95. Atgagcatcggcctcctgtgctgtgcagccttgtctctcctgtgggcaggtccagtgaatgctggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactgcagtgtgcccaggat atgaaccatgaatac atgtcctggtatcgacaagacccaggcatggggctgaggctgattcattac tcagttggtgctggtatcactgaccaaggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctcccagacatctgtgtacttc tgtgccagcagttacgtcgggaacaccggggagctgtttttt (SEQ ID NO: 95)

[0155] In certain embodiments, the TCR may comprise an amino acid sequence of a TCR beta chain variable region that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:96. GVTQTPKFQVLKTGQSMTLQCAQD MNHEY MSWYRQDPGMGLRLIHY SVGAGI TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF CASSYVGNTGELFF GEGSRLTVLE (SEQ ID NO: 96)

[0156] In certain embodiments, the TCR may comprise an amino acid sequence of a TCR beta chain constant region that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:97. DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 97)

[0157] In certain embodiments, the TCR may comprise a beta chain CDR1 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:98. MNHEY (SEQ ID NO: 98)

[0158] In certain embodiments, the TCR may comprise a beta chain CDR2 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:99. SVGAGI (SEQ ID NO: 99)

[0159] In certain embodiments, the TCR may comprise a beta chain CDR3 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:100. CASSYVGNTGELFF (SEQ ID NO: 100)

[0160] In certain embodiments, a TCR (e.g., TCR alpha, beta, delta, and / or gamma) chain can include a signal peptide. In certain embodiments, the signal peptide is encoded by a nucleic acid that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101 or SEQ ID NO: 102. In certain embodiments, the signal peptide is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:103 or SEQ ID NO:104. atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagc (SEQ ID NO: 101) atgagcatcggcctcctgtgctgtgcagccttgtctctcctgtgggcaggtccagtgaatgct (SEQ ID NO: 102) METLLGLLILWLQLQWVSS (SEQ ID NO: 103) MSIGLLCCAALSLLWAGPVNA (SEQ ID NO: 104)

[0161] In certain embodiments, the TCR is HLA-A * In the context of the 02 class I allele, the TCR recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1. In certain embodiments, the TCR may target an epitope characterized by the amino acid sequence according to SEQ ID NO: 105. SLLMWITQC (SEQ ID NO: 105)

[0162] One specific example of a TCR that can be utilized intracellularly is TCRpp65 alpha, and specific examples of sequences include at least the following (underlined indicates signal peptide sequence): ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGACAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCCAGTGATGTAGGCATCTACTTCTGTGCTGGACCCATGAAAACCTCCTACGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ ID NO: 27) MDSWTFCCVSLCILVAKHTDAG QQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGPMKTSYDKVIFGPGTSLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 28)

[0163] One specific example of a TCR that can be utilized intracellularly is TCRpp65beta, and specific examples of sequences include at least the following (underlined indicates signal peptide sequence): ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGA GTTATCCAGTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGTTCGGCAAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC (SEQ ID NO: 29) MDSWTFCCVSLCILVAKHTDAG VIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSSANYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 30)

[0164] TCRpp65ZFLGDEFL15

[0165] In certain embodiments, a construct (sometimes referred to as TCRpp65ZFLGDEFL15) can be utilized in which TCRpp65 is linked to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, full length CD3 epsilon, and also linked to IL-15. One representative sequence of such a construct is as follows:

[0166] In TCRpp65ZFLGDEFL15, the sequences of the corresponding components are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0167] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADATNFSLLKQAGDVEENPGP (SEQ ID NO: 75) (comprising a P2A sequence at its C-terminus) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 76)

[0168] TCRa - extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSEGRGSLLTCGDVEENPGP (SEQ ID NO: 77) (including the T2A sequence at its C-terminus) ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC (SEQ ID NO: 78)

[0169] TCR5: Designated TCRCgdZFLGDEFL15, it is the constant region of TCR gamma and delta, linked to full-length CD3 zeta, full-length CD3 gamma, full-length CD3 delta, and full-length CD3 epsilon; and IL-15. Representative sequences are as follows:

[0170] TCR steady-state gamma-delta (TCRCgd) MRWALLVLLAFLSPASQDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKSGSGATNFSLLKQAGDVEENPGPMILTVGFSFLFFYRGTLCSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO: 82)

[0171] CD3: (SEQ ID NO: 79)

[0172] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0173] TCR6: Also known as TCRCabZFLGDEFL15, is the constant region of TCR alpha and beta, linked to full-length CD3 zeta, full-length CD3 gamma, full-length CD3 delta, and full-length CD3 epsilon; and IL-15. Representative sequences are as follows:

[0174] TCR constant alpha-beta (TCRCab) METLLGLLILWLQLQWVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGATNFSLLKQAGDVEENPGPMSIGLLCCAALSLLWAGPVNADLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 83)

[0175] CD3: (SEQ ID NO: 79)

[0176] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO: 49)

[0177] In some embodiments, a TCR construct comprises a NY-ESO-specific TCR and a CD8 alpha / beta co-receptor molecule. In some embodiments, such a construct can comprise a TCR alpha chain variable region signal peptide, a TCR alpha chain variable region, a TCR alpha chain constant region, a 2A element (e.g., a P2A element), a TCR beta chain variable region signal peptide, a TCR beta chain variable region, a TCR beta chain constant region, a 2A element (e.g., an E2A element), a CD8-beta polypeptide, a 2A element (e.g., a T2A element), and a CD8-alpha polypeptide. In some embodiments, a TCR construct comprising a NY-ESO-specific TCR and a CD8 alpha / beta co-receptor molecule nucleotide coding sequence is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 124. In some embodiments, a TCR construct comprising a NY-ESO-specific TCR and a CD8 alpha / beta co-receptor molecule amino acid sequence is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 125.

[0178] In some embodiments, the CD8 alpha co-receptor molecule is transcriptionally linked to any TCR molecule disclosed herein. In some embodiments, the CD8 alpha co-receptor molecule nucleotide coding sequence is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 126. In some embodiments, the CD8 beta co-receptor molecule nucleotide coding sequence is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 127. In some embodiments, the CD8 alpha co-receptor amino acid sequence is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 128. In some embodiments, the CD8 beta co-receptor amino acid sequence is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 129. ATGAGGCCACGACTTTGGCTGCTGCTCGCTGCACAGTTGACTGTACTGCATGGCAATAGTGTGTTGCAGCAGACACCTGCATACATCAAGGTTCAGACAAATAAGATGGTTATGCTGAGTTGCGAGGCAAAAATTAGTTTGAGCAATATGCGGATCTACTGGTTGCGACAGAGACAGGCTCCCAGTAGTGATAGTCACCACGAATTCCTGGCTCTTTGGGATTCCGCAAAAGGAACGATTCATGGGGAAGAAGTAGAGCAGGAGAAGATTGCGGTTTTCCGCGATGCATCTCGCTTTATCCTTAATCTTACATCCGTTAAGCCTGAGGACAGTGGGATCTATTTTTGTATGATTGTAGGGTCCCCCGAATTGACATTTGGGAAGGGTACGCAGCTCTCCGTAGTTGACTTTCTGCCCACAACGGCACAACCCACTAAGAAGTCCACCCTGAAGAAGCGCGTCTGTCGCTTGCCCAGACCTGAAACCCAAAAGGGTCCACTCTGTTCCCCTATAACCCTGGGGTTGTTGGTGGCGGGCGTCTTGGTCCTGCTTGTTAGCTTGGGCGTAGCCATTCATCTGTGTTGCCGAAGACGCAGAGCCCGACTTAGATTTATGAAGCAATTCTATAAGTGA(SEQ ID NO: 126) ATGGCCTTGCCCGTCACTGCGCTTTTGCTCCCGCTCGCTCTTCTCCTGCATGCAGCCCGACCATCTCAATTTAGAGTTTCTCCACTCGACAGGACGTGGAACCTCGGCGAAACCGTCGAACTTAAATGTCAAGTACTTCTCTCAAATCCGACTTCTGGTTGCTCATGGCTCTTTCAGCCGAGAGGAGCAGCTGCCAGCCCCACCTTCCTGCTGTATCTCTCCCAGAACAAGCCGAAGGCCGCCGAAGGGCTCGATACTCAACGATTTAGCGGGAAGCGACTCGGGGACACGTTCGTTCTTACTCTCAGCGATTTTAGAAGAGAGAACGAGGGATATTATTTTTGTTCCGCACTCTCTAACAGCATCATGTACTTCAGTCATTTTGTACCAGTCTTTCTCCCTGCAAAACCAACGACTACTCCAGCACCAAGACCGCCCACTCCCGCACCTACTATTGCAAGCCAACCTTTGAGTCTCCGACCAGAGGCATGCAGACCTGCTGCTGGAGGTGCAGTACATACGCGAGGGTTGGATTTTGCCTGCGATATCTATATCTGGGCCCCCTTGGCCGGCACGTGCGGGGTGCTCCTGCTGAGTCTCGTAATTACTCTTTATTGTAATCATAGAAACCGCAGAAGGGTGTGTAAGTGTCCCCGGCCTGTCGTGAAAAGCGGGGATAAGCCCAGTTTGTCTGCTCGGTACGTC(SEQ ID NO: 127) MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK(SEQ ID NO: 128) MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 129)

[0179] In some embodiments, the TCR construct comprises a PRAME-specific TCR chain. In some embodiments, the TCR construct comprising a PRAME-specific TCR chain comprises a TCR alpha chain and a TCR beta chain found in PRAME-specific TCR clone 46, clone 54, and / or clone DSK3. In some embodiments, the TCR construct comprising a PRAME-specific TCR chain comprises a TCR alpha chain and a TCR beta chain that target the PRAME epitope SLLQHLIGL (SEQ ID NO: 131) and / or QLLALLPSL (SEQ ID NO: 132).

[0180] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 133 (e.g., TCR clone 46 TCR alpha) and / or SEQ ID NO: 134 (e.g., TCR clone 46 TCR beta). In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 135 (e.g., TCR clone 46 TCR alpha) and / or SEQ ID NO: 136 (e.g., TCR clone 46 TCR beta). (query number 133) (query number 134) MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGIPRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 135) MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTPWLAGGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 136)

[0181] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 137 (e.g., TCR clone 54 TCR alpha) and / or SEQ ID NO: 138 (e.g., TCR clone 54 TCR beta). In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 139 (e.g., TCR clone 54 TCR alpha) and / or SEQ ID NO: 140 (e.g., TCR clone 54 TCR beta). ATGCTGCTGCTGCTGGTGCCCGTGCTGGAAGTGATCTTCACCCTGGGCGGCACCAGAGCCCAGAGCGTGACACAGCTGGGCAGCCACGTGTCCGTGTCTGAGAGGGCCCTGGTGCTGCTGAGATGCAACTACTCTTCTAGCGTGCCCCCCTACCTGTTTTGGTACGTGCAGTACCCCAACCAGGGGCTGCAGCTGCTCCTGAAGTACACCAGCGCCGCCACACTGGTGAAGGGCATCAACGGCTTCGAGGCCGAGTTCAAGAAGTCCGAGACAAGCTTCCACCTGACCAAGCCCAGCGCCCACATGTCTGACGCCGCCGAGTACTTCTGTGCCGTGAGCGGCCAGACCGGCGCCAACAACCTGTTCTTCGGCACCGGCACCCGGCTGACAGTGATCCCTTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO: 137) ATGGGCTTCCGGCTGCTGTGCTGCGTGGCCTTTTGTCTGCTGGGAGCCGGACCTGTGGATAGCGGCGTGACCCAGACCCCCAAGCACCTGATCACCGCCACCGGCCAGAGAGTGACCCTGCGCTGCAGCCCTAGAAGCGGCGACCTGAGCGTGTACTGGTATCAGCAGAGCCTCGACCAGGGCCTGCAGTTCCTGATCCAGTACTACAACGGCGAGGAACGGGCCAAGGGCAACATCCTGGAACGGTTCAGCGCCCAGCAGTTCCCCGATCTGCACAGCGAGCTGAACCTGAGCAGCCTGGAACTGGGCGACAGCGCCCTGTACTTCTGCGCCAGCGCCAGATGGGATAGAGGCGGCGAGCAGTACTTCGGCCCTGGCACCAGACTGACCGTGACCGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO: 138) MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSERALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSGQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 139) MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASARWDRGGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 140)

[0182] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 141 (e.g., TCR clone DSK3 TCR alpha) and / or SEQ ID NO: 142 (e.g., TCR clone DSK3 TCR beta). In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 143 (e.g., TCR clone DSK3 TCR alpha) and / or SEQ ID NO: 144 (e.g., TCR clone DSK3 TCR beta). (query number 141) MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKDNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 142) (query number 143) MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSDGGGVYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 144)

[0183] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 145-152. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a TCR alpha chain and a TCR beta chain found in the PRAME-specific TCR clones T116-49 and / or T402-93 and / or modified versions thereof. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a TCR alpha chain and a TCR beta chain that target the PRAME epitope LYVDSLFFL (SEQ ID NO: 167). In some embodiments, the PRAME-specific TCR sequences, TCR variable domain sequences, CDR sequences, and / or TCR constant domain sequences are described in International Patent Application Publication No. WO 2022 / 063966 A1, which is incorporated herein by reference for purposes described herein. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 153-166. ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTTGGGCAGCTGGACCTGGGTCCGATCTCAGCCAGCCTTTCAGTCTCCTCCAGGCCGTGATCCTGAGAGAAGGCGAGCCGCGGTATCAACTGCAGCTTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCCGAGCCCCTGTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGCTTCAACGAGAAAGCAGCAGTCCAGCCCGTGACCTGGACCCAGCTGGAGCTCAGCGCCACCTACTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGCACCCTGCTGACCGTGAATCCCAATCCAATCCAATC ATGCTGCTGATCACCTCATGCTGGTGCTGGATGCAGCTGAGCCAAGTGATCAGATCCCTCAGTCAGCAGCACGTGCAAGAAGGCGAGGACTTCACCACCCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTTACAAGCAGCGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGTCAAGTCCGGCGCAAGTGAAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGCAAGGAAGACAGCAGCCCTGCACATCACCGCCACACAGACCACCACGATGTGGCACCTACTTTTGTGTGCGCCCTGCCCTGCCTAGAGCCGGCAGCTATCAACTAGCATTCGGCAAGGCACCAAGCTGAGCGGTGATCCCCAAC (sequence number 146) ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTGTGGCAGCTGACCTGGGTCCGATCTCAGCAGCCTGTTCAGTCTCCTCAGGCCGTGATCCTGAGAGAAGGCGAGGACGCCGTGATCAACTGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCGAGGCCCCTGTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGCTTCAACGAGAAGAAGCAGCAGTCCAGCCTGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGCACCTACTTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGGCACCCTGCTGACCGTGAATCCCAATATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO: 147) ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGAGCCAAGTGAACGGCCAGCAAGTGATGCAGATCCCTCAGTACCAGCACGTGCAAGAAGGCGAGGACTTCACCACCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTACAAGCAGCGGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGGTCAAGTCCGGCGAAGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAACAGCAGCCTGCACATCACCGCCACACAGACCACCGATGTGGGCACCTACTTTTGTGCTGGCGCCCTGCCTAGAGCCGGCAGCTATCAACTGACATTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAACATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO: 148) ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGACACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGGTTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCCAGCCAGAGCCGTGTCTCGCCATCAGCGACTACGAGGGGCACCAGGGCCTTTTTTGGCCAAGCACAAGACTGACCGTGGTG (sequence number 149) ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAAGTGTACCGTGGAAGGGCACCAGCAATCCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGGACTGCAGCTGCTGTTTTTACAGCCGTCGGCATCGCCAGATCAGCGAGGTTTCCACAGAATCTGAGCCGCAGACCCCAGGACAGACAGTTTATCCTGGAGCAGCAAAGCTGCTGCTGAGCGACACAGCGGCTTCTACCTGTGTGGCTTGGAGCCTCGGGAGCCGGCTACACCGACACAGACTATTTTGGCCCTGGCACCAGACTGACCGTGCTG(sequence number 150) ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGGACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGTGTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCAGCCAGACCAGCGTGTACTTCTGCGCCATCAGCGACTACGAGGGCACCGAGGCCTTTTTTGGCCAAGGCACAAGACTGACCGTGGTGGAAGATCTCCGGAACGTGACCCCCCCTAAAGTGACCCTGTTCGAACCCAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGAGTGTCCACCGACCCTCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAAGGCTCCCCCAAGCCCGTGACCCAGAATATCTCTGCCGAGGCCTGGGGCAGAGCCGACTGTGGAATTACCAGCGCCAGCTACCACCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAAGAAGAACAGC(SEQ ID NO: 151) ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAATGTACCGTGGAAGGCACCAGCAATCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGACTGCAGCTGCTGTTTTACAGCGTCGGCATCGGCCAGATCAGCAGCGAGGTTCCACAGAATCTGAGCGCCAGCAGACCCCAGGACAGACAGTTTATCCTGAGCAGCAAGAAGCTGCTGCTGAGCGACAGCGGCTTCTACCTGTGTGCTTGGAGCCTCGGAGCCGGCTACACCGACACACAGTATTTTGGCCCTGGCACCAGACTGACCGTGCTGGAAGATCTCCGGAACGTGACCCCCCCTAAAGTGACCCTGTTCGAACCCAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGAGTGTCCACCGACCCTCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAAGGCTCCCCCAAGCCCGTGACCCAGAATATCTCTGCCGAGGCCTGGGGCAGAGCCGACTGTGGAATTACCAGCGCCAGCTACCACCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAAGAAGAACAGC(SEQ ID NO: 152) METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTANSGGSNYKLTFGKGTLLTVNPN (SEQ ID NO: 153) MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGALPRAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 154) IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 155) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 156) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 157) METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTANSGGSNYKLTFGKGTLLTVNPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 158) MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGALPRAGSYQLTFGKGTKLSVIPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 159) MGTRLFFYVALCLLWTGHVDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISDYEGTEAFFGQGTRLTVV (SEQ ID NO: 160) MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLGAGYTDTQYFGPGTRLTVL (SEQ ID NO: 161) EDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 162) DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 163) EDLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 164) MGTRLFFYVALCLLWTGHVDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISDYEGTEAFFGQGTRLTVVEDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 165) MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLGAGYTDTQYFGPGTRLTVLEDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 166)

[0184] In some embodiments, the TCR construct comprises a gp100-specific TCR chain. In some embodiments, the TCR construct comprising a gp100-specific TCR chain comprises a TCR alpha chain and a TCR beta chain found in the gp100-specific TCR clone Sp(0.01)A and / or modified versions thereof. In some embodiments, the TCR construct comprising a gp100-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the gp100 epitope KTWGQYWQV (SEQ ID NO: 168). In some embodiments, the gp100-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in Patent Publication US8,216,565B2, which is incorporated herein by reference for purposes described herein.

[0185] In some embodiments, a TCR construct comprising a gp100-specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 169 and / or 170. In some embodiments, a TCR construct comprising a gp100-specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 171-174. ATGAAATCCTTGAGTGTTTCCCTAGTGGTCCTGTGGCTCCAGTTAAACTGGGTGAACAGCCAGCAGAAGGTGCAGCAGAGCCCAGAATCCCTCATTGTCCCAGAGGGAGCCATGACCTCTCTCAACTGCACTTTCAGCGACAGTGCTTCTCAGTATTTTGCATGGTACAGACAGCATTCTGGGAAAGCCCCCAAGGCACTGATGTCCATCTTCTCCAATGGTGAAAAAGAAGAAGGCAGATTCACAATTCACCTCAATAAAGCCAGTCTGCATTTCTCGCTACACATCAGAGACTCCCAGCCCAGTGACTCTGCTCTCTACCTCTGTGCAGCCAATAACTATGCCCAGGGATTAACCTTCGGTCTTGGCACCAGAGTATCTGTGTTTCCCTACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO: 169) ATGGGCTCCAGACTCTTCTTTGTGGTTTTGATTCTCCTGTGTGCAAAACACATGGAGGCTGCAGTCACCCAAAGTCCAAGAAGCAAGGTGGCAGTAACAGGAGGAAAGGTGACATTGAGCTGTCACCAGACTAATAACCATGACTATATGTACTGGTATCGGCAGGACACGGGGCATGGGCTGAGGCTGATCCATTACTCATATGTCGCTGACAGCACGGAGAAAGGAGATATCCCTGATGGGTACAAGGCCTCCAGACCAAGCCAAGAGAATTTCTCTCTCATTCTGGAGTTGGCTTCCCTTTCTCAGACAGCTGTATATTTCTGTGCCAGCAGCCCTGGGGGGGGGGGGGAACAGTACTTCGGTCCCGGCACCAGGCTCACGGTTTTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACGAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGAAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCATGA(SEQ ID NO: 170) MKSLSVSLVVLWLQLNWVNSQQKVQQSPESLIVPEGAMTSLNCTFSDSASQYFAWYRQHSGKAPKALMSIFSNGEKEEGRFTIHLNKASLHFSLHIRDSQPSDSALYLCAANNYAQGLTFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 171) MGSRLFFVVLILLCAKHMEAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSPGGGGEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKRKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 172) QQKVQQSPESLIVPEGAMTSLNCTFSDSASQYFAWYRQHSGKAPKALMSIFSNGEKEEGRFTIHLNKASLHFSLHIRDSQPSDSALYLCAANNYAQGLTFGLGTRVSVFPY (SEQ ID NO: 173) EAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSPGGGGEQYFGPGTRLTVL (SEQ ID NO: 174)

[0186] In some embodiments, the TCR construct comprises a MART-1-specific TCR chain. In some embodiments, the TCR construct comprising the MART-1-specific TCR chain comprises the TCR alpha chain and TCR beta chain found in the MART-1-specific TCR clone F4 and / or F5 and / or modified versions thereof. In some embodiments, the TCR construct comprising the MART-1-specific TCR chain comprises the TCR alpha chain and TCR beta chain targeting the MART-1 epitope AAGIGILTV (SEQ ID NO: 175). In some embodiments, the MART-1-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in Patent Publication US9,128,080B2, which is incorporated herein by reference for purposes described herein.

[0187] In some embodiments, TCR constructs comprising a MART-1 specific TCR chain comprise a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 176 to 179. In some embodiments, TCR constructs comprising a MART-1 specific TCR chain comprise an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 180 to 183. ATGTTGCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTACTTCTGTGCTGGTGGGACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAGGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ ID NO: 176) ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCCTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTTCTGTGCCATCAGTGAGGTAGGGGTTGGGCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO: 177) ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACTTCGGAGGAGGAAAGCTTATCTTCGGACAGGGAACGGAGTTATCTGTGAAACCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 178) ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTGTGCCAGCAGCCTAAGTTTCGGCACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO: 179) GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGGTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 180) DAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISEVGVGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 181) QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFGGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 182) IAGITQAPTSQILAAGRRMTLRCTQDMRHNAMYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDGYSVSRANTDDFPLTLASAVPSQTSVYFCASSLSFGTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 183)

[0188] In some embodiments, the TCR construct comprises a tyrosinase-specific TCR chain. In some embodiments, the TCR construct comprising the tyrosinase-specific TCR chain comprises a TCR alpha chain and a TCR beta chain found in the tyrosinase-specific TCR clone TIL1383I and / or modified versions thereof. In some embodiments, the TCR construct comprising the tyrosinase-specific TCR chain comprises a TCR alpha chain and a TCR beta chain that target the tyrosinase epitope represented by amino acids 368-376 of tyrosinase (reactive with the class I MHC (HLA-A2)-restricted epitope (368-376) of tyrosinase). In some embodiments, the tyrosinase-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in the publication Roszkowski et al., Cancer Res. 65(4):1570-6 (2005), which is incorporated herein by reference for purposes described herein.

[0189] In some embodiments, the TCR construct comprises a MAGE-A3-specific TCR chain. In some embodiments, the TCR construct comprising the MAGE-A3-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting amino acids 271-279 of MAGE-A3, e.g., epitope FLWGPRALV (SEQ ID NO: 184). In some embodiments, the TCR construct comprising the MAGE-A3-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting amino acids 112-120 of MAGE-A3, e.g., epitope KVAELVHFL (SEQ ID NO: 185). In some embodiments, the MAGE-A3-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publication No. WO2012 / 054825A1, which is incorporated herein by reference for purposes described herein. In certain embodiments, the anti-MAGE-A3 112-120 TCR comprises an A118T substitution relative to wild type (wherein position 118 of the alpha chain is threonine). In certain embodiments, the anti-MAGE-A3 112-120 TCR comprises an A118V substitution relative to wild type (wherein position 118 of the alpha chain is valine).

[0190] In some embodiments, a TCR construct comprising a MAGE-A3 specific TCR chain comprises a nucleotide coding sequence that is at least, or exactly, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 186-193. In some embodiments, a TCR construct comprising a MAGE-A3 specific TCR chain comprises an amino acid sequence that is at least, or exactly, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 194-201. ATGGGTCCTGTCACCTGCTCAGTTCTTGTGTCTCTCCTCAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTCTGGTATGTGTGCAACATCTCAATGAATCCCTCGGCTACTCCTGGAAGAGCTTCACAGAACAAAGGACCGAGCACCAAGGTTCCACGCCACTCTCCATAAGAGCAGCCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCTGTCAGACTCTGCCCGTACTACTGTGCTTTCGACAAATGCTTACAAAGTCATCTTT(sequence number 186) ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCTAGGAAACAGGCCTTGTGGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAAGATGGGAGAGAAGTTTTGCTGGAATGTGGGACAGGACATGAGCCATGAAACAATGTACTGTGATCGACAAGACCCTGGTCTGGGCTACAGCTGATTTATATCCATACGATGTTGATAGTAACAGCGAAGGACATCCCTAAAGGATACAGGGTCTCACGGAAGAGCGGGAGCATTTTCCCCTATTCTGGATTCTGCTAAAAACAAACCAGACATCTGTGTACTTCTGTGTAGCAGTTCAACAAACACAGAAGTCTTCTTT (sequence number 187) ATGGGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTTCTGGTATGTGCAACATCTCAATGAATCCCCTCGGCTACTCCTGAAGAGCTTCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTTTCGACACAAATGCTTACAAAGTCATCTTTGGAAAAGGGACACATCTTCATGTTCTCCCTAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACACCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO: 188) ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCCTAGGAACAGGCCTTGTGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAGAATGGGAGAGAATGTTTTGCTGGAATGTGGACAGGACATGAGCCATGAAACAATGTACTGGTATCGACAAGACCCTGGTCTGGGGCTACAGCTGATTTATATCTCATACGATGTTGATAGTAACAGCGAAGGAGACATCCCTAAAGGATACAGGGTCTCACGGAAGAAGCGGGAGCATTTCTCCCTGATTCTGGATTCTGCTAAAACAAACCAGACATCTGTGTACTTCTGTGCTAGCAGTTCAACAAACACAGAAGTCTTCTTTGGTAAAGGAACCAGACTCACAGTTGTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAACTCGTGA(SEQ ID NO: 189) ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCTGGAGCTCCAGATGCAGTTTATTCATACAGTGCAGCCACCTTACCTCTTTCTGTACGTGCAGTATCCTGGCCAGAGCCTCCAGATTTCTCCTCTCAAATACATCACAGAGACACACCGTTGTTAAAGGCACCAAGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCCGGATACAGGAAACTACAAATACGTCTT (sequence number 190) ATGGGCATCCAGACCCCTGTTGTGTGATCTTTTATGTTCTGATAGCAAATCACACAGATGCTGGAGTTACCCAGACCACACATGAGGTGGCAGAGAAAGGACAAACAATAATCCTGAAGTGTGAGCCAGTTTCAGGCCACAATGACCTTTTCTGGTACAGACAGCAAGATACAGGGACTAGAGTTGCTGAGCTACTTCCGCAGCAAGTCTCTTATGGGAAGATGGGTGGGCTTTTCAAGGATCGATTCAAAGCTGATGCTAAATTCATCCTTTCCCACTCTGAAGATTCAACCTACAGAACCCAGGGACTCAGCTGTGTATCTGTGGCCAGCGATTTTGGGACAGCTAGTGCAGAAACGCTGTATTTT (sequence number 191) ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCCTGGAGCTCAGATGCAGTTATTCATACAGTGCAGCACCTTACCTCTTCTGGTACGTGCAGTATCCTGGCCAGAGCCTCCAGTTTCTCCTCAAATACATCACAGGAGACACCGTTGTTAAAGGCACCAAGGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCGGATACAGGAAACTACAAATACGTCTTTGGAGCAGGTACCAGACTGAAGGTTATAGCACACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTCCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO: 192) ATGGGCATCCAGACCCTCTGTTGTGTGATCTTTTATGTTCTGATAGCAAATCACACAGATGCTGGAGTTACCCAGACACCCAGACATGAGGTGGCAGAGAAAGGACAAACAATAATCCTGAAGTGTGAGCCAGTTTCAGGCCACAATGACCTTTTCTGGTACAGACAGACCAAGATACAGGGACTAGAGTTGCTGAGCTACTTCCGCAGCAAGTCTCTTATGGAAGATGGTGGGGCTTTCAAGGATCGATTCAAAGCTGAGATGCTAAATTCATCCTTCTCCACTCTGAAGATTCAACCTACAGAACCCAGGGACTCAGCTGTGTATCTGTGTGCCAGCAGTTTTGGGACAGCTAGTGCAGAAACGCTGTATTTTGGCTCAGGAACCAGACTGACTGTTCTCGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCCTGACACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGAAACCACTTCCGCTGTCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGAATCACTTCAGCATCCTATCATCAGGGGGTTCTGTCTGCAACCATCCTCTATGAGATCCTACTGGGGAAGGCCACCCTATATGCTGTGCTGGTCAGTGGCCTGGTGCTGATGGCCATGGTCAAGAAAAAAAATTCCTGA(SEQ ID NO: 193) MGPVTCSVLVLLLMLRRSNGDGDSVTQTEGLVTLTEGLPVMLNCTYQTIYSNPFLFWYVQHLNESPRLLLKSFTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCAFDTNAYKVIF (SEQ ID NO: 194) MRVRLISAVVLCSLGTGLVDMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSTNTEVF (SEQ ID NO: 195) MGPVTCSVLVLLLMLRRSNGDGDSVTQTEGLVTLTEGLPVMLNCTYQTIYSNPFLFWYVQHLNESPRLLLKSFTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCAFDTNAYKVIFGKGTHLHVLPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNTTYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSL (SEQ ID NO: 196) MRVRLISAVVLCSLGTGLVDMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSTNTEVFFGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM (SEQ ID NO: 197) MVLVTILLLSAFFSLRGNSAQSVDQPDAHVTLSEGASLELRCSYSYSAAPYLFWYVQYPGQSLQFLLKYITGDTVVKGTKGFEAEFRKSNSSFNLKKSPAHWSDSAKYFCALEGPDTGNYKYV (SEQ ID NO: 198) MGIQTLCCVIFYVLIANHTDAGVTQTPRHEVAEKGQTIILKCEPVSGHNDLFWYRQTKIQGLELLSYFRSKSLMEDGGAFKDRFKAEMLNSSFSTLKIQPTEPRDSAVYLCASSFGTASAETLY (SEQ ID NO: 199) MVLVTILLLSAFFSLRGNSAQSVDQPDAHVTLSEGASLELRCSYSYSAAPYLFWYVQYPGQSLQFLLKYITGDTVVKGTKGFEAEFRKSNSSFNLKKSPAHWSDSAKYFCALEGPDTGNYKYVFGAGTRLKVIAHIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 200) MGIQTLCCVIFYVLIANHTDAGVTQTPRHEVAEKGQTIILKCEPVSGHNDLFWYRQTKIQGLELLSYFRSKSLMEDGGAFKDRFKAEMLNSSFSTLKIQPTEPRDSAVYLCASSFGTASAETLYFGSGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 201)

[0191] In some embodiments, the TCR construct comprises a MAGE-A4-specific TCR chain. In some embodiments, the TCR construct comprising the MAGE-A4-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the epitope GVYDGREHTV (SEQ ID NO: 202). In some embodiments, the TCR construct comprising the MAGE-A4-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the epitope FMNKFIYEI (SEQ ID NO: 203). In some embodiments, the MAGE-A4-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publication Nos. WO2017 / 174824A1 and WO2021 / 229212A1, each of which is incorporated herein by reference for purposes described herein. In certain embodiments, the anti-MAGE-A4 TCR alpha chain variable domain may have an M4V or M4L amino acid substitution. In certain embodiments, the anti-MAGE-A4 TCR beta chain variable domain may have an N10E amino acid substitution.

[0192] In some embodiments, a TCR construct comprising a MAGE-A4 specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of SEQ ID NOs: 204-205. In some embodiments, a TCR construct comprising a MAGE-A4 specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of SEQ ID NOs: 206-214. ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTCAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGCGGCAGCCGGGCCAAGAGA(SEQ ID NO: 204) ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGAGGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 205) MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKR (SEQ ID NO: 206) MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPD (SEQ ID NO: 207) MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 208) MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFFGPGTRLTVLE (SEQ ID NO: 209) MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF (SEQ ID NO: 210) MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIVTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF (SEQ ID NO: 211) MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF (SEQ ID NO: 212) MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYNEQFF (SEQ ID NO: 213) MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFF (SEQ ID NO: 214)

[0193] In some embodiments, the TCR construct comprises a Wilms tumor antigen (WT1) WT1-specific TCR chain. In some embodiments, the TCR construct comprising the WT1-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the epitope VLDFAPPGA (SEQ ID NO: 215). In some embodiments, the TCR construct comprising the WT1-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the epitope RMFPNAPYL (SEQ ID NO: 216). In some embodiments, the WT1-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publication Nos. WO2020 / 185796A1 and WO2021 / 034976A1, each of which is incorporated herein by reference for purposes described herein. In some embodiments, the leader sequence and / or signal peptide may be removed from the TCR amino acid sequence, and the percent sequence identity may be calculated based on the TCR amino acid sequence without the leader sequence and / or signal peptide.

[0194] In some embodiments, a TCR construct comprising a WT1-specific TCR chain comprises a nucleotide coding sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 217-256. In some embodiments, a TCR construct comprising a WT1-specific TCR chain comprises an amino acid sequence that is at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 257-291. ATGGAGACACTGCTGGGACTACTGATTCTGTGGCTGCAACTGCAATGGGTGAGCAGCAAACAGGAGGTTACCCAGATTCCTGCTGCTCTGTCTGTCTGAAGGCGGAGAATCTGGTGCTGAACTGCAGCTTCACAGATAGCGCCATCTACAACCTGCAGTGGTTCAGACAGGATCCTGGAAAAGGCCTGACAAGCCTGCTGCTGATTCAGAGCTCTCAGAGAGCAGACATCTGGAAGACTGAATGCTAGCCTGGACAAGTCTAGCGGCAGAAGCACCCTGTATATTGCCGCCTCTCCAACCTGGAGATTCTGCCACATACCTGTTGTGCTGTGAAGGAGACATCTGGCTCTAGACTGACCTTTGGCGAGGGAACACAACTGACCGTGAATCCTGAC(sequence number 217) ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCCTGAAATGTCTGTGCAGGAAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAACAACTACTACCTGTTTCTGGTACAAGCAGCCCCCCTCTAGGCAGATGATCCTGGTGATCAGACAGGAGCCCTATAAAACAGCAGAATGCCACAGAGAACCGTTCAGCGTGAACTTCCAGAAAGCGCCAAGAGCTTCAGCCCTGAAGATCTCTGATTCTCAGCTGGGCGATACAGCCATGTACTTTGCCCTTCATCTACCCCAGCTACACAAGCGGCACATACAAGGTCACATACAAGGTCAGATACAAGTACATCTTCGGCACCGGCACAAACTGAAGGTTCTGGCCAAC (sequence number 218) ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCAGAAACACGCCCCTCTCTGTCTGTGCAGGAAGGCAGAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTTGTGTACAAGAAGTACCCCGCGAGGCCCTACCTTCTGGATCAGCATCTAGCATCAAGGACAAGAACGAAGATGGCAGATTCACGAGTCACGTGTTCCTGAAACAAGAGCGCCAAGCCAGCTGAGCCTGCACATTGGCTTCTCAACCTGGAGATTCTGCCGGTACTTTTGTGCTGCCTTGGAACAGGCGAAGCTATATCCCCACATTTGGGAAGAGGAAACAAGCCTGATGTGTCACCCTTAC (sequence number 219) ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCCAGAACACGCCCTCTCTGTCTGTGTCAGGAAGGCGAAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTTGTGGTACAAGAAGTACCCCGCGAGGCCCTACCTTCTGGATCAGCATCTCTAGCATCAAGGACAAGAACGAAGGCAGATTACCCGTGTTCCTGAAACAAGAGCGCCAAGCCACCTGAGCCTGCACATTGTGCCTTCTCAACCTGGAGATTCTGCGTGTACTTTTGTGCTGCCTTGGCATTGGCGACTACAAACTGAGCTTTTGGAGCCGGCACAACAGTGACCGTTAGACCCAAT (sequence number 220) ATGGTGAAGATCCGGCAGTTCCTCCTGGCTATTCTGTGGCTGCAACTGTCTTGTGTGTCTGCTGCCAAGAATGAAGTGGAGCAGTCTCCCCAGAACCTTACAGCCCAGGAAGGCGAGTTTTATCACCATCAACTGCAGCTATTCTGTGGCATTAGCGCCCTGCATTGGCTGCAGCAACACCCTGGGAGGGAATTGTGTCTCTGTTTATGCTGTCTTCTGCCAAGAAGAAGCACGGCCGGCTGATTGCCACCATCAACATCCAGGAAGCACTCTTCTCTGCACATTACAGCCCTCATCCAGGGATTCTGCCGTGTACATCTGTGCCGTGGAACCAGCTACGATAAGGTGATTTTTCGGACCAGGCACCTCTCTGAGCGGTGATCCCCAAT (sequence number 221) ATGAAGAGCCTGAGAGTCCTGCTGGTGTGGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGTGGAGCGAATAGCGGCCCTCTGTTCCTGAAGGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGGAGTGGCAGGTTTACAGCCCAGCTGAAACAAGGCCAGCCAGTAGTTTTCTCTGCTGATCAGAGATAGCCAGCTAGCGATTCTGCCACCTACCTGTGTCGTGAACTTACTTGGAGCTACAGGATACTCTACACTACACTTCGGCAAAGGCACCATGCTGCTGGTGAGCCCTGAT(sequence number 222) ATGTGGGGCGTTTTCCTTCTTGTATGAGCATGAAGATGGGCGGCACAACAGGCCAGAACATCGATCAGCCCTACCGAGATGACAGCCACAGAAGGAGCTATTGTTCAGATCAACTGCACCTACCAGACAAGCGGCTTTCAACGGCCTGTTCTGGTACCAGCAGCATGCTGGAGAAGCTCCTACATTTCTGAGCTACAATGTGCTGGATGGCCTGGAGAGAAAAGCAGGTTTAGCAGCTTTCCTGAGCGAGTCAAGGGCTATTCTTATCTGCTGCTGAGGAGCTGCAGATGAAGGATTCCGCCAGCTACCTGTGTGCCGTTAGGGGCATCAATGATTACAAGCTGAGCTTTGGAGCCGGAACAACAGTGACCGTGAGAGCCAAC (sequence number 223) ATGGAGAAGATGCTGGAGTTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAAAGCTCTAGCCTGAACTGCAGCTACACAGGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGCGAGGAGAAGGAAGGAAGAGACTGAAAGCTACCCTGACCAAGAAGGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCCGTGATTACCGGCCTTTCAGAAGCTGGTGTTTCTCCCAAAT (sequence number 224) ATGAGACTGGTGGCACGCGTACTGTTTTCTGACCTTTGGCACCATCATCGATGCCAAGACAACCCAGCCTTACAAGCATGGACTGTGCCGAGGGAAGAGCTGCTAATCCTGCCATGTAATCACAGCACAATCAGCGGCAACGAGTACGGTACTACTGGTACCGGCAGATCTCACTCTCAAGGACCTCAGTACATCATTCATGGCCTGAAGAAACAACGAGACCAACGAGATGGCCAGCCGTGTCATCCTGATTCTGCCTCATGCTACACTGAGAGATACCGCCGTGTACTACTGCATTGCCGGAGTGGGAAGAGGCCAGAATTTCGTTTGGACCTTGGAACACTGAGCGTTCTGCCCTAT(sequence number 225) ATGGAGAAGAACCCCTTGGCAGCACCTCTGCTTATTCTGTGGTTCCACCTGGATTGTGTGAGCAGCATCCTGAATGTGGAGCAGTCTCCTCAGAGCTGCATGTGCAAGAAGGCGATAGCACCAATTTCACCTGCAGCTTTTCCAAGCAGCAACTTCTACGCCCTGCACTGGTACAGATGGGAAACCGCCAAATTCTCCTGAAGCCCTGTTTGTGATCACCTGAATGGCGACGAAGAAAGAGGACAATTAGCGCCACCCCTGAATACCAAGGAGGGCTACAGCTACCTGTACATCAAGGCTCTCAACCTGAGATTCTGCCACCCTTTGCGCCTTTACCCCAATTTCGGCAACGAGAAACTGACCTTTGGAACCCGGAACAAGGCTGACCATCATCCCCAAC (sequence number 226) ATGGAGAAGATGCTGGAGTTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAAAGCTCTAGCCTGAACTGCAGCTACACAGGTGTTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGCGAGGAGAAGGAAGGAAGACTGAAAGCTACCCTGACCAAGAAGGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATATCTGTGTGCTTTCAGCCTAGAGGAGATGGCTCTAGCAATACCGGCAAGCTGGATCTTGGCCAGGGAACAACACTGCAGGTGAAGCCGTGAAGCCGTGAAGCCGT(sequence number 227) ATCCAGAATCCCGATCCTGCTGTGTACCAGCTGCGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAGCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCGTGGCCTGGTCAACAAGAGCGACTTCGCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGACATTCTTCCCAAAGCCCCGAGAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCGGATCCTGCTCGGATCCTGCTGTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGCTTGGTCCAGCTGA(sequence number 228) CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACATCTCTTCTCTGTTGGGTGGTTCTGGGCTTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTATAAGGTGACCAAGAGGGGACAGGATGTGGCTCTGAGATGTGACCCTATTAGCGGACATGTGAGCCTGTACTGGTACAGACAAGCTCTGGGACAAGGACCCGAGTTTCTGACCTACTTCAACTATGAGGCCCAGCAGGACAAATCTGGACTGCCCAACGACAGATTCAGCGCCGAAAGACCAGAAGGCTCTATTAGCACACTGACCATCCAGAGAACAGAGCAGAGGGATTCTGCCATGTACAGATGCGCCAGCAGCTTAACAGGCTCTTACGAGCAGTACTTTGGACCTGGCACAAGACTGACAGTGACAGAG (SEQ ID NO: 229) CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCACACCTGAAGCCTCTAGCCCATATGAGCAGTACTTTGGCCCTGGCACCAGATTAACAGTGACAGAG(SEQ ID NO: 230) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCCATGGGACCTGGGACTGCTTCATTGGATGGCTCTGTGTTTGCTGGGAACAGGACATGGAGATGCTATGGTTGTCCAGAACCCCAGGTATCAGGTTACGGTGACCCAGTTTGGCAAACCAGTGACACTGAGCTGTTCTCAGACCCTGAACCACAACGTGATGTACTGGTACCAGCAGAAGTCTTCTCAGGCCCTAAGCTGCTTCCACTACTACGACAAGGACTTCAACAACGAGGCCGATACCCTGACAATTTCCAGAGAGGCCCAATACCAGCTTCGTTTCCTGGACATTAGAAGCCCTGGACTGGGAGATGCTGCCATGTACCTGTGTGCCACAGGCAATTTACAGGGAAGACAACCTCAGCACTTTGGCGATGGCACAAGGCTGTCTATCCTGGAG (sequence number 231) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGGCTGCTGGTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGTCTAGCAATCTCTAGACACCTGATCAAGGAGAGAGAGAACAGCCACCCTGAAGTGCTACCCCATCAGACACGATACAGTGTACTGGTATCAGCAAGGACCTGGGACAAAGTCCCCAGTTCCTGATCTGTCTCTACGAGAAGAGATGCAGAGCGACAAAGGCCATCCCAGACATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCCCTGGAACTGGCGATTCTGCTCTGTCTCTGGCCTTCTGAGACTGGGAAGAAACCCCAGTACTTTGGACCCCGGCCAAGACTCTGTTTCTGAG (sequence number 232) CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACAAGACTTCTCTGCTGGGTGGTGCTTGGATTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTACAAAGTGGCCAAGAGAGGACAGGATGTGGCTCTGAGATGTGACCCTATTAGCGGACATGTGAGCCTGTTTTGGTACCAGCAAGCTCTGGGACAAGGACCCGAGTTTCTGACCTACTTCCAGAATGAAGCCCAGCTGGATAAATCTGGACTGCCTAGCGACCGGTTCTTCGCCGAAAGACCTGAAGGATCTGTTAGCACCCTGAAGATTCAGAGAACACAGCAGGAGGACTCTGCCGTGTACCTGTGTGCCTCTTCTTTAGGACAGGCCTATGAGCAGTATTTTGGACCTGGCACCAGACTGACCGTGACAGAG (SEQ ID NO: 233) CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACAAGACTTCTCTGCTGGGTGGCCTTTTGTCTGCTGGTGGAAGAGCTGATTGAAGCTGGAGTTGTGCAGTCTCCTAGGTACAAGATCATCGAGAAGAAGCAGCCCGTGGCCTTCTGGTGTAATCCCATTTCTGGCCACAACACCCTGTACTGGTATCTGCAGAATCTGGGACAGGGCCCTGAACTGCTGATCAGATACGAGAACGAAGAAGCCGTGGACGATTCTCAACTGCCTAAGGACCGCTTTTCTGCCGAGAGGCTGAAAGGAGTGGATTCTACCCTGAAGATCCAACCTGCTGAACTGGGCGATTCTGCTGTGTACCTGTGCGCTTCTAGCCTGACAAGAGGAGCTGAAGCCTTTTTTGGACAGGGCACAAGACTGACAGTGGTGGAG (SEQ ID NO: 234) CTCAATAAAAGCCCACAACCCCCTCCACTCGGCGCGCACCATGGGACCTCAGCTTCTTGGATACGTTGTGCTGTGTCTGCTTGGAGCTCTTGAAGCTCAGGTTACCCAGAACCCCAGATACCTGATTACCGTGACCAGAGCAAAAAGCTGACCGTGACATGTAGCCAGAACATGAGCTCGGCAGGTACATGAGCTGGTACCGGCAGGATCCTGATGGCCTGAGACAGATCTACTACAGCATGAACGTGGAGGTGACCGATAAAGGCGACGTGCCTGAGGGATACAAGGTGAGCAGAAAGGAGAAGGAAGGAATTTCCCCTGAATCCTGGAAAGCCCAATCAGACAAGCCTGTCTTTGTGCCAGCAGCTTTTCTGGCGGCACATATGAGCAGTACTTCGGCCCTGGCACAAACTAGACAGTACAGTTACAGAG(sequence number 235) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGGCTGCTGGTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCATCAATCTCTAGACACCTGATCAAGGAGAGAGAGAACAGCCACCCTGAAGTGCTACCATCCCCCAGACACGATACAGTGTACTGGTATCACTCTGAGCTGGACAAGATCCCCAGTTCCTGATCCTCAGAGCGACAAAGGCAGCATCCCAGACAGATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCAGCCTGGAACTGGCGATTCTGCTCTGTCTCTGTGGCCAGCTATAGAGGAGGCAGCCACATATGAGCACATATTGAGCAGTACTTTGGCCCTGGCACAAGACTGACAGTGACAGAGACTGACAGTAGGACAGAGACTGACAGAGACTGACAGATGACAGAGGAGAGACTGAGTAGACAGTAGGACAGAGTAGACAGAGGAGAGACTGAGTAGACAGTAGAGAGAGGA CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGAGCACCAGACTCCTTTGCTGGATGGCTTTGTGTCTGCTTGGAGCTGAGCTGTCTGAAGCTGAAGTTGCCCAGTTCCCAGATACAAGATCACCGGAAAATCTCAGGCTTGGCCTTCTGGTGTGACCCTATTTCTGGACACACGCCACCTGTACTGGTATAGGCAAATTCTGGGACAAGGCCCTGAACTGCTGGTGCAATTTCGAGACAGGTTTTCTGCCGAGCGCGCTGAAAGGAGTTGATAGCACCCTGAAATCCAACCTGCTACTGGGCGATTCTGCTATGTACCTGTGCGCGCTTGTCTGGAAGAGATAGCCCTAACGAGAAGCTTTCTTTGGCTTGGAACCCAGCTGTTCTGTGGAAG(sequence number 237) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCTGTAGACTGTTGTGTGTGCTGTGCTGTGTCTGTGTGGAGCTGTGCCTATGGAAACAGGCGTTACCCAGACACCTAGACATCTGGTTATGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCATCTGGGCCATAACGGCCATGTACTGGTATAAGCAGAGCGCCAAGAAACCACTGGAACTGATGTTCGTGTACAGCCTGGAGGAGAGGTGGAGAATAATAAGCGTGCCCAGCAGATTTAGCCCTGAGTGCCCCAAATTCTTTCTCACCTGTTCTCCACCTGCACACATTACAGCCCGAGGATTCTGCCCTGTACCTGTGCTTCTTCTCAAGACCCTTACAAGCTGGAGCGGCAATACCATCTACTTCGGCGGAAGGCTCTTGGCTGACAGTGGTTGAA (sequence number 238) GATCTGAACAAGGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCTCCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTGCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGCGGCTTTACCTCCGTGTCCTATCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACTTC(SEQ ID NO: 239) GACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 240) (query number 241) ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTCAACTGCAGTTACACAGTCAGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGCTTTCTGCACATCACAGCCCCTAAACCTGAAGACTCAGCCACTTATCTCTGTGCTGTGCAGACCATGGACGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 242) (query number 243) ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAGTAATTATTATTTGTTCTGGTACAAACAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCAACCCTTGGGAGAACTATGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 244) ATGAAGAGCCTGAGAGTCCTGCTGGTGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGAAGTGGAGCAGAATAGCGGCCCTCTGTCTGTTCCTGAAGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGAGGATGGCAGGTTTACAGCCCAGCTGAACAAGGCCAGCCAGTATGTTTCTCTGCTGATCAGAGATAGCCAGCCTAGCGATTCTGCCACCTACCTGTGTGCCGTGAACATCGGAAATCACGACATGAGATTTGGAGCCGGCACAAGACTGACCGTGAAGCCCAATATCCAGAACCCTGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 245) ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCTGTGCAGACCATGGATGGCAACCAGTTCTACTTCGGCACAGGCACATCTCTGACCGTTATCCCCAATATCCAGAACCCTGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 246) ATGGCTTGTCCTGGATTCTTATGGGCTCTGGTGATCAGCACCTGTCTGGAGTTCTCTATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTGACACTGTCTTGCACCTACGATACAAGCGAGAGCGACTACTACCTGTTCTGGTACAAGCAGCCTCCCTCTAGGCAGATGATCCTGGTGATTAGACAGGAGGCCTACAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGAGATGCTGCCATGTACTTTTGTGCCAGCTCTCCAGGCACCTACAAGTACATTTTTGGCACCGGCACCAGACTGAAGGTGCTGGCCAATATCCAGAATCCCGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 247) ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAGCAACTACTACCTGTTCTGGTACAAGCAGCCCCCTTCTAGGCAGATGATCCTGGTGATCAGACAGGAGGCCTATAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGCGATACAGCCATGTACTTTTGTGCCTTCAACCCCTGGGAGAACTATGGCCAGAATTTCGTGTTCGGCCCTGGCACCAGACTGTCTGTTCTGCCTTATATCCAGAACCCCGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 248) ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGACACTGAAGTTACCCAGACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCACAGGGCTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACCTTCACCTACACGCCCTGCAGCCAGAAGACTCAGCCCTGTATCTCTGCGCCAGCAGCCAAGGGACTAGCGGGGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO: 249) ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTCTCTTTGGGACCTTCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO: 250) ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTTTCAGACGGGGGGGCTACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG (SEQ ID NO: 251) ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGACCCCATTCTCTCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG (SEQ ID NO: 252) (query number 253) ATGTCTATCGGTCTGCTGTGCTGTGCTGCTCTTTCTCTGCTTTGGGCTGGACCTGTGAATGCTGGAGTTACACAAACCCCCAAGTTCCAAGTGCTGAAGACAGGACAGAGCATGACCCTGCAGTGTGCTCAGGACATGAATCACGAGTACATGAGCTGGTACAGACAGGATCCTGGAATGGGCCTGAGGCTGATCCACTACTCTGTTGGAGCCGGAATTACAGATCAGGGAGAAGTGCCAAATGGCTACAACGTGAGCAGGAGCACAACCGAGGACTTCCCCTTAAGACTGTTGTCTGCTGCTCCATCTCAGACAAGCGTGTACTTTTGCGCCAGCTCCTACTCTCTGTGGGATCTGCAGGAAACCCAGTACTTTGGACCAGGCACAAGACTGTTAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 254) ATGGGCACATCTCTTCTCTGCTGGATGGCTCTTTGTCTGCTTGGAGCCGATCATGCCGATACAGGAGTTAGCCAGGATCCTAGACACAAGATCACCAAGAGAGGCCAGAATGTGACCTTCCGGTGCGATCCTATCTCTGAGCACAACAGGCTGTACTGGTACAGACAAACACTGGGACAAGGACCTGAGTTCCTGACCTACTTCCAGAACGAAGCCCAGCTGGAGAAGTCTAGACTTCTGAGCGACAGATTTAGCGCCGAGAGACCTAAAGGCAGCTTTAGCACCCTGGAGATCCAGAGAACAGAACAGGGCGATTCTGCCATGTACCTGTGTGCTAGCAGCTTTTCTGATGGAGGCGCCACCGATACACAGTATTTCGGACCTGGCACAAGACTGACAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 255) ATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCAGACCTCACAGCCTGACCGATACACAGTACTTTGGCCCTGGCACAAGACTGACAGTGTTAGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 256) METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVKETSGSRLTFGEGTQLTVNP (SEQ ID NO: 257) MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFIYPSYTSGTYKYIFGTGTRLKVLAN (sequence number 258) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGTGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 259) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGIGDYKLSFGAGTTVTVRAN (SEQ ID NO: 260) MVKIRQFLLAILWLQLSCVSAAKNEVEQSPQNLTAQEGEFITINCSYSVGISALHWLQQHPGGGIVSLFMLSSGKKKHGRLIATINIQEKHSSLHITASHPRDSAVYICAVRTSYDKVIFGPGTSLSVIPN (SEQ ID NO: 261) MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNLLGATGYSTLTFGKGTMLLVSP (SEQ ID NO: 262) MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVRGINDYKLSFGAGTTVTVRAN (SEQ ID NO: 263) MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVITGFQKLVFGTGTRLLVSPN (SEQ ID NO: 264) MRLVARVTVFLTFGTIIDAKTTQPTSMDCAEGRAANLPCNHSTISGNEYVYWYRQIHSQGPQYIIHGLKNNETNEMASLIITEDRKSSTLILPHATLRDTAVYYCIAGVGRGQNFVFGPGTRLSVLPY (SEQ ID NO: 265) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFHPNFGNEKLTFGTGTRLTIIPN (SEQ ID NO: 266) MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQPRGDGSSNTGKLIFGQGTTLQVKP (SEQ ID NO: 267) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 268) MGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLTGSYEQYFGPGTRLTVTE (SEQ ID NO: 269) MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSATPEASSPYEQYFGPGTRLTVTE (SEQ ID NO: 270) MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSNLQGRQPQHFGDGTRLSILE (SEQ ID NO: 271) MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLRLGRETQYFGPGTRLLVLE (SEQ ID NO: 272) MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLGQAYEQYFGPGTRLTVTE (SEQ ID NO: 273) MGTRLLCWVAFCLLVEELIEAGVVQSPRYKIIEKKQPVAFWCNPISGHNTLYWYLQNLGQGPELLIRYENEEAVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAVYLCASSLTRGAEAFFGQGTRLTVVE (SEQ ID NO: 274) MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSRDREQESPLHFGNGTRLTVTE (SEQ ID NO: 275) MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSFSGGTYEQYFGPGTRLTVTE (SEQ ID NO: 276) MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSYRGGSTYEQYFGPGTRLTVTE (SEQ ID NO: 277) MSTRLLCWMALCLLGAELSEAEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSQRDSPNEKLFFGSGTQLSVLE (SEQ ID NO: 278) MGCRLLCCAVLCLLGAVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQDPYKLSGNTIYFGEGSWLTVVE (SEQ ID NO: 279) DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 280) DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 281) MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPN (SEQ ID NO: 282) MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPN (SEQ ID NO: 283) MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCASSPGTYKYIFGTGTRLKVLAN (SEQ ID NO: 284) MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPY (SEQ ID NO: 285) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 286) MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFGPGTRLTVLE (SEQ ID NO: 287) MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFGPGTRLLVLE (SEQ ID NO: 288) MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSDGGATDTQYFGPGTRLTVLE (SEQ ID NO: 289) MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFGPGTRLTVLE (SEQ ID NO: 290) DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 291)

[0195] In some embodiments, the TCR construct comprises a human papillomavirus (HPV)-specific TCR chain. In some embodiments, the TCR construct comprising the HPV-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the HPV 18 E6 protein and / or the HPV 18 E7 protein. In some embodiments, the HPV 18 E6 epitope is amino acids 121-135 and / or amino acids 77-91 of the HPV 18 E6 protein. In some embodiments, the TCR construct comprising the HPV-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the HPV 18 E7 protein. In some embodiments, the HPV 18 E7 epitope is amino acids 11-19. In some embodiments, the HPV-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publication No. WO 2015 / 009604 A1, which is incorporated herein by reference for purposes described herein.

[0196] B.NK cells NK cells modified to express the TCR / CD3 receptor complex can be obtained from any suitable source, including fresh or frozen. In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte products (PBSCs), NK cell lines (e.g., NK-92), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art. Specifically, NK cells can be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines, or a mixture thereof. In certain embodiments, NK cells are isolated from pooled CBs. CBs can be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. NK cells can be autologous or allogeneic with respect to the recipient individual. Isolated NK cells can be haplotype-matched or mismatched to the subject receiving the cell therapy. For example, NK cells can be detected by specific surface markers such as CD16 and CD56 in humans.In some cases, the source of NK cells is umbilical cord blood, and NK cells can exist in umbilical cord blood as a heterogeneous cell mixture, and can be depleted of certain cells that express CD3.In other methods, umbilical cord blood is used to drive NK cells by isolating CD34+ cells.

[0197] NK cells may be preactivated with one or more inflammatory cytokines and may or may not be expanded. In some cases, NK cells are preactivated before being modified to express CD3±TCR or after being modified to express the CD3±TCR complex. In specific embodiments, preactivation of NK cells may include culturing isolated NK cells in the presence of one or more cytokines. NK cells may be stimulated with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, etc.). In certain embodiments, the preactivation cytokine may be selected from the group consisting of IL-12, IL-15, IL-18, and combinations thereof. One or more additional cytokines may be used in the preactivation step. Pre-activation may be for a short period of time such as 5 to 72 hours, for example 10 to 50 hours, particularly 10 to 20 hours, for example 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, particularly about 16 hours. The pre-activation culture may comprise IL-12 at a concentration of 0.1 to 150 ng / mL, for example 0.5 to 50 ng / mL, particularly 1 to 20 ng / mL, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng / mL, particularly about 10 ng / mL. The pre-activation culture may comprise IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, for example 40-60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, especially about 50 ng / mL.

[0198] In some cases, NK cells are expanded before or after being modified to express a CD3±TCR complex. Preactivated NK cells can be expanded in the presence of artificial antigen-presenting cells (aAPCs). Preactivated NK cells can be washed, for example, two, three, four, or five times, particularly three times, before expansion. aAPCs can be engineered to express CD137 ligand and / or membrane-bound cytokines. The membrane-bound cytokine can be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, aAPCs are engineered to express CD137 ligand and mIL-21. aAPCs can be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. They can express ICAM-1 (CD54) and LFA-3 (CD58). In particular, the aAPCs may be K562 cells, e.g., K562 cells engineered to express CD137 ligand and mIL-21. The aAPCs may be irradiated. Engineering may be by any method known in the art, such as retroviral transduction. Expansion may be for approximately 2 to 30 days, e.g., 3 to 20 days, particularly 12 to 16 days, e.g., 12, 13, 14, 15, 16, 17, 18, or 19 days, particularly about 14 days. The preactivated NK cells and aAPCs may be present in a ratio of approximately 3:1 to 1:3, e.g., 2:1, 1:1, 1:2, particularly about 1:2. The expansion culture may further contain cytokines that promote expansion, such as IL-2. IL-2 may be present at a concentration of approximately 10 to 500 U / mL, e.g., 100 to 300 U / mL, particularly about 200 U / mL. IL-2 can be replenished during expansion, for example, every 2-3 days. aAPCs can be added to the culture for at least a second time, for example, after about 7 days of expansion.

[0199] In certain embodiments, NK cells are transfected or transduced with one or more membrane-bound cytokines, including IL-21, IL-12, IL-18, IL-23, IL-7, or IL-15, secreted by the NK cells or anchored to the NK cell membrane. In such cases, the membrane-bound cytokines may be anchored to the NK cell membrane at a particular transmembrane domain, such as the transmembrane domains of CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, or DAP12.

[0200] After preparation, the modified NK cells may be immediately infused (containing an effective amount of one or more bispecific or multispecific antibodies), or the NK cells may be preserved, such as by cryopreservation. In certain embodiments, the cells can be expanded ex vivo as a bulk population within about 1, 2, 3, 4, or 5 days for days, weeks, or months.

[0201] III. Heterologous Proteins In a specific embodiment, NK cells are engineered to express not only one or more components of the TCR / CD3 complex, but also one or more other heterologous proteins that can enhance the activity of the NK cells in any way, including at least their activation, persistence, expansion, homing, and / or cytotoxicity.

[0202] A. Bispecific or Multispecific Antibodies In some embodiments, the NK cells are engineered to express one or more bispecific or multispecific antibodies, while in other cases the NK cells do not express antibodies, but antibodies are utilized in conjunction with the NK cells.

[0203] When NK cells are engineered to express antibodies, the antibodies can act as engagers, bridging specific immune effector cells with specific target cells to destroy them. In the present disclosure, engineered NK cells can be used in conjunction with standard T cell engagers (BiTEs), because engineered NK cells are often engineered to express CD3, the T cell antigen to which BiTE engagers bind. In such cases, BiTEs used in the present invention can also target cancer antigens or viral antigens tailored to the medical condition of the intended recipient individual. For example, BiTEs can be tailored to bind to cancer antigens characteristic of cancer cells of the individual's cancer. The anti-CD3 antibody in the BiTE can target the CD3γ chain, CD3δ chain, CD3ε chain, or CD3ζ chain.

[0204] In some cases, in addition to expressing a CD3 complex (with or without a TCR) that allows NK cells to be used as a therapeutic agent using BiTEs, NK cells can be engineered to express (or not express, but instead use in combination) one or more bispecific NK engagers (BiKEs). BiKEs include antibodies that bind to surface proteins on NK cells, including surface proteins naturally expressed on NK cells, and also include antibodies that bind to desired target antigens. BiKEs can target NK cells via antibodies to NK surface proteins, such as CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCRs, and KIRs. In such cases, BiKEs used in the present invention can also target cancer antigens or viral antigens tailored to the medical condition of the intended recipient individual. For example, BiKEs can be tailored to bind to cancer antigens characteristic of cancer cells of the individual's cancer.

[0205] In embodiments in which NK cells express a CD3 complex (with or without a TCR) and one or more BiKEs, one or more vectors may be utilized to transfect or transduce the CD3 complex components (with or without a TCR) and one or more BiKEs into the cells. In some cases, the one or more CD3 complex components (with or without a TCR) and BiKEs may or may not be on the same multicistronic vector.

[0206] B. Engineered receptors In a specific embodiment, NK cells are engineered to express one or more engineered receptors.In some cases, the engineered receptor is an engineered antigen receptor that targets any kind of cancer antigen or virus antigen.The receptor can be adjusted to target the desired antigen based on the medical condition of the intended recipient individual.

[0207] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). NK cells may be modified to encode at least one CAR, and the CAR may be, for example, a first-generation, second-generation, or third-generation or later generation. A CAR may or may not be bispecific for two or more different antigens. A CAR may include one or more costimulatory domains. Each costimulatory domain may include one or more costimulatory domains, such as, for example, a member of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40, or a combination thereof. In a specific embodiment, the CAR includes CD3 zeta. In certain embodiments, the CAR lacks one or more specific costimulatory domains; for example, the CAR may lack 4-1BB and / or lack CD28.

[0208] In certain embodiments, the intracellular CAR polypeptide comprises an extracellular spacer domain, sometimes referred to as a hinge, that connects the antigen-binding domain and the transmembrane domain. The extracellular spacer domain includes, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, artificial spacers composed of polypeptides such as CD8-alpha hinge, CD28, and Gly3, or the CH1 and CH3 domains of IgG (e.g., human IgG1 or IgG4). In certain cases, the extracellular spacer domain can comprise (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8-alpha or CD4, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vii) the hinge and CH2 of IgG1, (viii) the hinge region of CD28, or a combination thereof. In specific embodiments, the hinge is derived from IgG1, and in certain aspects, the CAR polypeptide comprises a particular IgG1 hinge amino acid sequence or is encoded by a particular IgG1 hinge nucleic acid sequence.

[0209] The transmembrane domain of the CAR may be derived from a natural or synthetic source. If the source is natural, the domain is derived from any membrane-bound or transmembrane protein in some aspects. The transmembrane region includes those derived from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules, such as DAP10 or DAP12 (i.e., at least the transmembrane region thereof). Alternatively, the transmembrane domain is synthetic in some embodiments. In some aspects, the synthetic transmembrane domain primarily comprises hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine may be found on either side of the synthetic transmembrane domain.

[0210] In some embodiments, the engineered receptor utilizes one or more homing receptors (which may home to a target without necessarily releasing a signal, such as in events utilizing adhesion molecules) and / or one or more chemokine receptors. Examples of chemokine receptors include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors. In certain cases, the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD62L), CXCR1, CXCR2, or CX3CR1.

[0211] C. Cytokines In some embodiments, the NK cell-expressing cells are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with the one or more cytokines on the same vector as other genes.

[0212] One or more cytokines can be co-expressed from the vector, such as as a separate polypeptide from any component of the TCR / CD3 complex. For example, interleukin-15 (IL-15) is tissue-restricted and is only observed at any level in serum or systemically under pathological conditions. IL-15 has several desirable properties for adoptive therapy. IL-15 is a homeostatic cytokine that promotes the eradication of established tumors by inducing the development and proliferation of natural killer cells and relieving the functional suppression of tumor-resident cells, thereby inhibiting activation-induced cell death (AICD). In addition to IL-15, other cytokines are also contemplated. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. NK cells expressing IL-15 are capable of continuing supportive cytokine signaling, which is beneficial for survival after infusion.

[0213] In a specific embodiment, the cells express one or more exogenously supplied cytokines. By way of example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokines may be exogenously supplied to NK cells by being expressed from an expression vector within the cells. Alternatively, endogenous cytokines within the cells are upregulated by manipulating the expression of the endogenous cytokine, such as by genetic modification of the cytokine's promoter site. When cytokines are supplied to cells on an expression construct, the cytokines may be encoded from the same vector as one or more components of the CD3 complex, with or without the TCR complex.

[0214] In some embodiments, a specific sequence of IL-15 is utilized as shown below (underlined denotes the signal peptide sequence): ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCC GGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGT GATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49) MRISKPHLRSISIQCYLCLLLNSHFLTEA GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 48)

[0215] D. Antigen The modified NK cells of the present disclosure are utilized in conjunction with bispecific or multispecific antibodies that target one or more specific antigens. In addition, NK cells can be modified with engineered antigen receptors that target one or more specific antigens. When NK cells are modified with one or more engineered antigen receptors, the antigens targeted by the bispecific or multispecific antibodies and the antigens targeted by the one or more engineered antigen receptors may or may not be the same antigen. In some cases, the antigens targeted by the bispecific or multispecific antibodies and the antigens targeted by the one or more engineered antigen receptors are different antigens but are associated with the same type of cancer.

[0216] Some antigens targeted by antibodies and / or engineered antigen receptors are expressed in association with the disease, condition, or cell type targeted via adoptive cell therapy. Diseases and conditions include proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors of the immune system, such as blood cancers, lymphomas, leukemias, and / or myelomas, e.g., B, T, myeloid leukemia, lymphoma, and multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0217] Any suitable antigen can be targeted in this method. In some cases, the antigen may be associated with certain cancer cells but not with non-cancer cells. Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, autologous / self-antigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In certain embodiments, antigens include NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, and CD23. , CD30, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19 ... 6, MAGE-A10, MAGE-A12, MART-1, melanoma-associated antigen, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, MC1R, mda-7, gp75, Gp100, PSA, PSM, tyrosinase, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phosphoinositide 3 kinase Enzyme kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II, CDC27 / m,TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences are known in the art, for example in the GENBANK® database: CD19 (accession number NG_007275.1),EBNA (accession number NG_002392.2), WT1 (accession number NG_009272.1), CD123 (accession number NC_000023.11), NY-ESO (accession number: NC_000023.11), EGFRvIII (accession number: NG_007726.3), MUC1 (accession number: NG_029383.1), HER2 (accession number: NG_007503.1), CA-125 (Accession number NG_055257.1), WT1 (Accession number NG_009272.1), Mage-A3 (Accession number NG_013244.1), Mage-A4 (Accession number NG_013245.1), Mage-A10 (Accession number NC_000023.11), TRAIL / DR4 (Accession number NC_000003.12), and / or CEA (Accession number NC_000019.10).

[0218] For example, tumor-associated antigens can be derived from prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain tumor, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-stage membrane epithelial antigen of the prostate (STEAP).

[0219] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. Additionally, tumor antigens may be self-peptide hormones, such as full-length gonadotrophin-releasing hormone (GnRH), a short 10-amino acid peptide that is useful in the treatment of many cancers.

[0220] Antigens can include epitope regions or epitope peptides derived from genes mutated or transcribed at different levels in tumor cells compared to normal cells, e.g., aberrantly expressed intronic sequences such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitope regions or epitope peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0221] E. Suicide gene In certain embodiments, suicide genes are utilized in conjunction with NK cell therapy to control their use and allow for termination of cell therapy at a desired event and / or time. Suicide genes are used in transduced cells to induce the death of the transduced cells when necessary. Cells of the present disclosure modified to carry one or more vectors encompassed by the present disclosure, which may contain one or more suicide genes. In some embodiments, the term "suicide gene," as used herein, is defined as a gene that, upon administration of a prodrug or other drug, results in the transition of the gene product to a compound that will kill the host cell. In other embodiments, the suicide gene encodes a gene product that, if desired, is targeted by an agent (e.g., an antibody) that targets the suicide gene product.

[0222] In some cases, cell therapy may be subject to the use of one or more suicide genes of any type when the individual receiving and / or the individual receiving cell therapy exhibits or is considered at risk of developing one or more symptoms, including imminent development, of one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (for example). The use of a suicide gene may be part of a planned protocol for treatment, or may be used only when its need is recognized. In some cases, cell therapy is terminated using an agent targeting the suicide gene or its gene product because treatment is no longer required.

[0223] The use of suicide genes can be initiated when at least one adverse event occurs to an individual, and the adverse event can be recognized by any means, including routine monitoring, whether or not it is continuous from the start of cell therapy. Adverse events can be detected by examinations and / or tests. If an individual has cytokine release syndrome (sometimes called cytokine storm), the individual may have, for example, elevated inflammatory cytokines (by way of example only: interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, IL-6, and TNF-alpha); fever; fatigue; hypotension; hypoxia; tachycardia; nausea; capillary leakage; cardiac / renal / liver dysfunction; or a combination thereof. If an individual experiences neurotoxicity, the individual may have confusion, delirium, aplasia, and / or seizures. In some cases, the individual is tested for markers related to the onset and / or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF-alpha, and / or ferritin.

[0224] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-alpha mutant polypeptides (see PCT / US19 / 62009, incorporated herein by reference in its entirety), which may be affected by delivery of antibodies that bind to TNF-alpha mutants. Examples of suicide gene / prodrug combinations that can be used include herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. The so-called suicide gene, purine nucleoside phosphorylase from E. coli, which converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, can be utilized. Examples of other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), EGFRv3, and thymidine phosphorylase (TP).

[0225] IV. Administration of Therapeutic Compositions The CD3-expressing NK cells and the bispecific or multispecific antibody are administered to an individual in need thereof, including in a manner that brings them into close proximity so that the anti-CD3 antibody of the bispecific or multispecific antibody can bind to CD3 on the CD3-expressing NK cells. In some cases, the two components are administered separately to the individual, while in other cases, the two components are complexed together prior to administration, such as by ex vivo methods. In another embodiment, the NK cells express the antibody. In some cases, the two components are not pre-complexed prior to administration, but are co-administered by any suitable route, such as by co-infusion into the patient.

[0226] Embodiments of the present disclosure relate to methods for using compositions comprising NK cells and antibodies provided herein to treat or prevent a medical disease or disorder. The methods include administering therapeutically effective amounts of CD3 (± TCR)-modified NK cells and antibodies to a subject, thereby treating or preventing the disease in the subject, including reducing the risk of the disease, reducing the severity of the disease, and / or delaying the onset of the disease. In certain embodiments of the present disclosure, cancer or infectious diseases are treated by the transfer of a composition comprising a population of NK cells and a corresponding antibody. In at least some cases, due to the release of proinflammatory cytokines, NK cells can augment the adaptive immune response by reversing the anti-inflammatory tumor microenvironment and promoting the differentiation, activation, and / or recruitment of accessory immune cells to malignant tumor sites.

[0227] The cancers for which the treatment method of the present invention is useful include all malignant cell types, such as those found in solid tumors or blood tumors.Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, malignant tumors of T cells or B cells, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0228] The cancer may be of the following histological types, among others, but is not limited to: neoplasm, malignant tumor; carcinoma; carcinoma, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumor; carcinoid tumor, malignant; lobular-alveolar adenocarcinoma; papillary adenocarcinoma; chromatophore carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; condylar carcinoma Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrial carcinoma; Adnexal carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Keratin adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma (malignant); Ovarian stromal tumor (malignant); Sarcoma (malignant); Granulosa cell tumor (malignant); Androblastoma (malignant); Sertoli cell carcinoma; Leydig cell tumor (malignant); Lipid cell Cystoma (malignant); Paraganglioma (malignant); Extramammary paraganglioma (malignant); Pheochromocytoma; Angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna melanoma; Lentigo acuminata melanoma; Nodular melanoma; Malignant melanoma of giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumor (malignant); Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Mesenchymoma (malignant); Brenner tumor (malignant); Philodes tumor (malignant); Synovial sarcoma; Mesothelioma (malignant); dysgerminoma; embryonal carcinoma; teratoma (malignant); ovarial goiter (malignant); choriocarcinoma; mesostosis, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; soft cortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor (malignant); ameloblastoma; ameloblastoma (malignant); ameloblastoma fibrosarcoma; pinealoma (malignant); chordoma; glioma (malignant); ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma;Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Paragranuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; High-grade lymphoblastic NHL ;High-grade small non-necrotic cell NHL;Bulky disease NHL;Mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroid leukemia;Lymphocytic cell leukemia;Myeloid leukemia;Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryocytic leukemia;Myeloid sarcoma;Hairy cell leukemia;Chronic lymphocytic leukemia (CLL);Acute lymphoblastic leukemia (ALL);Acute myeloid leukemia (AML);And chronic myeloblastic leukemia.

[0229] Therapies provided herein may include the administration of a combination of therapeutic agents, such as a first cancer therapeutic and a second cancer therapeutic. The therapeutic agents can be administered by any suitable method known in the art. For example, the first cancer treatment and the second cancer treatment can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered in separate compositions. In some embodiments, the first and second cancer treatments are in the same composition. Embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapeutic agents can be administered in one composition or in two or more compositions, for example, two compositions, three compositions, or four compositions. Various combinations of agents can be employed. Examples of therapies other than those disclosed herein include surgery, chemotherapy, drug therapy, radiation therapy, hormone therapy, immunotherapy (other than those disclosed herein), or combinations thereof.

[0230] The therapeutic agents of the present disclosure may be administered via the same or different routes of administration. In some embodiments, the cancer therapeutic agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0231] Treatment may include various "unit doses." A unit dose is defined as containing a predetermined amount of therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the discretion of a clinical technician. A unit dose does not have to be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose includes a single administrable dose.

[0232] The amount to be administered, i.e., both the number of treatments and the unit dose, depends on the desired treatment effect. It is understood that an effective dose refers to the amount required to achieve a specific effect. In certain embodiments, it is contemplated that a dose ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, it is contemplated that doses include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times per day and / or on multiple days, weeks, or months.

[0233] In certain embodiments, an effective amount of the pharmaceutical composition is one that can provide blood levels of about 1 μM to 150 μM. In other embodiments, an effective dose provides blood levels of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose may provide the following blood levels of drug resulting from the therapeutic agent administered to a subject: about, at least about, or up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 6, 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, or 100 μM, or any range derivable therein. In certain embodiments, a therapeutic agent administered to a subject is metabolized in the body to become a metabolic therapeutic agent, in which case blood levels may refer to the amount of that agent. Alternatively, to the extent that a therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0234] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dosage include the physical and clinical condition of the patient, the route of administration, the intended treatment goal (palliative versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatments the subject may be undergoing.

[0235] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood levels), e.g., 4 μM to 100 μM. It will also be understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions regarding uptake and concentration measurements are well known, and one skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies, and results described herein.

[0236] V. Kit Certain aspects of the present disclosure also relate to kits comprising compositions of the invention or compositions for carrying out methods of the invention. In certain embodiments, the kits comprise NK cells, which may be fresh or frozen, pre-activated or expanded or not. The NK cells may or may not already express one or more components of the TCR / CD3 complex. If the NK cells do not already express one or more components of the TCR / CD3 complex, the kits may include reagents for corresponding transfection or transduction of the NK cells, including vectors expressing the components, primers for amplifying the components, and other reagents. In some cases, the NK cells may or may not express one or more heterologous proteins as defined herein; if they do not express the components, the kits may include vectors expressing the heterologous proteins, primers for amplifying the heterologous proteins, and the like.

[0237] The kits may include components individually packaged or placed in containers such as tubes, bottles, vials, syringes, or other suitable container means. Individual components may also be provided in the kit in concentrated amounts, and in some embodiments, components are provided individually so that they are at the same concentration as the other components when present in solution. Component concentrations may be provided at 1x, 2x, 5x, 10x, or 20x or more. [Example]

[0238] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should recognize that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and can therefore be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0239] Example 1 Preparation and effective use of CD3-expressing NK cells This example relates to cancer immunotherapy as a strategy to redirect the specificity of NK cells for one or more target antigens by "arming" or pre-conjugating them with bispecific or multispecific antibodies, for example, prior to infusion or by separately co-injecting the two products. The NK cells are transduced with one or more CD3 chains, including the CD3ζ chain, CD3γ chain, CD3δ chain, and CD3ε chain, and can be from any source. The cells may or may not be expanded, may be pre-activated with one or more inflammatory cytokines, such as IL-12 / 15 / 18, and / or may be genetically modified to express one or more heterologous proteins, including, for example, engineered antigen receptors, such as chimeric antigen receptors or TCRs, cytokine genes, and / or homing / chemokine receptors.

[0240] 1A and 1B illustrate different embodiments of NK cells engineered to be utilized with bispecific or multispecific antibodies. As shown in FIG. 1A, in first-generation NK cells, the cells are engineered to express CD3 that can be activated with a bispecific or multispecific antibody, including a bispecific T cell engager (BiTE) comprised of an anti-CD3 antibody that binds to heterologous CD3 expressed on the surface of the NK cell. In another embodiment, the CD3-expressing NK cells are capable of being bound by a BiTE comprising an anti-CD3 antibody, and the NK cells also express one or more specific cytokines (e.g., IL-15 and / or IL-21), resulting in increased efficacy and potency that is particularly useful for treating solid tumors. In another embodiment, NK cells are engineered to express only CD3 so that they can be activated by BiTEs containing anti-CD3 antibodies, but are also utilized in conjunction with bispecific or multispecific antibodies (e.g., bispecific NK cell engagers, e.g., BiKEs) containing antibodies that bind to naturally occurring surface antigens on NK cells, such as CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCRs, or KIRs. In this way, NK cells respond to both NK engagers and T cell engagers. In another embodiment, NK cells express an engineered antigen receptor, such as a CAR or engineered TCR, in addition to expressing CD3 to engage with T cell engagers.

[0241] Figure 1B illustrates a different embodiment in which NK cells have been engineered to express both CD3 and TCR. On the right, a T cell TCR is illustrated, with an α chain and a β chain containing the antigen-binding site, and the TCR forms a complex with CD3ζ to signal. The T cell TCR is co-complexed with two CD3ε chains, a CD3δ chain, and a CD3γ chain. In some embodiments, the NK cells express a TCR in which one or more of the cytoplasmic domains of any of the CD3 molecules are heterologous intracellular domains, such as those from CD16, NKG2D, DAP10, DAP12, NCR, and DNAM-1. As shown on the left side of Figure 1B, the NK cells are engineered to express a CD3 co-receptor component; in one example, the CD3 component is CD3ε. In such cases, standard BiTEs (top left, including an antibody against a tumor antigen and an antibody against CD3) typically utilized with T cells that naturally express CD3 can be utilized in conjunction with the CD3-expressing NK cells. In this particular example, the NK cell expresses a polypeptide comprising the extracellular domain of CD3ε (although the extracellular domain of other CD3 components may also be utilized), which is linked to the transmembrane and / or cytoplasmic domain of another molecule, such as the transmembrane and / or cytoplasmic domain of CD3ζ, CD16, NKG2D, DAP10, DAP12, NCR, or DNAM-1.

[0242] As mentioned above, Figure 1C shows a schematic diagram of the generation of a surface-expressible single chimeric CD3 construct that can be used in conjunction with an anti-CD3 BiTE. In one example, the CD3 epsilon extracellular domain (ECD) is fused to the CD28, CD16, or NKG2D transmembrane domain (TM), with or without the CD3 zeta and / or DAP10 intracellular domain, and the CD28, CD16, or NKG2D intracellular domain (ICD). In one example, the construct is packaged within a Moloney murine virus-derived SFG retroviral vector backbone, which can be used with a packaging plasmid for virus production. When a CD3-BiTE is used with such a construct in Figure 1C, the antibody binds to the extracellular domain of CD3ε.

[0243] Embodiments of the present disclosure utilize some or all of the CD3 receptor complex. As illustrated in Figures 2A and 2B, NK cells may be transfected or transduced with full-length CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon. In such cases, the full-length CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon each comprise an extracellular domain, a transmembrane domain, and an intracellular domain. When different components of the receptor are expressed from the same vector, they may be configured to be produced as separate polypeptides, such as by using an IRES or 2A element. In either case, any expression construct may be configured to express one or more cytokines, including at least IL-15.

[0244] Figure 4 demonstrates CD3 expression on NK cells 4 days after transduction with CMV TCR complexes. This figure is a FACS plot showing CD3 expression on NK cells 4 days after CMV TCR complex transduction. Non-transduced (NT) NK cells (CD56+ CD3-) serve as a negative control, and T cells (CD3+ CD56-) serve as a positive control.

[0245] Figure 5 demonstrates TCR expression on NK cells 4 days after transduction of NK with the CMV TCR complex. In particular, a FACS plot showing TCRa / b expression on NK cells 4 days after CMV TCR complex transduction is provided. Non-transduced (NT) NK cells (CD56+ CD3- TCRa / b-) serve as a negative control, and T cells (CD3+ TCRa / b+ CD56-) serve as a positive control.

[0246] Figure 6 shows TCR / CD3 expression in NK cells 6 days after CMV TCR complex transduction. Specifically, the FACS plot shows dual expression of CD3 and TCRa / b in NK cells 6 days after CMV TCR complex transduction. Non-transduced (NT) NK cells (CD56+ CD3- TCRa / b-) serve as a negative control, and T cells (CD3+ TCRa / b+ CD56-) serve as a positive control.

[0247] Figure 7 shows the CD3 / TCR-mediated binding of CD3-CD19 BiTEs to NK cells at different concentrations. Specifically, various cells (non-transduced NK cells, T cells, or three different NK-TCR cells) were incubated with the CD3-CD19 bispecific engager (BiTe) blinatumomab at two different concentrations (0.5 μg / μl or 4 μg / μl) for 1 hour at 37°C. Biotin-labeled CD19 antigen (Miltenyi CD19 CAR Detection Reagent) was then added for 20 minutes, followed by the addition of anti-biotin antibody at room temperature for 15 minutes. This strategy was used to detect BiTe engagement with CD3+ cells. The histograms in Figure 7 show the level of CD19 binding to the CD3-CD19 bispecific engager (BiTe) correlating with CD3 expression on NK-TCR and T cells.

[0248] Figure 8 shows NK-TCR cytokine production after stimulation with plate-bound CD3 antibody. Specifically, 20 μg / ml of the CD3-OKT3 clone was incubated overnight at 4°C in a flat-bottom 96-well plate to form plate-bound antigens. The following day, T cells or NK cells (NT or TCR-transduced) were added to the wells for 4 hours, followed by the addition of Brefeldin A (which prevents cytokine release and traps them in the cytoplasm for detection by intracellular cytokine staining). They were then harvested and subjected to surface and intracellular staining to assess cytokine production and degranulation (TNFα and CD107a). The FACS plot in Figure 8 shows a TNFα and CD107a double-positive population in TCR-transduced NK cells. Non-transduced (NT) NK cells (CD56+ CD3-) served as a negative control, and T cells (CD3+ CD56-) served as a positive control.

[0249] Figure 9 demonstrates the phosphorylation of CD3ζ in NK TCR / CD3 cells after CD3 crosslinking. The various cells tested included non-transduced (NT) NK cells; non-transduced (NT) T cells; or three different CD3-TCR-transduced NK cells (where CD1, CD2, or CD3 represent different donors). Each NK cell population was transduced with CD3ZFLGDEFL15 (see Figures 2A and 2B). NK cells were incubated with CD3 OKT3 clone (Miltenyi, 130-093-387) at a concentration of 20 μg / ml on ice for 20 minutes. Cells were then crosslinked with Fab2 IgG1 antibody at various time points and stained to check for CD3z phosphorylation. This analysis of CD3ζ is useful because it can be crosslinked with CD3 monoclonal antibodies only if NK cells express it as an internalization signal from the surface. Non-CD3 transduced NK cells do not show any phosphorylation or activation after stimulation.

[0250] CD3-TCR-transduced NK cells also increased upon stimulation with CD3 OKT3 and displayed basal levels of tonic signaling similar to those of T cells, whereas untransduced NK cells did not exhibit CD3ζ phosphorylation either basally or upon CD3 OKT3 stimulation.

[0251] Figure 10 shows that pre-incubation of CD3-CD19 BiTEs with TCR / CD3-expressing NK cells increased their killing activity against Raji cells. NK cells were transduced with either CD3-TCR#1 (CD3ZFLGDEFL15 (see Figures 2A and 2B)) or CD3-TCR#2 (Z2, also known as CD3ZGDEFL8SP21CD8, which contains full-length CD3ζ, full-length CD3γ, full-length CD3δ, and full-length CD3ε linked to membrane-bound IL21 (with the CD8 transmembrane domain for membrane-bound IL21). NK cells transduced with the CD3 / TCR constructs or untransduced NK cells were loaded with blinatumumab, incubated for 1 hour, and washed with PBS. They were then co-cultured with CD19+ B-cell lymphoma cells at different effector:target cell ratios (1:1 ratio in Figure 10A and 1:5 ratio in Figure 10B) for various time points. As used herein, effector cells are CD3-TCR NK cells and target cells are Raji cells. CD3-TCR-transformed NK cells loaded with blinatumomab showed enhanced anti-tumor activity compared to untransduced NK cells or CD3 / TCR-transduced NK cells loaded with blinatumomab, but not loaded with blinatumomab, at any E:T ratio.

[0252] Example 2 NY-ESO TCR on NK cells Examples of the present invention relate to the generation and use of NY-ESO TCR in NK cells. Figure 11 shows an example of cell production. This schematic diagram illustrates an example in which NK cells are first transduced with a uTNK15 construct incorporating signaling domains from the CD3 complex, NK costimulatory molecules, and IL-15, followed by a second transduction step to introduce TCR molecules, generating NK cells co-expressing CD3 and NK signaling molecules, IL-15, and the TCR complex. In one embodiment, NK cells were derived from umbilical cord blood and expanded in complete medium using irradiated (100 Gy) universal antigen-presenting cell (uAPC) feeder cells (feeder cell:NK ratio 2:1) and recombinant human IL-2 (200 U / ml). To generate universal T cell-like NK cells capable of secreting IL-15 (uTNK15 cells), NK cells were purified and transduced with a retroviral construct containing the CD3 complex with NK costimulatory molecules and IL-15 genes 4 days after isolation. Forty-eight hours after the initial transduction, NK cells expressing uTNK15 were transduced with TCRs targeting the antigen of choice.

[0253] Figure 12 shows the expression of NY-ESO TCR on uTNK15-transduced NK cells. NK cells were derived from umbilical cord blood and expanded in complete medium using irradiated (100 Gy) universal antigen-presenting cell (uAPC) feeder cells (feeder cell:NK ratio 2:1) and recombinant human IL-2 (200 U / ml). To generate universal T cell-like NK cells capable of secreting IL-15, NK cells were purified and transduced 4 days after isolation with a retroviral construct containing a CD3 complex carrying NK costimulatory molecules and the IL-15 gene. Forty-eight hours after the initial transduction, uTNK15 cells were transduced with a TCR complex targeting the selected antigen. Forty-eight hours later, flow cytometry was used to evaluate the expression of CD3 and NY-ESO TCR on the various uTNK15 constructs. Non-transduced (NT) NK cells served as a negative control. CD3 and NY-ESO TCRs were highly expressed on all uTNK15 cells compared to NT NK cells. The number of tumor-specific TCR molecules expressed on TCR-engineered NK cells using various TCR constructs is provided in Figure 13, with NT NK cells used as a negative control.

[0254] Figure 14 demonstrates NY-ESO TCR expression in untransduced and transduced T cells. T cells were isolated from umbilical cord blood (from the same donor as the NK cells to serve as a paired positive control) and activated with CD3 / CD28 microbeads at a concentration of 25 μl / million in RPMI complete medium for 48 hours. T cells were then transduced with a retroviral construct containing the NY-ESO TCR. 48 hours after transduction, flow cytometry revealed that the NY-ESO TCR was highly expressed in the transduced T cells compared to the untransduced T cells.

[0255] NK cells transduced with the NY-ESO TCR kill target cells pulsed with the NY-ESO peptide in a dose-dependent manner (Figure 15). 51TCR killing assays were performed 7 days after TCR transduction to determine the killing ability of TCR-engineered NK and T cells against LCL cells loaded with different concentrations of NY-ESO peptide for 2 hours. NY-ESO TCR-transduced uTNK15 cells exhibit increased killing of peptide-pulsed LCL cells compared to untransduced NK cells. NY-ESO TCR-transduced T cells exhibit increased killing of peptide-pulsed LCL cells compared to untransduced T cells.

[0256] Figure 16 shows that NY-ESO is endogenously expressed in myeloma, sarcoma, and melanoma cell lines. Flow cytometry was used to determine NY-ESO expression in U266 (myeloma), Saos-2 (sarcoma), and A375 (melanoma) cell lines. The U266, Saos-2, and A375 cell lines showed higher levels of NY-ESO expression than the negative control Raji cell line.

[0257] NY-ESO TCR-transduced T cells kill NY-ESO-expressing tumor targets at higher E:T ratios (Figure 17). 51 TCR killing assays were performed 7 days after TCR transduction to determine the killing ability of NY-ESO TCR-engineered T cells against NY-ESO-expressing myeloma, osteosarcoma, and melanoma cell lines. NY-ESO TCR-transduced T cells exhibit increased killing of NY-ESO-positive cell lines compared with non-transduced T cells.

[0258] Figure 18 demonstrates that NY-ESO TCR-transduced NK cells kill NY-ESO-expressing tumor targets even at low E:T ratios. 51 CR killing assays were performed 7 days after TCR transduction to determine the killing ability of NY-ESO TCR-engineered NK cells against NY-ESO-expressing myeloma, osteosarcoma, and melanoma cell lines. NY-ESO TCR-transduced NK cells exhibit increased killing of NY-ESO-positive cell lines compared to untransduced NK cells, even at low effector:target ratios.

[0259] Figure 19 shows that NY-ESO-transduced NK cells have a similar phenotype to NT NK cells. CytoF imaging revealed that non-transduced NK cells and NY-ESO TCR-transduced uTNK15 cells share a similar phenotype. Figure 19A shows a u-map plot with similar clusters, and Figure 19B shows a heatmap with similar expression of various markers in NT cells and NY-ESO TCR-transduced uTNK15 cells.

[0260] Figure 20 provides a table depicting the percentage of CD3+ and CD3+TCR+ NK cells in each uTNK15 product. Flow cytometry was used to assess the composition of single positive CD3 NK cells (CD3+) and double positive CD3 / TCR NK cells (CD3+TCR+). While untransduced NK cells consist of less than 1% CD3+ and CD3+TCR+ NK cells, TCR-transduced uTNK15 cell products consist of more than 60% CD3+ and more than 25% CD3+TCR+ NK cells.

[0261] Figure 21A provides a FACS plot showing successful CD3 expression on NK cells 4 days after transduction with TCR constant alpha-beta (TCRCab; TCR6 construct). Non-transduced (NT) NK cells (CD56+ CD3-) served as a negative control. In Figure 21B, NT NK cells and uTNK15 NK cells were incubated with the CD3-CD19 bispecific engager (BiTe), blinatumumab, at 10 μg / μl for 1 hour at 37°C. Biotin-labeled CD19 antigen (Miltenyi CD19 CAR Detection Reagent) was then added for 20 minutes, followed by the addition of anti-biotin antibody for 15 minutes at room temperature. This strategy was used to detect BiTe engagement with CD3+ cells. The histogram in this figure shows the level of CD19 binding to the CD3-CD19 bispecific engager (BiTe), which correlates with CD3 expression on uTINK15 NK cells. In Figure 21C, CD3 / TCR-transduced or non-transduced NK cells were loaded with blinatumomab, incubated for 1 hour, and washed with PBS. They were then co-cultured with LCL cells at different E:T ratios (A. 1:1, B. 1:5) for various time points. CD3-TCR-transduced NK cells loaded with blinatumomab showed enhanced antitumor activity compared with non-transduced NK cells loaded with blinatumomab or CD3 / TCR-transduced NK cells not loaded with blinatumomab at any E:T ratio.

[0262] Example 3 NY-ESO TCR on CD3-expressing NK cells in vivo As shown in Figures 22A-22C, NK cells containing the constructs described herein were tested in vivo and found to robustly inhibit tumor growth. A schematic outlining the experimental procedures performed is shown in Figure 22A. Briefly, NSG mice were irradiated with 300 cGy on day -1, and on day 0, individual mice were injected with 0.5 x 10 NK cells transduced with FireFlyluciferase (FFluc). 6U266-B1 cells (a myeloma cell line expressing both HLA-A2 and NY-ESO antigens) were injected into the tail vein of the mice, and on day 3, 5 × 10 6 Effector cells (NY-ESO TCR NK cells carrying the UT-NK15-NY ESO TCR construct, designated WT, #A, or #B, respectively; WT refers to wild-type CD3 molecules carrying IL-15; #A refers to CD3-CD28 (e.g., UT-NK15-28) carrying IL-15; and #B refers to CD3-DAP10 (e.g., UT-NK15-DAP10) carrying IL-15; or NY-ESO TCR T cells) were injected, and the animals were then monitored over time and sacrificed appropriately (N=5 mice per group). Figure 22B displays the results of the monitoring experiment described in Figure 22A as a function of bioluminescence imaging over time (shown are representative images from days 1, 7, 14, and 21, respectively). FIG. 22C is a graphical quantification of the bioluminescence mean radiance displayed in FIG. 22B, with the Y-axis representing mean radiance in p / s / cm 2 / sr, and the X axis represents time.

[0263] As shown in Figures 23A-B, the in vivo activity of effector cells (e.g., NK cells or T cells) containing the NY-ESO1-targeting TCR and the UT-NK15 construct was tested. Figure 22A shows images of spheroids formed by the osteosarcoma tumor cell line Saos-2, which were used to test the cytotoxic activity of NK cells and T cells expressing the NY-ESO1-specific TCR. Saos-2 cells were stably transduced to express GFP and seeded overnight at 10,000 cells per well in a 96-well plate, followed by the addition of 40,000 NK cells or T cells. Images of the co-culture were scanned over time and analyzed using an IncuCyte cell analysis system. Figure 22B shows a graph displaying the percentage of effector cell cytotoxicity (Y axis) captured from representative images after 3 days of co-culture. NK cells were co-transduced with the NY-ESO-TCR and a UT-NK15 signaling complex co-expressing a different costimulatory molecule (e.g., UTNK-15-28 or UTNK-15-DAP10) fused to the CD3ζ signaling chain. T cells were transduced with the NY-ESO TCR alone. Abbreviations in the graph are as follows: 28 = CD3ζ fused to the CD28 costimulatory domain; 10 = CD3ζ fused to the Dap10 costimulatory domain; 8 = CD8 alpha / beta co-receptor as part of the NY ESO TCR construct; and wo IL-15 = construct containing only CD3 zeta, epsilon, gamma, and delta without costimulation or IL-15. The best in vitro cytotoxicity was observed with TCR NK cells expressing UTNK15 with CD28 or DAP10 costimulatory domains fused to CD3ζ (e.g., UTNK-15-28, or UTNK-15-DAP10; SEQ ID NO: 121 and SEQ ID NO: 119, respectively) compared with NK cells transduced with the CD3 complex alone or with UT-NK15 without the costimulatory domain. Addition of CD8 alpha / beta co-receptors to the TCR did not significantly improve NK or T cell cytotoxicity.

[0264] As shown in Figures 24A-D, the in vivo activity of effector cells (e.g., NK cells or T cells) containing the NY-ESO-targeting TCR and the UT-NK15 construct was tested. Figure 24A shows the in vivo study design for testing the activity of NK cells and T cells transduced with different NY-ESO TCRs. Ten-week-old NSG mice were irradiated (300 cGy) and the following day injected with 500,000 U266 cells (an HLA-A2-positive, NY-ESO-expressing myeloma cell line) via the tail vein. Three days later, the mice received 5 million TCR-transduced T cells or TCR-transduced NK cells. The mice were then monitored for tumor control by BLI imaging. Figure 24B shows the BLI imaging results of the study outlined and performed according to Figure 24A. Mice were injected with U266 tumor cells alone, or with T cells transduced with NY-ESO-specific TCR, or with NK cells co-transduced with NY-ESO TCR and UT-NK15, bearing CD3ζ fused to CD28 (labeled as NY-ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15 CD28 NK cells). Figure 24C shows quantification of the mean brightness intensity of the regions of interest in animals tested according to Figure 24A and imaged in Figure 24B. Figure 24D shows a graph depicting the survival curves of the cohorts of the aforementioned animals. Results demonstrated that NY-ESO TCR T and NY-ESO TCR UTNK-15-CD28 NK cells mediated potent antitumor activity in vivo.

[0265] As shown in Figure 25, we tested the in vivo activity of effector cells (e.g., NK cells) containing the NY-ESO TCR and CD3 complex with or without IL-15. NSG mice were irradiated (300 cGy) and the following day, 500,000 U266 cells (an HLA-A2-positive, NY-ESO-expressing myeloma cell line) were injected via the tail vein. Three days later, the mice received 5 million TCR-transduced T cells or NK cells. Tumor control in mice was monitored by BLI imaging. NK cells were transduced with the NY-ESO-specific TCR and co-transduced with the CD3 complex without IL-15, or with the UT-NK15 expressing CD3ζ fused to CD28 (UT-NK15-28) or UT-NK15 expressing CD3ζ fused to DAP10 (UT-NK15-DAP10) costimulatory molecules, with or without expression of the D8 alpha / beta co-receptor. The results showed that the absence of IL-15 resulted in reduced antitumor activity in vivo.

[0266] Taken together, these results demonstrate that effector cells (e.g., NK cells) containing the constructs described herein (e.g., NY-ESO TCR constructs and / or CD3 constructs such as UT-NK15 or modified versions thereof, e.g., UT-NK-15-28 or UT-NK15-DAP10) are sufficient to robustly inhibit tumor growth in vivo.

[0267] Example 4 PRAME TCR in CD3-expressing NK cells in vitro As shown in Figures 26A-C, NK cells containing constructs targeting the melanoma preferentially expressed antigen (PRAME) antigen described herein were tested in vitro and found to robustly inhibit tumor cell growth. Figure 26A shows the expression of both UT-NK15 (x-axis, CD3) and PRAME-specific TCR (y-axis, TCR) on NK cells (TCR clones 46, 54, or DSK3, respectively) or the expression of PRAME-specific TCR on T cells transduced with it (TCR clones 46 or 54). PRAME-specific TCR expression on NK cells was confirmed using antibodies against the TCR and CD3. PRAME-specific TCR expression on T cells was confirmed by tetramer staining using a 46 / 54 peptide / MHC-specific tetramer. Figure 26B shows the in vitro cytotoxicity of NK cells expressing the PRAME-specific TCR against the U266 myeloma cell line. Using Incucyte live-cell imaging, we measured the cytotoxicity of T cells transduced with PRAME-specific TCRs and NK cells transduced with UT-NK15 and PRAME-specific TCRs against U266 myeloma cells. GFP-expressing U266 cells were cocultured with PRAME-specific TCR-expressing T cells or NK cells at a 1:1 effector:target ratio (50,000 effector cells and 50,000 target cells were seeded in each well of a 96-well plate). A reduction in GFP expression indicated cell death. After 26 hours, 50,000 tumor cells (labeled "reload") were added to each well in duplicate for tumor reload assays. NK cells expressing UT-NK15 and PRAME-specific TCR clone 46 or PRAME-specific TCR clone 54 exerted the best antitumor activity when re-challenged with U266 cells and showed superior cytotoxicity when compared with control T cells transduced with PRAME-specific TCR clone 46 or 54, respectively. Figure 26C shows the in vitro cytotoxicity of NK cells expressing PRAME-specific TCR against the UA375 melanoma cell line.We used Incucyte live-cell imaging to measure the cytotoxicity of T cells transduced with PRAME-specific TCRs and NK cells transduced with UT-NK15 and PRAME-specific TCRs against UA375 melanoma cells. GFP-expressing UA375 cells were cocultured with PRAME-expressing T cells or NK cells at a 1:1 effector:target ratio (50,000 effector cells and 50,000 target cells were seeded in each well of a 96-well plate). A reduction in GFP expression indicated cell death. 26 hours later, 50,000 tumor cells were added in duplicate to each well for tumor rechallenge assays. Open symbols represent T cells, and closed symbols represent NK cells. NT = not transduced. NK cells expressing UT-NK15 and PRAME-specific TCR clone 46 (TCR-46), PRAME-specific TCR54 (TCR-54), or PRAME-specific TCR clone DSK3 (DSK) exerted potent antitumor activity and showed superior cytotoxicity when rechallenged with UA375 cells when compared with control T cells transduced with PRAME-specific TCR clones 46, 54, or DSK3, respectively.

[0268] Taken together, these results demonstrate that effector cells (e.g., NK cells) containing the constructs described herein (e.g., PRAME-specific TCR constructs) are sufficient to robustly inhibit tumor growth in vivo. Furthermore, NK cells containing the CD3 constructs described herein linked to PRAME-specific TCR constructs exhibited increased cytotoxicity when compared to T cell control cells containing the same TCR constructs, particularly upon continued and / or rechallenge with tumor cells.

[0269] Example 5 TCR on CD3-expressing NK cells in vivo NK cells containing the constructs described herein have been tested in vivo and show robust inhibition of tumor growth. Experiments are performed according to the schematics and experimental procedures described herein. Briefly, NSG mice are irradiated (e.g., with approximately 300 cGy) on day -1, and then on day 0, individual mice are transduced with cancer cells (e.g., 0.5 x 10 6 Mice are injected via tail vein with approximately 5 x 10 cells, e.g., cells expressing (naturally harboring and / or transduced with) an antigen described herein, and on day 3, mice are treated with approximately 5 x 10 effector cells transduced with a transgenic TCR (e.g., a TCR construct comprising a gamma / delta TCR chain and / or an alpha / beta TCR chain and targeting, e.g., an antigen described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.), with or without other constructs described herein (e.g., UT-NK15 constructs with or without IL-15, with or without CD3 fusions to costimulatory molecules, and / or with or without additional control constructs). 6 TCR NK cells) were injected into the animals. The animals were then monitored over time and sacrificed as appropriate. The results of the monitoring of the above experiment were recorded, for example, as a function of bioluminescence imaging over time (e.g., day 1, day 7, day 14, day 21, etc.).

[0270] Test the in vitro activity of effector cells (e.g., NK cells or T cells) containing a TCR (e.g., a TCR construct comprising a gamma / delta TCR chain and / or an alpha / beta TCR chain targeting an antigen described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.) and the UT-NK15 construct. Test the in vitro activity of effector cells (e.g., NK cells or T cells) containing an appropriate tumor cell line (e.g., 0.5 x 10) containing the antigen of interest. 6The cytotoxic activity of specific TCR-expressing NK and / or T cells is tested using spheroids formed by cells, e.g., cells expressing (naturally and / or transduced with) the antigens described herein. Cancer cells are stably transduced to express an appropriate marker (e.g., GFP, FFluc, etc.), and a number of these cells (e.g., approximately 10,000 cells) are seeded overnight in a 96-well plate per well, followed by the addition of a number of effector cells (e.g., approximately 40,000 cells). Images of the co-culture are scanned over time and analyzed using an appropriate system (e.g., IncuCyte cell analysis system). The percentage of cytotoxicity of effector cells is determined from representative images after several days of co-culture (e.g., 1 day, 3 days, 7 days, etc.). NK cells are co-transduced with a UT-NK15 signaling complex co-expressing a TCR targeting an antigen and a different costimulatory molecule fused to the CD3ζ signaling chain (e.g., UTNK-15-28 or UTNK-15-DAP10). Appropriate control cells are transduced with the appropriate construct described herein. Superior in vitro cytotoxicity is observed with TCR NK cells expressing UTNK15 with a CD28 or DAP10 costimulatory domain fused to CD3ζ (e.g., UTNK-15-28 or UTNK-15-DAP10; e.g., SEQ ID NO: 121 and SEQ ID NO: 119, respectively) compared to NK cells transduced with only the CD3 complex or UT-NK15 without the costimulatory domain.

[0271] The in vivo activity of effector cells (e.g., NK cells or T cells) containing an antigen-specific TCR (e.g., a TCR construct comprising a gamma / delta TCR chain and / or an alpha / beta TCR chain targeting an antigen described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.) and the UT-NK15 construct is tested. Assays for in vivo analysis of effector cells (e.g., NK cells or T cells) containing the engineered constructs are performed similarly to the experimental design described in Figure 24. Briefly, NSG mice of an appropriate age (e.g., 10-week-old NSG mice) are irradiated (e.g., with approximately 300 cGy) and the next day injected with tumor cells containing the target antigen of interest (e.g., approximately 500,000 cells; e.g., naturally expressing and / or transduced with an antigen described herein) via the tail vein. Three days later, mice receive a bolus of effector cells (e.g., 5 million TCR-transduced T cells and / or TCR-transduced NK cells). The mice are then monitored for tumor control (e.g., by BLI imaging). The mean radiance of the region of interest is measured and quantified, and animals containing test constructs containing a TCR targeting the antigen of interest and a UT-NK15 construct, with or without CD3 fusion and / or IL-15 expression, show improved survival and / or reduced mean radiance relative to control animals. The results demonstrate that TCR UTNK-15 NK cells mediate potent antitumor activity in vivo.

[0272] The in vivo activity of effector cells (e.g., NK cells) comprising a TCR (e.g., a TCR construct comprising a gamma / delta TCR chain and / or an alpha / beta TCR chain targeting an antigen described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.) and a CD3 complex is tested, with or without IL-15. NSG mice are irradiated (e.g., with approximately 300 cGy), and the next day, they are injected via the tail vein with antigen-expressing tumor cells (e.g., approximately 500,000 cells; e.g., cells naturally expressing and / or transduced with an antigen described herein). Three days later, the mice receive a bolus of effector cells (e.g., 5 million TCR-transduced T cells and / or TCR-transduced NK cells). The mice are monitored for tumor control (e.g., by BLI imaging). NK cells were transformed with antigen-specific TCR and co-transduced with the CD3 complex without IL-15, or the UT-NK15 expressing CD3ζ fused to CD28 (UT-NK15-28) or UT-NK15 expressing CD3ζ fused to DAP10 (UT-NK15-DAP10) costimulatory molecules, with or without expression of the CD8 alpha / beta co-receptor. Results show that the absence of IL-15 results in reduced antitumor activity in vivo.

[0273] Taken together, these results demonstrate that effector cells (e.g., NK cells) containing the constructs described herein (e.g., TCR constructs and / or CD3 constructs such as UT-NK15 or modified versions thereof, e.g., UT-NK-15-28 or UT-NK15-DAP10) are sufficient to robustly inhibit tumor growth in vivo.

[0274] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods and to the steps or the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More particularly, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

Claims

**Claim 1**: A composition comprising NK cells modified to express a single-chain or any combination of part or all of CD3ζ, CD3δ, CD3ε, or CD3γ. **Claim 2** The composition according to claim 1, wherein the NK cells are modified to express one or more of TCRα chain, TCRβ chain, TCRγ chain, and TCRδ chain. **Claim 3** The composition according to claim 1, wherein any one or more of the CD3ζ, CD3ε, CD3δ, and CD3γ are heterologously linked to one or more intracellular signaling domains. **Claim 4** The composition according to claim 3, wherein the intracellular signaling domain is selected from the group consisting of CD28, DAP10, CD16, NKG2D, DAP12, 2B4, 4-1BB, CD2, and combinations thereof. **Claim 5** The composition according to claim 4, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 115; an amino acid sequence that is at least about 85% identical to SEQ ID NO: 116; or an amino acid sequence that is at least about 85% identical to SEQ ID NO:

117. **Claim 6** The composition according to claim 1, further comprising one or more bispecific antibodies or multispecific antibodies, wherein the bispecific antibody or multispecific antibody comprises an anti-CD3 antibody. **Claim 7** The composition according to claim 6, wherein the NK cells express an antibody and / or the antibody forms a complex with the NK cells. **Claim 8** The composition according to claim 1, wherein the NK cells are modified to express one or more heterologous proteins selected from engineered antigen receptors, cytokines, homing receptors, or chemokine receptors. **Claim 9** The composition according to claim 8, wherein the engineered antigen receptor is a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). **Claim 10** The composition according to claim 9, wherein the engineered antigen receptor is an engineered T cell receptor (TCR) that targets the NY-ESO antigen or a PRAME antigen epitope. **Claim 11** The composition according to claim 10, wherein the T cell receptor comprises a sequence that is at least 85% identical to SEQ ID NO: 25 and a sequence that is at least 85% identical to SEQ ID NO:

26. **Claim 12** The composition according to claim 10, wherein the target PRAME antigen epitope is SLLQHLIGL (SEQ ID NO: 131) and / or QLLALLPSL (SEQ ID NO: 132).

13. The composition according to claim 10, wherein the T cell receptor comprises: (i) a sequence having at least 85% identity to SEQ ID NO: 135 and a sequence having at least 85% identity to SEQ ID NO: 136; (ii) a sequence having at least 85% identity to SEQ ID NO: 139 and a sequence having at least 85% identity to SEQ ID NO: 140; or (iii) a sequence having at least 85% identity to SEQ ID NO: 143 and a sequence having at least 85% identity to SEQ ID NO:

144.

14. The composition according to claim 8, wherein the heterologous protein is a cytokine, and the cytokine is selected from the group consisting of: (i) IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, or a combination thereof; or (ii) IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, or a combination thereof, wherein the cytokine is membrane-bound and comprises a transmembrane domain from CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, or DAP12.

15. The composition according to claim 6, wherein the bispecific antibody comprises an antibody targeting a cancer antigen.

16. A composition comprising a complex, (1) NK cells modified to express some or all of the CD3 receptor complex and optionally modified to express the T cell receptor (TCR) αβ chain or TCRγδ chain, and (2) a bispecific or multispecific antibody that binds to CD3 on the NK cells A composition comprising.

17. The composition according to claim 16, wherein the NK cells are modified to express a TCRαβ chain having at least 85% identity to SEQ ID NO: 25 and SEQ ID NO: 26, the TCRαβ chain targets the NY-ESO antigen, and the bispecific antibody is blinatumomab.

18. The composition according to claim 16, wherein any one or more of CD3ζ, CD3ε, CD3δ, and CD3γ are heterologously linked to one or more intracellular signaling domains.

19. The composition according to claim 18, wherein the intracellular signaling domain is selected from the group consisting of CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

20. The composition according to claim 18, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 115, an amino acid sequence that is at least about 85% identical to SEQ ID NO: 116, or an amino acid sequence that is at least about 85% identical to SEQ ID NO:

117.

21. The composition according to claim 1, which is used for the treatment of cancer.

22. A composition for use in the treatment of cancer, comprising: (1) NK cells that express some or all of the CD3 receptor complex and optionally (2) some or all of the T cell receptor (TCR) αβ chain or TCRγδ chain, wherein the specificity of the NK cells is redirected towards a cancer antigen.

23. The NK cells are modified to express some or all of CD3ζ, CD3ε, CD3δ, and CD3γ, The composition according to claim 22, wherein any one or more of CD3ζ, CD3ε, CD3δ, and CD3γ are heterologously linked to one or more intracellular signaling domains.

24. The composition according to claim 23, wherein the intracellular signaling domain is selected from the group consisting of CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

25. The composition according to claim 24, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO: 115; an amino acid sequence that is at least about 85% identical to SEQ ID NO: 116; or an amino acid sequence that is at least about 85% identical to SEQ ID NO:

117.

26. The composition according to claim 22, wherein the NK cells express some or all of the TCRαβ chain or TCRγδ chain, and the TCRαβ chain or TCRγδ chain targets the NY-ESO antigen or a PRAME antigen epitope.

27. The composition according to claim 26, wherein the TCR chain is the TCRαβ chain and is at least 85% identical to SEQ ID NO: 25 and at least 85% identical to SEQ ID NO:

26.

28. The composition according to claim 26, wherein the target PRAME antigen epitope is SLLQHLIGL (SEQ ID NO: 131) and / or QLLALLPSL (SEQ ID NO: 132).

29. The composition according to claim 26, wherein the TCR chain comprises the following: (i) a sequence having at least 85% identity to SEQ ID NO: 135 and a sequence having at least 85% identity to SEQ ID NO: 136; (ii) a sequence having at least 85% identity to SEQ ID NO: 139 and a sequence having at least 85% identity to SEQ ID NO: 140; or (iii) a sequence having at least 85% identity to SEQ ID NO: 143 and a sequence having at least 85% identity to SEQ ID NO:

144.

30. The composition according to claim 22, wherein the NK cells are modified to express one or more additional heterologous proteins.

31. A polynucleotide or polypeptide comprising a sequence having at least 85% identity to any one or more of SEQ ID NOs: 118 to 123.

Images