CN118055770A

CN118055770A - CD3 expressing natural killer cells with enhanced function for adoptive immunotherapy

Info

Publication number: CN118055770A
Application number: CN202280061681.5A
Authority: CN
Inventors: 刘恩莉; K·雷兹瓦尼; R·巴萨尔; 刘斌; D·马林科斯达
Original assignee: University of Texas System
Current assignee: University of Texas System
Priority date: 2021-07-23
Filing date: 2022-07-22
Publication date: 2024-05-17

Abstract

Embodiments of the invention include methods and compositions wherein NK cells are artificially modified to express T cell receptors and CD3 co-receptors on NK cells that do not naturally express T cell receptors and CD3 co-receptors. The modified NK cells function effectively with bispecific or multispecific antibodies tailored to comprise anti-CD 3 antibodies that bind to the modified NK cells, thereby triggering signaling, activation, and cytotoxicity of target cells to which the antibodies also bind. Thus, NK cells are specifically configured to work effectively with bispecific NK cell binding molecules (bikes) as well as bispecific T cell binding molecules (bikes).

Description

CD3 expressing natural killer cells with enhanced function for adoptive immunotherapy

The present application claims priority to U.S. provisional patent application Ser. No. 63/225,281 filed on 7.23 of 2021, and also claims priority to U.S. provisional patent application Ser. No. 63/310,526 filed on 2.15 of 2022, and also claims priority to U.S. provisional patent application Ser. No. 63/344,931 filed on 5.23 of 2022, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to at least the fields of immunology, cell biology, molecular biology and medicine, including at least cancer medicine.

Background

Natural Killer (NK) cells have been investigated as potential anti-tumor effectors, but many obstacles limit their therapeutic utility, mainly related to their lack of antigen specificity. One approach to overcome this is to transduce NK cells with Chimeric Antigen Receptors (CARs) or engineered T Cell Receptors (TCRs) to target the desired antigen. In T cells, bispecific or multispecific antibodies, such as bispecific T cell engagement molecules (bites), may be utilized that bind CD3 on the T cell surface and also bind antigens on the cancer cell surface. CD3 consists of four distinct chains, in mammals, the complex comprises one CD3 gamma chain, one CD3 delta chain and two CD3 epsilon chains. These chains bind to the T Cell Receptor (TCR) and zeta chains (zeta chains) to generate activation signals in T lymphocytes. However, NK cells do not naturally express the CD3 receptor complex or TCR.

The present invention meets a long felt need in the art for improved immunotherapy, including immunotherapy with NK cells.

Disclosure of Invention

Embodiments of the invention include methods and compositions for treating individuals with cancer using adoptive cell therapies. In particular embodiments, a therapeutically effective amount of dichotomy therapy (bipartite therapy) is provided to an individual that includes modified NK cells and antibodies that are capable of binding NK cells to initiate signaling, activation, and killing of target cells. The present invention relates to NK cells that have been modified to express a variety of proteins that are not naturally expressed in NK cells and work together, including heterologous proteins that are not naturally present in NK cells on the surface of NK cells.

In particular embodiments, NK cells are engineered to express one or more proteins from the CD3 co-receptor complex and optionally the TCR receptor complex, and each protein is typically present on the T cell surface. Such engineering provides greater versatility for use of NK cells with a variety of bispecific or multispecific antibodies, including antibodies comprising anti-CD 3 antibodies (e.g., anti-CD 3 scFv). In particular embodiments, the modified NK cells are administered to an individual in need thereof with one or more bispecific or multispecific antibodies each having one antibody that targets CD3 and one antibody that binds a desired antigen (e.g., a cancer antigen). As a result, in certain cases, NK cells expressing CD3 are able to bind to the anti-CD 3 antibody portion of the bispecific or multispecific antibody, and antibodies that bind to the cancer antigen on the surface of the cancer cell. This coordinated binding between NK cells and antibodies results in activation of cytotoxicity against the target cancer antigen.

In particular embodiments, the invention relates to modified NK cells expressing all or part of the CD3 complex with or without a TCR, and in certain cases, a single CD3 chain is heterologously linked to an NK-associated signaling domain, all of which allow the use of modified NK cells with various bispecific antibodies.

Embodiments of the invention include compositions comprising NK cells modified to express a portion or all or any combination of single strands of cd3δ, cd3ε, cd3γ, or cd3ζ. In some cases, NK cells are modified to express a T Cell Receptor (TCR) αβ chain or a tcrγδ chain. NK cells may be modified to express some or all of cd3ζ, two cd3ε, cd3δ, and cd3γ. In some cases, NK cells are modified to express the full length: cd3ζ, cd3ε, cd3δ and/or cd3γ. In particular instances, any one or more of cd3ζ, cd3ε, cd3δ, and cd3γ is heterologously linked to one or more intracellular signaling domains. The intracellular signaling domain may be selected from the group consisting of CD16, NKG2D, DAP, 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 include other costimulatory signals related to NK cell function, such as but not limited to 2B4, DNA, 4-1BB, DAP12, NKG2D, and the like. In certain embodiments, the composition further comprises one or more bispecific or multispecific antibodies, wherein the bispecific or multispecific antibodies comprise an anti-CD 3 antibody. NK cells may express and/or complex with antibodies. In some embodiments, the TCR is directed against a cancer antigen or a viral antigen. In particular embodiments, the NK cells are derived from umbilical Cord Blood (CB), peripheral Blood (PB), bone marrow, stem cells, or mixtures thereof. In some embodiments, the TCR is directed against an NY-ESO antigen. In some embodiments, the TCR is directed against a PRAME antigen. NK cells may be pre-activated, for example with one or more cytokines including: such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or combinations thereof. In some embodiments, NK cells are expanded, for example in the presence of IL-2. In particular embodiments, NK cells are modified to express one or more heterologous proteins, such as one or more engineered antigen receptors, one or more cytokines, one or more homing receptors, and/or one or more chemokine receptors. In particular instances, 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., a cytokine 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 comprise a transmembrane domain from CD8, CD28, CD27, B7H3, igG1, igG4, CD4, DAP10 or DAP 12. In certain instances, NK cells express chimeric antigen receptors and cytokines. In some cases, the bispecific antibody comprises an antibody that targets a cancer antigen.

Embodiments of the present invention include compositions comprising a complex comprising: (1) NK cells modified to express part or all of the CD3 receptor complex and optionally modified to express a T Cell Receptor (TCR) αβ chain or a tcrγδ chain; and (2) a bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD 3 antibody which binds to CD3 on NK cells. In particular embodiments, the complex is contained in a pharmaceutically acceptable excipient. The complex may be contained in a delivery device.

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

Embodiments of the invention include a method of redirecting NK cells specific for a cancer antigen with a bispecific or multispecific anti-CD 3 antibody for use in treating an individual, the method comprising the step of administering the antibody and optionally NK cells expressing part or all of a CD3 receptor complex and optionally expressing part or all of a tcrαβ chain or tcrγδ chain to the individual. In certain embodiments, the method further comprises the step of modifying the NK cells to express part or all of the CD3 receptor complex. In particular embodiments, the method further comprises the step of modifying the NK cell to express a tcrαβ chain or a tcrγδ chain. In some cases, the method further comprises the step of modifying the NK cells to express one or more heterologous proteins.

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 the specific examples, while indicating specific embodiments of the 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.

Drawings

The following drawings form a 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.

FIG. 1A shows various embodiments of NK cells engineered to express CD3, including use with various heterologous proteins such as cytokines, bispecific NK cell binding molecules, and engineered antigen receptors (CARs and/or TCRs). Figure 1B shows NK cells carrying CD3 and TCR for optimal cancer immunotherapy. FIG. 1C shows an example of a single chimeric CD3 construct.

FIG. 2A shows one example of an expression construct for the CD3 receptor complex component of transduced or transfected NK cells. FIG. 2B shows an example of a plasmid map of a representative expression construct.

FIG. 3 provides a table of various TCR/CD3 expression construct designs for NK-TCR engineering.

Figure 4 shows expression of CD3 on engineered NK cells on day 4 after transduction with one example of a CMV-guided TCR complex.

FIG. 5 shows expression of TCR on engineered NK cells on day 4 after CMV-directed TCR complex transduction.

FIG. 6 shows expression of TCR/CD3 on engineered NK cells on day 6 after CMV-directed TCR complex transduction into cells.

FIG. 7 shows the binding of one example of CD3-CD19 BiTE on NK cells at different concentrations by CD3/TCR complex on NK cells.

FIG. 8 shows TNF alpha and CD107a cytokine production of NK-TCR following stimulation with plate-bound CD3 antibody.

FIG. 9 shows the phosphorylation of CD3z in NK TCR/CD3 cells after crosslinking CD 3.

FIGS. 10A-10B show that pre-culturing NK cells expressing TCR/CD3 with CD3-CD19 BiTE increases their killing activity against Raji cells. FIG. 10A shows the effector to target ratio of 1:1, and FIG. 10B shows the effector to target ratio of 1:5.

FIG. 11 provides a schematic representation of the transduction of multiple retroviruses to produce NK cells expressing CD3, IL-15 and TCR complexes.

FIG. 12 shows expression of NY-ESO TCR on NK cells transduced with uTNK. WT refers to a wild-type CD3 molecule with IL-15; a refers to CD3-CD28 with IL-15; b is CD3-DAP10 with IL-15; c refers to CD3-CD28-Dap10 with IL-15.

FIG. 13 shows the number of TCR molecules expressed on NK cells (per cell). WT refers to a wild-type CD3 molecule 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. Phycoerythrin fluorescent quantification kit (BD Biosciences) was used to determine the number of NY-ESO TCR molecules on NK cells.

FIG. 14 shows expression of NY-ESO TCR on T cells.

FIG. 15 shows that NK cells transduced with NY-ESO TCR kill NY-ESO peptide-loaded target cells in a dose-dependent manner. WT refers to a wild-type CD3 molecule with IL-15; a refers to CD3-CD28 with IL-15; b is CD3-DAP10 with IL-15; c refers to CD3-CD28-Dap10 with IL-15.

FIG. 16 shows the expression of endogenous NY-ESO on human tumor cell lines.

FIG. 17 shows that NY-ESO TCR transduced T cells kill tumor targets expressing NY-ESO.

FIG. 18 provides the results that NY-ESO TCR transduced NK cells can kill NY-ESO expressing tumor targets even at low E:T ratios. WT refers to a wild-type CD3 molecule with IL-15; a refers to CD3-CD28 with IL-15; b is CD3-DAP10 with IL-15; c refers to CD3-CD28-Dap10 with IL-15.

FIGS. 19A and 19B show that NY-ESO transduced NK cells have a similar phenotype (19A) and expression pattern (19B) as NT NK cells. WT refers to a wild-type CD3 molecule with IL-15; a refers to CD3-CD28 with IL-15; b is CD3-DAP10 with IL-15; c refers to CD3-CD28-Dap10 with IL-15.

FIG. 20 provides a table showing the cellular composition of the amplified uTNK products. WT refers to a wild-type CD3 molecule 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.

Figure 21A shows that NK cells can be transduced successfully with CD3 and TCR constant α - β (TCRCab) (referred to as TCR6 construct) and engineered NK cells can bind Blinatumumab (figure 21B) and selectively kill cd19+ lymphoma targets (figure 21C).

FIGS. 22A-22C show in vivo activity of effector cells (e.g., NK cells or T cells) comprising NY-ESO targeted TCRs. Fig. 22A is a schematic diagram of experimental steps performed. FIG. 22B shows bioluminescence imaging over time (day 1, day 7, day 14 and day 21) of mice implanted with firefly luciferase (FFluc) -transduced U266B.1 cells with control, NY-ESO TCR NK cells or NY-ESO TCR T cells (NK cells containing WT, #A or #BUT-NK 15-NY ESO TCR constructs, respectively; WT refers to wild-type CD3 molecule with IL-15; #A refers to CD3-CD28 with IL-15; #B refers to CD3-DAP10 with IL-15). Fig. 22C is a graphical quantification of the bioluminescence mean radiation shown in fig. 22B. These results indicate that effector cells comprising the NY-ESO TCR constructs described herein strongly inhibit tumor growth in vivo.

FIGS. 23A-B show in vitro activity of effector cells (e.g., NK cells or T cells) comprising NY-ESO targeted TCR and UT-NK15 constructs. FIG. 23A is an image of a cell sphere formed by osteosarcoma tumor cell line Saos-2 stably transduced to express GFP, used to test the cytotoxic activity of NK cells and T cells expressing NY-ESO1 specific TCR. Fig. 23B is a graph showing the percent cytotoxicity (Y-axis) of representative images after 3 days of co-culture. NK cells were co-transduced with NY-ESO-TCR and UT-NK15 signaling complexes, with UT-NK15 signaling complexes co-expressing different co-stimulatory molecules fused to the CD3 zeta signaling chain or a TCR complex without IL-15. T cells were transduced with NY-ESO TCR alone. Abbreviations in the figures: 28 Cd3ζ fused to CD28 costimulatory domain; 10 Cd3ζ fused to Dap10 costimulatory domain; 8 = CD8 a/β co-receptor as part of NY ESO TCR construct; wo IL-15 = the construct contained only cd3ζ, ε, γ and δ TCR complexes, without co-stimulation or IL-15.

FIGS. 24A-D show in vivo activity of effector cells (e.g., NK cells or T cells) comprising NY-ESO targeted TCR and UT-NK15 constructs. FIG. 24A depicts a plan for in vivo studies to test the activity of different NY ESO TCR transduced NK cells and T cells. FIG. 24B depicts BLI imaging results from the test described and performed in FIG. 24A, mice were injected with U266 tumor cells, and three days later received T cells transduced with NY ESO-specific TCR or NK cells co-transduced with NY-ESO TCR and UT-NK15 (CD 3 zeta fused with CD28 in UT-NK15, labeled NY-ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15CD28 NK cells). The tumor group alone served as a control. Fig. 24C depicts the average radiation intensity of a region of interest of an animal tested according to fig. 24A and imaged in fig. 24B. Fig. 24D is a graph depicting survival curves for the above-described animal groups.

FIG. 25 shows in vivo activity of effector cells (e.g., NK cells) engineered to express NY ESO TCR and CD3 complexes, either with or without IL-15 transgene in the constructs. NSG mice were irradiated (300 cGy) and the next day by tail vein injection of 500,000U 266 cells (HLA-A 2 positive, NY-ESO expressing myeloma cell line). Three days later, mice received 500 ten thousand TCR-transduced T cells or NK cells. Tumor control in mice was monitored by BLI imaging. NK cells were transduced with NY-ESO specific TCR (with or without expression of CD 8. Alpha./beta. Co-receptor), co-transduced with CD3 complex without IL-15 transgene, or co-transduced with UT-NK15 expressing CD3 zeta fused with CD28 co-stimulatory molecule (UT-NK 15 CD 28) or UT-NK15 expressing CD3 zeta fused with DAP10 co-stimulatory molecule (UT-NK 15 DAP 10).

Figures 26A-C show in vitro expression of antigens preferentially expressed in melanoma (PRAME) TCRs on effector cells (e.g., NK cells or T cells) and in vitro activity of the cells. FIG. 26A shows the expression of UT-NK15 (x-axis, CD 3) and PRAME-specific TCR (y-axis, TCR) in NK cells (TCR clone 46, 54 or DSK3, respectively), or PRAME-specific TCR in T cells transduced with the same (TCR clone 46 or 54). Figure 26B shows in vitro cytotoxicity of NK cells expressing PRAME-specific TCRs against the U266 myeloma cell line. Cytotoxicity of U266 myeloma cells on T cells transduced with PRAME-specific TCR and NK cells transduced with UT-NK15 and PRAME-specific TCR was measured using Incucyte live cell imaging. GFP-expressing U266 cells were co-cultured with PRAME-specific TCR-expressing T cells or NK cells at a 1:1 effector to target ratio. A decrease in GFP expression indicates cell death. After 26 hours, a second round of 50,000 tumor cells (referred to as "re-challenge") was added to each well for the tumor re-challenge assay. The open symbols represent T cells, while the filled symbols represent NK cells. Nt=untransduced. Figure 26C shows in vitro cytotoxicity of NK cells expressing PRAME-specific TCRs against UA375 melanoma cell lines. Cytotoxicity of UA375 melanoma cells with T cells transduced with PRAME-specific TCRs and NK cells transduced with UT-NK15 and PRAME-specific TCRs (PRAME-specific TCR clone 46 (TCR-46), PRAME-specific TCR clone 54 (TCR-54) or PRAME-specific TCR clone DSK3 (DSK)) was measured using intucyte live cell imaging. GFP-expressing UA375 cells were co-cultured with PRAME-expressing T cells or NK cells at a ratio of effector to target of 1:1. A decrease in GFP expression indicates cell death. After 26 hours, a second round of 50,000 tumor cells were added to each well for tumor re-challenge assays. The open symbols represent T cells, while the filled symbols represent NK cells. Nt=untransduced.

Detailed Description

The words "a" and "an" when used in this specification (including the claims) with the word "comprising" mean "one or more" in accordance with the long-standing patent law convention. 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 of the methods or compositions described herein may be practiced with respect to any other of the methods or compositions described herein, and that different embodiments may be combined.

Throughout this specification, unless the context requires otherwise, the words "comprise," "comprising," and "include" will be understood to imply the inclusion of a stated 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 … …" is intended to include and be limited to anything in the middle of the phrase "consisting of … …". Thus, the phrase "consisting of … …" indicates that the listed elements are required or optional, and that no other elements may be present. "consisting essentially of … …" is intended to include any element listed in the middle of the phrase and is limited to other elements that do not interfere with or contribute to the activity or effect specified in the present invention. Thus, the phrase "consisting essentially of … …" indicates that the recited element is essential or optional, but that no other element is optional and may or may not be present depending on whether it affects the activity or action of the recited element.

Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an additional embodiment," or "another embodiment," or a combination thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase above in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the terms "or" and/or "are used to describe a plurality of components that are combined or are mutually exclusive. For example, "x, y, and/or z" may refer to "x" alone, "y" alone, "z," "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 embodiments.

In the present application, the term "about" is used in accordance with its ordinary and customary meaning in the art of cell and molecular biology to indicate the standard deviation of a value comprising the error of a device or method for determining the value.

As used herein, the term "CD3 receptor complex" or "CD3 co-receptor complex" refers to a protein complex that essentially acts as a T cell co-receptor and consists of a cd3ζ chain, a cd3γ chain, one cd3δ chain, and two cd3ε chains (although only one cd3ε chain is used in the alternative).

The term "engineered" as used herein refers to artificially created entities, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some instances, the engineered entity is synthetic and contains elements that do not naturally occur or are configured in the manner in which they are used in the present disclosure. In certain embodiments, the vector is engineered by recombinant nucleic acid techniques and the cells are engineered by transfection or transduction of the engineered vector. The cell may be engineered to express a heterologous protein that the cell does not naturally express, either because the heterologous protein is recombinant or synthetic, or because the cell does not naturally express the protein.

The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when properly administered to an animal (e.g., a human). In accordance with the present invention, the person skilled in the art will be aware of the preparation of pharmaceutical compositions comprising antibodies or additional active ingredients. Furthermore, for animal (e.g., human) administration, it is understood that the preparation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA office of biological standards.

As used herein, "pharmaceutically acceptable carrier" includes: any and all aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral vehicles such as sodium chloride, ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquids, and nutritional supplements, such as materials and combinations thereof, as known to those of ordinary skill in the art. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to well known parameters.

The term "subject" as used herein generally refers to an individual having or suspected of having cancer. The subject may be any organism or animal subject as a method or material object, including mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), domestic pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. The subject may be a patient, e.g., suffering from or suspected of suffering from a disease (which may be referred to as a medical condition), e.g., benign or malignant tumor or cancer. The subject may be receiving or has received treatment. The subject may be asymptomatic. The subject may be a healthy individual, but it is desirable to prevent cancer. The term "individual" may be used interchangeably, at least in some instances. As used herein, a "subject" or "individual" may or may not be located in a medical facility, and may be treated as an outpatient to the medical facility. An individual may be receiving one or more pharmaceutical compositions via the internet. Individuals may include human or non-human animals of any age, and thus include adults and adolescents (i.e., children) as well as infants, and include intrauterine individuals. The term does not mean that medical treatment is required and thus, whether clinical or supporting basic scientific research, an individual may voluntarily or involuntarily become part of an experiment.

As used herein, "treating" or "treatment" 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 the amount of one or more measurable markers of the disease or condition being treated (e.g., cancer). Treatment may involve optionally alleviating or ameliorating a symptom of a disease or condition, or delaying the progression of a disease or condition. "treating" does not necessarily mean complete eradication or cure of a disease or condition or associated symptoms. Treatment may mean alleviation of at least one symptom of a disease or condition.

As used herein, "TCR/CD3 complex" refers to a protein complex naturally occurring on the surface of T cells and comprises T cell receptor alpha and beta chains and/or T cell receptor gamma and delta chains, as well as cd33ζ, cd3γ, cd3δ and cd3ε chains.

I. Embodiments of the invention

Natural Killer (NK) cells are an emerging cellular immunotherapy for hematological malignancies and solid tumor patients. The invention relates in particular to modified NK cells, which have enhanced function for use in immunotherapy compared to NK cells not so modified. These modifications allow NK cells to have greater versatility in use with other therapeutic agents and, at least in some embodiments, T cell-like activity by utilizing the CD3/TCR receptor complex. In particular embodiments, NK cells are modified to express: (i) A single CD3 chain (cd3ζ, cd3ε, cd3δ, or cd3γ) or a portion or all of a human CD3 receptor complex (including any combination of cd3δ, ε (one or two copies of ε), γ, and ζ); or (ii) a single CD3 chain or a human CD3 receptor complex (including any combination of cd3δ, ε (one or two molecules), γ, and ζ) as a full-length protein or as a partial protein, 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 invention relates to the use of NK cells expressing CD3 in the diagnosis and treatment of diseases comprising the use of cells in combination with bispecific or multispecific antibodies, wherein one epitope of the antibody binds CD3 on NK cells expressing CD3. NK cells expressing CD3 can be pre-complexed ex vivo with bispecific antibodies to redirect their specificity to a target antigen and/or combined in vivo. In diagnostic embodiments, the labeled NK cells may be loaded with any kind of bispecific or multispecific antibody, including antibodies comprising at least an anti-CD 3 antibody, and the loaded labeled NK cells may be monitored for trafficking to the site of the target antigen to which the other antibody on the bispecific or multispecific antibody binds.

II compositions of the invention

The present invention relates to a composition comprising at least modified NK cells expressing at least part of the TCR/CD3 complex. In some cases, the composition further includes bispecific or multispecific antibodies, including in the same formulation, although in alternative embodiments, NK cells and antibodies are used as physically separate compositions.

NK cell TCR/CD3 modification

In particular embodiments, provided herein are compositions comprising NK cells artificially modified to express part or all of a TCR receptor complex and part or all of a CD3 co-receptor complex. In particular embodiments, NK cells are modified to include all components of the CD3 complex, including cd3ζ, cd3ε, cd3γ, and cd3δ. Although in particular cases full length cd3ζ, cd3ε, cd3γ, and cd3δ are used, including their extracellular, transmembrane, and intracellular domains, in alternative embodiments only portions of one or more of cd3ζ, cd3ε, cd3γ, and cd3δ are used, each of which may or may not be combined with one or more intracellular signaling domains, such as CD16, NKG2D, DAP, DAP12, CD28, 41BB, 2B4, CD27, OX40, or any combination thereof. NK cells may also be modified to express TCR receptor complexes, although in alternative embodiments no component of the TCR receptor complex is used.

In certain embodiments, an amino acid sequence (e.g., a polypeptide) may comprise an amino acid 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 (gin, 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).

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, or 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).

In certain embodiments, any specific sequence of a CD3 receptor component is used, including wild-type or mutant of the component, provided that the CD3 receptor with the mutant is capable of allowing signaling through the CD3 complex, thereby resulting in activation and killing of the target. In some cases, NK cells are modified using examples of the following sequences of CD3 epsilon, CD3 delta, CD3 gamma, and CD3 zeta.

CD3 ε (UniProtKB-P07766 (CD3E_human))

Signal peptides

Extracellular domain

sp|P07766|23-126

Transmembrane domain

sp|P07766|127-152

Intracellular domains

sp|P07766|153-207

One example of Homo sapiens CD3E molecule (CD 3E) mRNA is located in NCBI reference sequence: accession number NM-000733.4

Examples of the entirety of the respective nucleic acid and amino acid CD3 epsilon sequences are as follows (underlined refers to the signal peptide sequence):

CD3 delta (UniProtKB-P04234 (CD3D_human))

Signal peptides

Extracellular domain

sp|P04234|22-105

Transmembrane domain

sp|P04234|106-126

Intracellular domains

sp|P04234|127-171

Chile CD3d molecule, delta (CD 3-TCR complex), mRNA (cDNA clone MGC:88324IMAGE: 3041045), intact CDsBC070321.1

Examples of the entirety of the respective nucleic acid and amino acid CD3 delta sequences are as follows (underlined refers to the signal peptide sequence):

CD3 gamma (T cell surface glycoprotein CD3 gamma chain Gene CD3G P09693)

Signal peptides

Extracellular domain

sp|P09693|23-116

Transmembrane domain

sp|P09693|117-137

Intracellular domains

sp|P09693|138-182

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Homo sapiens CD3G molecule (CD 3G), mRNA; NM-000073.3:81-629 Chinesian CD3G molecule (CD 3G), mRNA

Examples of the entirety of the respective nucleic acid and amino acid CD3 gamma sequences are as follows (underlined refers to the signal peptide sequence):

CD3ζ

Signal peptides

sp|P20963|SP

Extracellular domain

sp|P20963|22-30ECD

Transmembrane domain

sp|P20963|31-51tmd

Intracellular domains

sp|P20963|52-164ICD

Examples of the entirety of the respective nucleic acid and amino acid cd3ζ sequences are as follows (underlined refers to the signal peptide sequences):

homo sapiens CD247 molecule (CD 247; also referred to as CD3 zeta), transcript variant 1, mRNA

NCBI reference sequence NM-198053.3

NM-198053.3:65-559 Chile CD247 molecule (CD 247), transcript variant 1, mRNA

In particular embodiments, NK cells are modified to express one or more of a TCR a chain, a TCR β chain, a TCR γ chain, and a TCR δ, and any combination thereof may be used. In particular cases, NK cells are modified to express a T Cell Receptor (TCR) αβ chain or a tcrγδ chain. In certain instances, NK cells are modified to express part or all of only the constant regions of one or more of the TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain. NK cells may be modified to express part or all of the constant region only of the T Cell Receptor (TCR) αβ chain or tcrγδ chain. Where a partial constant region is used, the portion of the constant region can be at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids, including consecutive amino acids of any constant region. Portions of the constant region can comprise 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.

In certain instances, any of the sequences contained herein are used to modify NK cells, although in other instances, sequences related to these in terms of identity are used. For example, related sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to any of the sequences contemplated herein may be used in the present invention.

Specific constructs for expressing various TCR/CD3 proteins in NK cells may be used, and may be used in various configurations. In particular instances, NK cells can be transduced or transfected with one or more vectors to express any of a variety of proteins encompassed herein, including at least one or more components of the TCR/CD3 complex. In certain cases, one or more of the vectors may or may not themselves be polycistronic, as they ultimately produce more than one isolated polypeptide. Where one or more polycistronic vectors are used, they may utilize one or more Internal Ribosome Entry Sites (IRES) and/or one or more sites for 2A self-cleaving peptides. Where one or more 2A sequences are used, the following sequences may be used, where GSG is an optional linker:

In the case of polycistronic vectors expressing multiple protein components, the order of the 5 'to 3' direction on the polynucleotide vector may be any order, although in the alternative they are present on the vector in a particular order. The polycistronic vector may express the multiple components of the CD3 receptor complex without expressing other heterologous proteins, or the polycistronic vector may express the multiple components of the CD3 receptor complex and one or more other heterologous proteins. The polycistronic vector may express the multiple components of the TCR receptor complex without expressing other heterologous proteins, or the polycistronic vector may express the multiple components of the TCR receptor complex and one or more other heterologous proteins. The polycistronic 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 particular embodiments, the polycistronic vector comprises one or more components of a CD3 receptor complex and one or more heterologous proteins, such as cytokines and engineered antigen receptors, such as CARs.

An example of a polycistronic vector is shown in FIG. 2A, in which full-length CD3 epsilon, CD3 delta, CD3 gamma and CD3 zeta are present and separated by the same or different sites of the 2A self-cleaving peptide. As shown in the plasmid map of fig. 2B, the polycistronic vector may comprise signal peptides, extracellular domains, transmembrane domains, and intracellular domains for each of CD3 epsilon, CD3 delta, CD3 gamma, and CD3 zeta.

FIG. 3 provides a table showing examples of various TCR expression constructs for engineering NK cells expressing TCRs. In a particular embodiment of the invention, the CD3 receptor component and the TCR receptor component are expressed in NK cells by different vectors. In any case, the vector may express a TCR directed against a particular antigen (e.g., a cancer antigen or a viral antigen). The TCR may or may not comprise at least part of cd3ζ, including the intracellular domain of cd3ζ, except that NK cells also express cd3ζ as a separate molecule from the TCR and as part of the CD3 receptor complex. Likewise, the CAR may or may not contain at least part of cd3ζ, including the intracellular domain of cd3ζ, except that NK cells also express cd3ζ as part of the molecule and CD3 receptor complex that is separate from the TCR.

In particular embodiments, the TCR of the modified NK cell is not necessarily used as an aspect of the cell's therapy, but rather as a structural support or scaffold to facilitate the function or enhanced function of the CD3 receptor complex. That is, the TCR may be any TCR, and may not be used to target the ability of a particularly desired antigen. In this case, as an example, TCRs targeting viral antigens may be used for NK cells, which will be used for cancers not necessarily associated with that particular virus. In other cases, TCRs are selected for their ability to target a particular cancer antigen. Examples of antigens against which TCRs may be directed are provided elsewhere herein.

In fig. 3, an example of the following constructs is noted:

TCR1: refers to TCRpp65 (TCR for HLA-A2 restricted CMVpp 65) linked to the intracellular cd3ζ domain and full-length cd3γ, full-length cd3δ and full-length cd3ε, and this construct may also be referred to as TCRpp65ZicdGDEFL, which may comprise the following sequences:

MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:39)

In TCRpp65ZicdGDEFL, the sequences of the corresponding components are as follows, although these specific sequences or other sequences may be used in this and/or other constructs:

TCRb ectodomain:

MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD(SEQ ID NO:40)

ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO:41)

cd3ζ intracellular domain (Z-ICD):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO:42)( Wherein the P2A sequence is located at the C-terminal end

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc(SEQ ID NO:43)( Wherein the lower case letter sequence is a P2A sequence

TCRa extracellular domain:

MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS(SEQ ID NO:44)

ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO:45)

CD3γδε(CD3GDE)：

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:46)( Wherein the E2A sequence is located at the C-terminal end

ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGcagtgtactaattatgctctcttgaaattggctggagatgttgagagcaatcccgggccc(SEQ ID NO:47)( Wherein the lower case letter is an E2A sequence)

IL-15：

MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:48)

ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

TCR2 is TCRpp attached to full length CD3 zeta, full length CD3 gamma, full length CD3 delta and full length CD3 epsilon; it lacks IL-15. Representative sequences are as follows:

CTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGACGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCCATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCG(SEQ ID NO:50)

LEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSEGRGSLLTCGDVEENPGPMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGP(SEQ ID NO:51)

TCR 3-TCRpp which is linked to the intracellular CD3z domain and IL-15-can also be referred to as TCRpp65 Zicd-its representative sequence is as follows:

MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:52)

In TCRpp65Zicd, the sequences of the corresponding components are as follows, although these specific sequences or other sequences may be used for this and/or other constructs:

TCRb ectodomain:

Cd3ζ intracellular domain (Z-ICD):

TCRa extracellular domain:

Cd3ζ intracellular domain (Z-ICD) (in particular embodiments, two or more Z-ICD sequences may be used):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:53)

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:54)

IL-15:

TCR 4-TCRpp-also known as TCRpp. Beta. Alpha., representative sequence is as follows :MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:55)

For TCRpp beta alpha, the sequences of the corresponding components are as follows, although these specific sequences or other sequences may be used for this and/or other constructs:

TCRb ectodomain

Cd3ζ intracellular domain (Z-ICD):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO:42)AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:54)

TCRa extracellular domain:

cd3ζ intracellular domain (Z-ICD):

IL-15：

Another representative sequence of TCRpp βα is as follows:

ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGAGTTATCCAGTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGTTCGGCAAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCCAGTGATGTAGGCATCTACTTCTGTGCTGGACCCATGAAAACCTCCTACGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:56)

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSSANYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFEGRGSLLTCGDVEENPGPMLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGPMKTSYDKVIFGPGTSLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*(SEQ ID NO:57)

z1 refers to full length cd3ζ, full length cd3γ, full length cd3δ, and full length cd3ε (see fig. 2A and 2B), also referred to as CD3ZFLGDEFL15, linked to IL15, and representative sequences are as follows:

MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:58)

ATGCTCGAGATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:59)

Z2: refers to full length cd3ζ, full length cd3γ, full length cd3δ, and full length cd3ε, also referred to as CD3ZGDEFLSP821CD28, linked to membrane-bound IL21 (membrane-bound IL21 has a CD8 transmembrane domain), representative sequences are as follows:

MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRICLTSDRLAPAAGLAAPRRQAVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWV*(SEQ ID NO:60)

For CD3ZGDEFLSP821CD28, the sequences of the corresponding components are as follows, although these specific sequences or other sequences may be used for this and/or other constructs:

CD3：

MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:61)

ATGCTCGAGATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCC(SEQ ID NO:62)

SP CD8：

MRICLTSDRLAPAAGLAAPRRQAV(SEQ ID NO:63)

atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg(SEQ ID NO:64)

IL-21:

HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:65)

CATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAACGGATAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT(SEQ ID NO:66)

CD8 hinge:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:67)

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ ID NO:68)

CD28 transmembrane domain:

FWVLVVVGGVLACYSLLVTVAFIIFWV*(SEQ ID NO:69)

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCC TTTATTATTTTCTGGGTG(SEQ ID NO:70)

Z3: refers to full length cd3ζ, full length cd3γ, full length cd3δ, and full length cd3ε, also referred to as CD3ZGDEFL SP21CD8, linked to membrane bound IL21 (membrane bound IL21 has a CD28 transmembrane domain), which is represented by the following sequence:

MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRICLTSDRLAPAAGLAAPRRQAVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT(SEQ ID NO:71).

for CD3ZGDEFL8SP21CD8, the sequences of the corresponding components are as follows, although these specific sequences or other sequences may be used for this and/or other constructs:

CD3：

MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:61)ATGCTCGAGATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCC(SEQ ID NO:62)

SP CD8:

MRICLTSDRLAPAAGLAAPRRQAV(SEQ ID NO:63)

IL-21：

cataaatcttcctctcaaggtcaggaccgccatatgattcgaatgcggcagctgattgacatagtcgatcaactgaagaactatgtgaatgatcttgtgcccgagtttttgccagcccctgaagacgtagaaactaattgtgagtggagtgccttttcctgctttcaaaaggcacagctgaaatccgccaacacgggcaataacgaacggataattaacgtatccattaagaagctgaagcggaagccgccctcaaccaatgcgggacggcggcaaaagcatcgcttgacctgtccgtcatgcgacagctacgagaaaaagcccccgaaggagttcttggaacgcttcaagagtctccttcagaaaatgattcaccagcacctgtcctcacggacgcacggaagcgaggacagt(SEQ ID NO:65)

CD8 hinge:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:67)

CD8 transmembrane domain:

IYIWAPLAGTCGVLLLSLVIT*(SEQ ID NO:72)

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC(SEQ ID NO:73)

in certain embodiments, the CD3 constructs provided herein comprise fusions having intracellular co-stimulatory domains derived from CD16, NKG2D, DAP, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, or any combination thereof. In certain embodiments, the intracellular co-stimulatory domain is fused to cd3δ, cd3ε, cd3γ, and/or cd3ζ. In certain embodiments, such CD3 fusion constructs comprise cd3ζ fused to a DAP10 intracellular co-stimulatory domain. In certain embodiments, such a CD3 fusion construct comprises cd3ζ fused to a CD28 intracellular co-stimulatory domain. In certain embodiments, such CD3 fusion constructs comprise cd3ζ fused to DAP10 and CD28 intracellular co-stimulatory domains. In certain embodiments, CD3ζ fused to an intracellular co-stimulatory domain of DAP10 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, CD3 zeta fused to the intracellular co-stimulatory domain of CD28 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, the cd3ζ fused to the DAP10 intracellular co-stimulatory domain and the CD28 intracellular co-stimulatory 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 intracellular co-stimulatory domain of DAP10 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, CD3 zeta fused to the intracellular co-stimulatory domain of CD28 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, the cd3ζ fused to the DAP10 intracellular co-stimulatory domain and the CD28 intracellular co-stimulatory 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, the cd3ζ fused to the intracellular domain may not comprise the C-terminal 2A domain. In certain embodiments, the cd3ζ fused to the intracellular domain may not comprise 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)

In certain embodiments, the intracellular co-stimulatory domain of DAP10 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 co-stimulatory 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 co-stimulatory domain and the CD28 intracellular co-stimulatory domain are represented by nucleotide 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. 114. In certain embodiments, the intracellular co-stimulatory domain of DAP10 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 co-stimulatory 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 co-stimulatory domain and the CD28 intracellular co-stimulatory 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)

UTNK15-DAP10: refers to a fusion of full length cd3ζ, comprising full length cd3δ, and full length cd3δ linked to IL15 with an intracellular co-stimulatory domain derived from DAP10, which can 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, UTNK-DAP 10 amino acid sequences can be 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 119 .ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:118)

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQ.DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:119)

UTNK15-28: refers to a fusion of full length cd3ζ comprising a full length cd3ζ and an intracellular co-stimulatory domain derived from CD28, full length cd3γ, full length cd3δ, and full length cd3ε linked to IL15, 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 UTNK 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.

ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:120)

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQ.DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:121)

UTNK15-28-DAP10: refers to a fusion of full length cd3ζ comprising 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 with an intracellular co-stimulatory domain derived from DAP10 and an intracellular co-stimulatory domain derived from CD28, full length cd3γ, full length cd3δ, and full length cd3ε linked to IL 15. In certain embodiments, UTNK-28-DAP 10 amino acid sequences can be 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. 123.

ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCCATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:122)

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAI ILLQGTLAQS IKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATI SGFLFAEIVS IFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKI PIEELEDRVFVNCNTS ITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGI IVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQ.DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:123)

As shown in FIG. 3 and described above, the term "linked" refers to the presence on the same polynucleotide vector and does not necessarily mean that two polypeptides are expressed as one polypeptide. For example, cytokines produced by the vectors of the invention may ultimately be produced as isolated molecules from components of any one or more of the TCR/CD3 receptor complexes. However, the term "fused" or "fusion" refers to two polypeptides comprising peptide bonds linking two molecules, i.e., the two polypeptides are covalently bound by an amide bond and are not separated by a cleavage element (e.g., a 2A element).

One specific example of a TCR that may be used in a cell is an NY-ESO TCR, specific examples of sequences include at least the following:

TCRα:

XQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:25)

TCRβ:

GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:26)

In certain embodiments, the TCR may comprise a TCR a 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.

In certain embodiments, the TCR may comprise a TCR a 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.

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In certain embodiments, the TCR may comprise a TCR a 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.

In certain embodiments, the TCR may comprise a TCR a chain variable region 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. 88.

In certain embodiments, a TCR may comprise an amino acid sequence of a TCR a 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.

In certain embodiments, the TCR can comprise an amino acid sequence of the alpha chain CDR1 that is at least or exactly 80% or 100% identical to SEQ ID NO 90.

In certain embodiments, the TCR can comprise an amino acid sequence of the alpha chain CDR2 that is at least or exactly 80% or 100% identical to SEQ ID NO 91.

In certain embodiments, the TCR can comprise an amino acid sequence of the alpha chain CDR3 that is at least or exactly 80% or 100% identical to SEQ ID NO. 92.

In certain embodiments, the TCR may comprise a TCR β 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. 93.

In certain embodiments, the TCR may comprise a TCR β 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.

In certain embodiments, the TCR may comprise a TCR β 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.

In certain embodiments, the TCR may comprise a TCR β chain variable region 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. 96.

In certain embodiments, a TCR may comprise a TCR β chain constant region 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. 97.

In certain embodiments, the TCR can comprise an amino acid sequence of the β chain CDR1 that is at least or exactly 80% or 100% identical to SEQ ID NO 98.

In certain embodiments, the TCR can comprise an amino acid sequence of the β chain CDR2 that is at least or exactly 80% or 100% identical to SEQ ID NO 99.

In certain embodiments, the TCR can comprise an amino acid sequence of the β chain CDR3 that is at least or exactly 80% or 100% identical to SEQ ID NO. 100.

In certain embodiments, the TCR (e.g., TCR α, β, δ, and/or γ) chains can comprise 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.

In certain embodiments, the TCR recognizes a peptide corresponding to amino acid residues 157-165 of human cancer testis Ag NY-ESO-1 in the context of an HLA-A 02I allele. In certain embodiments, the TCR can target an epitope characterized by the amino acid sequence of SEQ ID NO. 105.

One specific example of a TCR that can be used in a cell is TCRpp65 a, and specific examples of sequences include at least the following (underlined refers to the sequence of the signal peptide):

One specific example of a TCR that can be used in a cell is TCRpp65 β, and specific examples of sequences include at least the following (underlined refers to the sequence of the signal peptide):

TCRpp65ZFLGDEFL15

In certain embodiments, constructs in which TCRpp is linked to full length cd3ζ, full length cd3γ, full length cd3δ, full length cd3ε, and also to IL-15 (and may be referred to as TCRpp65ZFLGDEFL 15) may be utilized. One representative sequence of such a construct is as follows:

MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSEGRGSLLTCGDVEENPGPMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:74).

In TCRpp65ZFLGDEFL, the sequences of the corresponding components are as follows, although these specific sequences or other sequences may be used for this other construct and/or other constructs:

TCRb ectodomain:

MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADATNFSLLKQAGDVEENPGP(SEQ ID NO:75)( And includes a P2A sequence at its C-terminus

ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO:76)

TCRa extracellular domain:

MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSEGRGSLLTCGDVEENPGP(SEQ ID NO:77)( And includes a T2A sequence at its C-terminus

ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC(SEQ ID NO:78)

TCR5: designated TCRCgdZFLGDEFL, is the constant region of tcrγ and δ linked to full length cdζ, full length cd3γ, full length cd3δ and full length cd3ε; IL-15. Representative sequences are as follows:

TCR constant gamma delta (TCRCgd)

ATGCGGTGGGCCCTACTGGTGCTTCTAGCTTTCCTGTCTCCTGCCAGTCAGGATAAACAACTTGATGCAGATGTTTCCCCCAAGCCCACTATTTTTCTTCCTTCGATTGCTGAAACAAAACTCCAGAAGGCTGGAACATACCTTTGTCTTCTTGAGAAATTTTTCCCAGATATTATTAAGATACATTGGCAAGAAAAGAAGAGCAACACGATTCTGGGATCCCAGGAGGGGAACACCATGAAGACTAACGACACATACATGAAATTTAGCTGGTTAACGGTGCCAGAAGAGTCACTGGACAAAGAACACAGATGTATCGTCAGACATGAGAATAATAAAAACGGAATTGATCAAGAAATTATCTTTCCTCCAATAAAGACAGATGTCACCACAGTGGATCCCAAATACAATTATTCAAAGGATGCAAATGATGTCATCACAATGGATCCCAAAGACAATTGGTCAAAAGATGCAAATGATACACTACTGCTGCAGCTCACAAACACCTCTGCATATTACACGTACCTCCTCCTGCTCCTCAAGAGTGTGGTCTATTTTGCCATCATCACCTGCTGTCTGCTTAGAAGAACGGCTTTCTGCTGCAATGGAGAGAAATCAGGAAGCGGAGCTACTAACTTTAGCCTGCTGAAGCAGGCTGGAGATGTGGAGGAGAACCCTGGACCTATGATTCTTACTGTGGGCTTTAGCTTTTTGTTTTTCTACAGGGGCACGCTGTGTAGTCAGCCTCATACCAAACCATCCGTTTTTGTCATGAAAAATGGAACAAATGTCGCTTGTCTGGTGAAGGAATTCTACCCCAAGGATATAAGAATAAATCTCGTGTCATCCAAGAAGATAACAGAGTTTGATCCTGCTATTGTCATCTCTCCCAGTGGGAAGTACAATGCTGTCAAGCTTGGTAAATATGAAGATTCAAATTCAGTGACATGTTCAGTTCAACACGACAATAAAACTGTGCACTCCACTGACTTTGAAGTGAAGACAGATTCTACAGATCACGTAAAACCAAAGGAAACTGAAAACACAAAGCAACCTTCAAAGAGCTGCCATAAACCCAAAGCCATAGTTCATACCGAGAAGGTGAACATGATGTCCCTCACAGTGCTTGGGCTACGAATGCTGTTTGCAAAGACTGTTGCCGTCAATTTTCTCTTGACTGCCAAGTTATTTTTCTTGTAA(SEQ ID NO:81)

MRWALLVLLAFLSPASQDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKSGSGATNFSLLKQAGDVEENPGPMILTVGFSFLFFYRGTLCSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL(SEQ ID NO:82)

CD3:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:79)

ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAGGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCCATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATCGGACCGCAGTGTACTAATTATGCTCTCTTGAAATTGGCTGGAGATGTTGAGAGCAATCCCGGGCCC(SEQ ID NO:80)

IL-15：

TCR6: also known as TCRCabZFLGDEFL, is the constant region of tcra and β, linked to full length cd3ζ, full length cd3γ, full length cd3δ and full length cd3ε; IL-15. Representative sequences are as follows:

TCR constant alpha-beta (TCRCab)

METLLGLLILWLQLQWVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGATNFSLLKQAGDVEENPGPMSIGLLCCAALSLLWAGPVNADLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:83)

ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCGGAAGCGGAGCTACTAACTTTAGCCTGCTGAAGCAGGCTGGAGATGTGGAGGAGAACCCTGGACCTATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGACCTGAAAAACGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAA(SEQ ID NO:84)

CD3：

IL-15：

In some embodiments, the TCR construct comprises an NY ESO-specific TCR and a CD8 a/β co-receptor molecule. In some embodiments, such constructs may comprise a TCR a chain variable region signal peptide, a TCR a chain variable region, a TCR a chain constant region, a 2A element (e.g., a P2A element), a TCR β chain variable region signal peptide, a TCR β chain variable region, a TCR β chain constant region, a 2A element (e.g., an E2A element), a CD8- β polypeptide, a 2A element (e.g., a T2A element), and a CD 8-a polypeptide. In some embodiments, a TCR construct comprising NY ESO-specific TCR and CD8 a/β co-receptor molecule nucleotide coding sequences 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 NY ESO-specific TCR and a CD8 a/β 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.

In some embodiments, the CD 8a co-receptor molecule is transcriptionally linked to any TCR molecule disclosed herein. In some embodiments, the nucleotide coding sequence of the CD 8. Alpha. Co-receptor molecule 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 nucleotide coding sequence of the CD8 beta co-receptor molecule 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 amino acid sequence of the CD 8. Alpha. Co-receptor 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 amino acid sequence of the CD8 beta co-receptor 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.

ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAG

GAGGTGACACAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGC

AGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTC

ACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCG

CTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCC

ACCTACCTCTGTGCTGTGAGGCCCCTTTATGGAGGAAGCTACATACCTACATTTGGAAGAGGA

ACCAGCCTTATTGTTCATCCGTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGAC

TCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCA

CAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGAC

TTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTC

AACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAG

CTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGG

TTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCC

AGCGGAAGCGGAGCTACTAACTTTAGCCTGCTGAAGCAGGCTGGAGATGTGGAGGAGAACCCT

GGACCTATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTG

AATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTG

CAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGG

CTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGC

TACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCC

CAGACATCTGTGTACTTCTGTGCCAGCAGTTACGTCGGGAACACCGGGGAGCTGTTTTTTGGA

GAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAAACGTGTTCCCACCCAAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCAGTGGACAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCCATGGCCTTGCCCGTCACTGCGCTTTTGCTCCCGCTCGCTCTTCTCCTGCATGCAGCCCGACCATCTCAATTTAGAGTTTCTCCACTCGACAGGACGTGGAACCTCGGCGAAACCGTCGAACTTAAATGTCAAGTACTTCTCTCAAATCCGACTTCTGGTTGCTCATGGCTCTTTCAGCCGAGAGGAGCAGCTGCCAGCCCCACCTTCCTGCTGTATCTCTCCCAGAACAAGCCGAAGGCCGCCGAAGGGCTCGATACTCAACGATTTAGCGGGAAGCGACTCGGGGACACGTTCGTTCTTACTCTCAGCGATTTTAGAAGAGAGAACGAGGGATATTATTTTTGTTCCGCACTCTCTAACAGCATCATGTACTTCAGTCATTTTGTACCAGTCTTTCTCCCTGCAAAACCAACGACTACTCCAGCACCAAGACCGCCCACTCCCGCACCTACTATTGCAAGCCAACCTTTGAGTCTCCGACCAGAGGCATGCAGACCTGCTGCTGGAGGTGCAGTACATACGCGAGGGTTGGATTTTGCCTGCGATATCTATATCTGGGCCCCCTTGGCCGGCACGTGCGGGGTGCTCCTGCTGAGTCTCGTAATTACTCTTTATTGTAATCATAGAAACCGCAGAAGGGTGTGTAAGTGTCCCCGGCCTGTCGTGAAAAGCGGGGATAAGCCCAGTTTGTCTGCTCGGTACGTCGGAAGCGGTGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGACCCATGAGGCCACGACTTTGGCTGCTGCTCGCTGCACAGTTGACTGTACTGCATGGCAATAGTGTGTTGCAGCAGACACCTGCATACATCAAGGTTCAGACAAATAAGATGGTTATGCTGAGTTGCGAGGCAAAAATTAGTTTGAGCAATATGCGGATCTACTGGTTGCGACAGAGACAGGCTCCCAGTAGTGATAGTCACCACGAATTCCTGGCTCTTTGGGATTCCGCAAAAGGAACGATTCATGGGGAAGAAGTAGAGCAGGAGAAGATTGCGGTTTTCCGCGATGCATCTCGCTTTATCCTTAATCTTACATCCGTTAAGCCTGAGGACAGTGGGATCTATTTTTGTATGATTGTAGGGTCCCCCGAATTGACATTTGGGAAGGGTACGCAGCTCTCCGTAGTTGACTTTCTGCCCACAACGGCACAACCCACTAAGAAGTCCACCCTGAAGAAGCGCGTCTGTCGCTTGCCCAGACCTGAAACCCAAAAGGGTCCACTCTGTTCCCCTATAACCCTGGGGTTGTTGGTGGCGGGCGTCTTGGTCCTGCTTGTTAGCTTGGGCGTAGCCATTCATCTGTGTTGCCGAAGACGCAGAGCCCGACTTAGATTTATGAAGCAATTCTATAAGTGA(SEQ ID NO:124)

METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGATNFSLLKQAGDVEENPGPMSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGSGQCTNYALLKLAGDVESNPGPMALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYVGSGEGRGSLLTCGDVEENPGPMRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK*(SEQ ID NO:125)

ATGAGGCCACGACTTTGGCTGCTGCTCGCTGCACAGTTGACTGTACTGCATGGCAATAGTGTG

TTGCAGCAGACACCTGCATACATCAAGGTTCAGACAAATAAGATGGTTATGCTGAGTTGCGAG

GCAAAAATTAGTTTGAGCAATATGCGGATCTACTGGTTGCGACAGAGACAGGCTCCCAGTAGT

GATAGTCACCACGAATTCCTGGCTCTTTGGGATTCCGCAAAAGGAACGATTCATGGGGAAGAA

GTAGAGCAGGAGAAGATTGCGGTTTTCCGCGATGCATCTCGCTTTATCCTTAATCTTACATCC

GTTAAGCCTGAGGACAGTGGGATCTATTTTTGTATGATTGTAGGGTCCCCCGAATTGACATTT

GGGAAGGGTACGCAGCTCTCCGTAGTTGACTTTCTGCCCACAACGGCACAACCCACTAAGAAG

TCCACCCTGAAGAAGCGCGTCTGTCGCTTGCCCAGACCTGAAACCCAAAAGGGTCCACTCTGT

TCCCCTATAACCCTGGGGTTGTTGGTGGCGGGCGTCTTGGTCCTGCTTGTTAGCTTGGGCGTA

GCCATTCATCTGTGTTGCCGAAGACGCAGAGCCCGACTTAGATTTATGAAGCAATTCTATAAGTGA(SEQ ID NO:126)

ATGGCCTTGCCCGTCACTGCGCTTTTGCTCCCGCTCGCTCTTCTCCTGCATGCAGCCCGACCA

TCTCAATTTAGAGTTTCTCCACTCGACAGGACGTGGAACCTCGGCGAAACCGTCGAACTTAAA

TGTCAAGTACTTCTCTCAAATCCGACTTCTGGTTGCTCATGGCTCTTTCAGCCGAGAGGAGCA

GCTGCCAGCCCCACCTTCCTGCTGTATCTCTCCCAGAACAAGCCGAAGGCCGCCGAAGGGCTC

GATACTCAACGATTTAGCGGGAAGCGACTCGGGGACACGTTCGTTCTTACTCTCAGCGATTTT

AGAAGAGAGAACGAGGGATATTATTTTTGTTCCGCACTCTCTAACAGCATCATGTACTTCAGT

CATTTTGTACCAGTCTTTCTCCCTGCAAAACCAACGACTACTCCAGCACCAAGACCGCCCACT

CCCGCACCTACTATTGCAAGCCAACCTTTGAGTCTCCGACCAGAGGCATGCAGACCTGCTGCT

GGAGGTGCAGTACATACGCGAGGGTTGGATTTTGCCTGCGATATCTATATCTGGGCCCCCTTG

GCCGGCACGTGCGGGGTGCTCCTGCTGAGTCTCGTAATTACTCTTTATTGTAATCATAGAAAC

CGCAGAAGGGTGTGTAAGTGTCCCCGGCCTGTCGTGAAAAGCGGGGATAAGCCCAGTTTGTCTGCTCGGTACGTC(SEQ ID NO:127)

MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSS

DSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTF

GKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK(SEQ ID NO:128)

MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGA

AASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFS

HFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV(SEQ ID NO:129)

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 TCR a and TCR β chains found in PRAME-specific TCR clone 46, clone 54, and/or clone DSK 3. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises TCR alpha and TCR beta chains that target PRAME epitope SLLQHLIGL (SEQ ID NO: 131) and/or QLLALLPSL (SEQ ID NO: 132).

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 tcra) and/or SEQ ID NO:134 (e.g., TCR clone 46 tcra). 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 tcra) and/or SEQ ID NO:136 (e.g., TCR clone 46 tcra).

ATGCTTCTGGAACACCTGCTGATTATCCTGTGGATGCAACTCACGTGGGTCTCCGGGCAACAACTGAATCAAAGCCCCCAATCCATGTTTATACAGGAGGGAGAGGACGTAAGTATGAATTGCACATCTTCATCTATCTTTAACACCTGGCTGTGGTACAAACAAGACCCCGGAGAAGGTCCTGTACTTCTCATCGCACTTTACAAAGCAGGTGAGCTTACCAGTAACGGGAGACTCACCGCACAGTTCGGTATTACAAGAAAGGATTCCTTTCTCAACATCTCCGCTTCTATCCCTTCAGACGTCGGAATTTATTTTTGTGCTGGTATCCCTCGAGACAATTACGGTCAAAACTTTGTATTTGGGCCTGGGACTCGGCTGTCAGTTTTGCCGTATATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:133)

ATGGGCATTAGGCTGCTGTGCAGAGTAGCATTTTGCTTTCTGGCAGTAGGATTGGTCGATGTAAAGGTTACACAGTCCTCACGGTACTTGGTAAAGCGCACTGGTGAAAAGGTCTTTCTGGAATGTGTACAAGATATGGATCACGAAAATATGTTTTGGTACAGGCAAGATCCCGGCCTTGGACTTAGACTGATATATTTCTCCTACGATGTTAAAATGAAGGAGAAGGGCGATATTCCAGAAGGATATTCCGTGAGCCGCGAAAAGAAGGAGCGATTCAGTTTGATACTCGAAAGTGCCTCCACAAACCAAACCTCTATGTACCTTTGCGCGTCAACGCCGTGGCTGGCCGGTGGCAATGAACAATTCTTCGGGCCGGGTACGCGCCTCACTGTCCTGGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:134)

MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGIPRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:135)

MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTPWLAGGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:136)

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 tcra) and/or SEQ ID NO:138 (e.g., TCR clone 54 tcra). 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 tcra) and/or SEQ ID NO:140 (e.g., TCR clone 54 tcra).

ATGCTGCTGCTGCTGGTGCCCGTGCTGGAAGTGATCTTCACCCTGGGCGGCACCAGAGCCCAGAGCGTGACACAGCTGGGCAGCCACGTGTCCGTGTCTGAGAGGGCCCTGGTGCTGCTGAGATGCAACTACTCTTCTAGCGTGCCCCCCTACCTGTTTTGGTACGTGCAGTACCCCAACCAGGGGCTGCAGCTGCTCCTGAAGTACACCAGCGCCGCCACACTGGTGAAGGGCATCAACGGCTTCGAGGCCGAGTTCAAGAAGTCCGAGACAAGCTTCCACCTGACCAAGCCCAGCGCCCACATGTCTGACGCCGCCGAGTACTTCTGTGCCGTGAGCGGCCAGACCGGCGCCAACAACCTGTTCTTCGGCACCGGCACCCGGCTGACAGTGATCCCTTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:137)ATGGGCTTCCGGCTGCTGTGCTGCGTGGCCTTTTGTCTGCTGGGAGCCGGACCTGTGGATAGCGGCGTGACCCAGACCCCCAAGCACCTGATCACCGCCACCGGCCAGAGAGTGACCCTGCGCTGCAGCCCTAGAAGCGGCGACCTGAGCGTGTACTGGTATCAGCAGAGCCTCGACCAGGGCCTGCAGTTCCTGATCCAGTACTACAACGGCGAGGAACGGGCCAAGGGCAACATCCTGGAACGGTTCAGCGCCCAGCAGTTCCCCGATCTGCACAGCGAGCTGAACCTGAGCAGCCTGGAACTGGGCGACAGCGCCCTGTACTTCTGCGCCAGCGCCAGATGGGATAGAGGCGGCGAGCAGTACTTCGGCCCTGGCACCAGACTGACCGTGACCGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:138)

MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSERALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSGQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:139)

MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASARWDRGGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:140)

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 tcra) and/or SEQ ID NO:142 (e.g., TCR clone DSK3 tcra). 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 tcra) and/or SEQ ID NO:144 (e.g., TCR clone DSK3 tcra).

ATGAAGAGCCTGAGGGTACTGCTGGTGATATTGTGGCTTCAGCTTAGTTGGGTCTGGTCACAACAAAAGGAAGTTGAGCAAAACTCAGGACCACTGAGTGTACCCGAGGGCGCTATAGCATCACTGAACTGTACCTACTCAGATCGGGGAAGCCAATCCTTTTTCTGGTACAGACAGTATTCCGGGAAGAGTCCTGAGTTGATCATGTTTATATACTCCAATGGCGATAAGGAGGATGGACGCTTCACCGCTCAGCTTAATAAAGCGTCACAGTATGTATCCCTCCTGATTCGGGACTCACAACCATCTGACTCTGCAACATACCTTTGTGCCGTAAAGGACAACGCCGGGAACATGCTCACTTTTGGAGGAGGTACCCGGCTTATGGTAAAACCACATATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:141)

MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKDNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:142)

ATGGGATTCCGGCTTCTTTGTTGTGTGGCATTTTGTCTGTTGGGTGCGGGTCCAGTCGATAGTGGTGTAACTCAGACACCAAAACACCTTATCACGGCAACTGGGCAACGAGTGACGCTCCGCTGTAGCCCGAGGTCCGGTGATTTGAGTGTGTACTGGTACCAGCAATCTTTGGACCAGGGCTTGCAGTTCCTCATACAGTATTACAATGGTGAAGAAAGAGCGAAGGGTAATATCCTGGAAAGATTCTCCGCACAACAGTTTCCTGATCTCCACAGCGAACTGAACCTGAGTTCTCTCGAGCTCGGGGATAGTGCTTTGTACTTCTGCGCGTCATCCGACGGTGGCGGAGTCTATGAACAATATTTCGGCCCAGGGACTAGGCTTACGGTGACGGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:143)

MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSDGGGVYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:144)

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 TCR a and TCR β chains found in 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 TCR alpha and TCR beta chains that target PRAME epitope LYVDSLFFL (SEQ ID NO: 167). In some embodiments, PRAME-specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in international patent application publication WO 2022/063966 A1, which is incorporated herein by reference for the 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.

ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTGTGGCAGCTGACCTGGGTCCGATCT

CAGCAGCCTGTTCAGTCTCCTCAGGCCGTGATCCTGAGAGAAGGCGAGGACGCCGTGATCAAC

TGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCGAGGCCCCT

GTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGC

TTCAACGAGAAGAAGCAGCAGTCCAGCCTGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGC

ACCTACTTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGGCACCCTGCTGACCGTGAATCCCAAT(SEQ ID NO:145)

ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGAGCCAAGTGAACGGCCAGCAA

GTGATGCAGATCCCTCAGTACCAGCACGTGCAAGAAGGCGAGGACTTCACCACCTACTGCAAC

AGCAGCACCACACTGAGCAACATCCAGTGGTACAAGCAGCGGCCTGGCGGACACCCTGTGTTT

CTGATCCAGCTGGTCAAGTCCGGCGAAGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGC

GAGGCCAAGAAGAACAGCAGCCTGCACATCACCGCCACACAGACCACCGATGTGGGCACCTAC

TTTTGTGCTGGCGCCCTGCCTAGAGCCGGCAGCTATCAACTGACATTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAAC(SEQ ID NO:146)

ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTGTGGCAGCTGACCTGGGTCCGATCT

CAGCAGCCTGTTCAGTCTCCTCAGGCCGTGATCCTGAGAGAAGGCGAGGACGCCGTGATCAAC

TGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCGAGGCCCCT

GTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGC

TTCAACGAGAAGAAGCAGCAGTCCAGCCTGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGC

ACCTACTTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGGC

ACCCTGCTGACCGTGAATCCCAATATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGAC

CCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCC

AAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGAC

AGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTC

AAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAG

TCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:147)

ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGAGCCAAGTGAACGGCCAGCAAGTGATGCAGATCCCTCAGTACCAGCACGTGCAAGAAGGCGAGGACTTCACCACCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTACAAGCAGCGGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGGTCAAGTCCGGCGAAGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAACAGCAGCCTGCACATCACCGCCACACAGACCACCGATGTGGGCACCTACTTTTGTGCTGGCGCCCTGCCTAGAGCCGGCAGCTATCAACTGACATTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAACATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:148)

ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGGACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGTGTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCAGCCAGACCAGCGTGTACTTCTGCGCCATCAGCGACTACGAGGGCACCGAGGCCTTTTTTGGCCAAGGCACAAGACTGACCGTGGTG(SEQ ID NO:149)

ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAATGTACCGTGGAAGGCACCAGCAATCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGACTGCAGCTGCTGTTTTACAGCGTCGGCATCGGCCAGATCAGCAGCGAGGTTCCACAGAATCTGAGCGCCAGCAGACCCCAGGACAGACAGTTTATCCTGAGCAGCAAGAAGCTGCTGCTGAGCGACAGCGGCTTCTACCTGTGTGCTTGGAGCCTCGGAGCCGGCTACACCGACACACAGTATTTTGGCCCTGGCACCAGACTGACCGTGCTG(SEQ ID NO: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)

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSI IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:156)

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSI IPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(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)

In some embodiments, the TCR construct comprises a gp 100-specific TCR chain. In some embodiments, the TCR construct comprising a gp 100-specific TCR chain comprises TCR a and TCR β chains found in gp 100-specific TCR clone Sp (0.01) a and/or modified versions thereof. In some embodiments, a TCR construct comprising a gp 100-specific TCR chain comprises TCR alpha and TCR beta chains targeting the gp100 epitope KTWGQYWQV (SEQ ID NO: 168). In some embodiments, gp 100-specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in patent publication US 8,216,565 B2, which publication is incorporated herein by reference for the purposes described herein.

In some embodiments, a TCR construct comprising a gp 100-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 NO:169 and/or SEQ ID NO: 170. In some embodiments, a TCR construct comprising a gp 100-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.

ATGAAATCCTTGAGTGTTTCCCTAGTGGTCCTGTGGCTCCAGTTAAACTGGGTGAACAGCCAG

CAGAAGGTGCAGCAGAGCCCAGAATCCCTCATTGTCCCAGAGGGAGCCATGACCTCTCTCAAC

TGCACTTTCAGCGACAGTGCTTCTCAGTATTTTGCATGGTACAGACAGCATTCTGGGAAAGCC

CCCAAGGCACTGATGTCCATCTTCTCCAATGGTGAAAAAGAAGAAGGCAGATTCACAATTCAC

CTCAATAAAGCCAGTCTGCATTTCTCGCTACACATCAGAGACTCCCAGCCCAGTGACTCTGCT

CTCTACCTCTGTGCAGCCAATAACTATGCCCAGGGATTAACCTTCGGTCTTGGCACCAGAGTA

TCTGTGTTTCCCTACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCT

CAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATG

GAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGC

AATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACC

AACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAA

ACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(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)

In some embodiments, the TCR construct comprises a MART-1 specific TCR chain. In some embodiments, a TCR construct comprising a MART-1 specific TCR chain comprises TCR a and TCR β chains found in MART-1 specific TCR clone F4 and/or F5 and/or modified versions thereof. In some embodiments, a TCR construct comprising a MART-1 specific TCR chain comprises TCR alpha and TCR beta chains targeting MART-1 epitope AAGIGILTV (SEQ ID NO: 175). In some embodiments, MART-1 specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in patent publication US 9,128,080 B2, which publication is incorporated herein by reference for the purposes described herein.

In some embodiments, a TCR construct comprising a MART-1 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: 176-179. In some embodiments, a TCR construct comprising a MART-1 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.

ATGTTGCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAG

CTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACT

TCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTC

TTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGT

ATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTAC

TTCTGTGCTGGTGGGACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATT

CCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAG

TCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGAT

GTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCT

GTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCA

GAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTT

GAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTG

AAGGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ ID NO:176)

ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCT

GGAATCACCCAGAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGT

CACCAGACTGAGAACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGG

CTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCAGATGGCTATAGT

GTCTCTAGATCAAAGACAGAGGATTTCCTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACA

TCTGTGTACTTCTGTGCCATCAGTGAGGTAGGGGTTGGGCAGCCCCAGCATTTTGGTGATGGG

ACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAG

CCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTC

TTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGC

ACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGC

CGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAG

TTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATC

GTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTG

TCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGC

GCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO:177)

ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGC

CAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCT

CTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGG

AAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACA

GCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGAT

TCAGCCACCTACCTCTGTGCCGTGAACTTCGGAGGAGGAAAGCTTATCTTCGGACAGGGAACG

GAGTTATCTGTGAAACCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCT

AAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAA

AGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTC

AAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAAC

AACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTG

GTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTC

CGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

TGA(SEQ ID NO:178)

ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCT

GGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGT

ACCCAGGATATGAGACATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGG

CTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGT

GTCTCCAGAGCAAACACAGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACA

TCTGTGTACTTCTGTGCCAGCAGCCTAAGTTTCGGCACTGAAGCTTTCTTTGGACAAGGCACC

AGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCA

TCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTC

CCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACG

GACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGC

CTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTC

TACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTC

AGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCT

GCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO:179)

GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTA

QFGITRKDSFLNISASIPSDVGIYFCAGGTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKS

SDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS

IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:180)

DAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDG

YSVSRSKTEDFLLTLESATSSQTSVYFCAISEVGVGQPQHFGDGTRLSILEDLNKVFPPEVAV

FEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL

SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:181)

QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTA

QLNKASQYVSLLIRDSQPSDSATYLCAVNFGGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSK

SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNN

SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:182)

IAGITQAPTSQILAAGRRMTLRCTQDMRHNAMYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDG

YSVSRANTDDFPLTLASAVPSQTSVYFCASSLSFGTEAFFGQGTRLTVVEDLNKVFPPEVAVF

EPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS

SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:183)

In some embodiments, the TCR construct comprises a tyrosinase-specific TCR chain. In some embodiments, the TCR construct comprising a tyrosinase-specific TCR chain comprises TCR a and TCR β chains found in tyrosinase-specific TCR clone TIL 1383I and/or modified versions thereof. In some embodiments, a TCR construct comprising a tyrosinase-specific TCR chain comprises TCR a and TCR β chains that target a tyrosinase epitope represented by amino acids 368-376 of tyrosinase (reactive towards MHC class I (HLA-A 2) restriction epitopes (368-376) of tyrosinase). In some embodiments, tyrosinase-specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in publication Roszkowski et al, cancer Res.65 (4): 1570-6 (2005), which is incorporated herein by reference for the purposes described herein.

In some embodiments, the TCR construct comprises a MAGE-A3 specific TCR chain. In some embodiments, a TCR construct comprising a MAGE-A3 specific TCR chain comprises TCR alpha and TCR beta chains targeting amino acids 271-279 of MAGE-A3, such as epitope FLWGPRALV (SEQ ID NO: 184). In some embodiments, a TCR construct comprising a MAGE-A3 specific TCR chain comprises TCR alpha and TCR beta chains targeting amino acids 112-120 of MAGE-A3, such as epitope KVAELVHFL (SEQ ID NO: 185). In some embodiments, MAGE-A3 specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in international patent application publication WO 2012/054825 A1, which is incorporated herein by reference for the purposes described herein. In certain embodiments, the anti-MAGE-A3112-120 TCR comprises an A118T substitution relative to wild type (wherein threonine is at position 118 in the alpha chain). In certain embodiments, the anti-MAGE-A3 112-120TCR comprises an A118V substitution relative to wild type (wherein valine is at position 118 in the alpha chain).

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 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.

ATGGGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGGAGGAGCAATGGCGAT

GGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTG

AACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTTCTGGTATGTGCAACATCTCAAT

GAATCCCCTCGGCTACTCCTGAAGAGCTTCACAGACAACAAGAGGACCGAGCACCAAGGGTTC

CACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTTTCGACACAAATGCTTACAAAGTCATCTTT(SEQ ID NO:186)

ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCCTAGGAACAGGCCTTGTGGACATG

AAAGTAACCCAGATGCCAAGATACCTGATCAAAAGAATGGGAGAGAATGTTTTGCTGGAATGTGGACAGGACATGAGCCATGAAACAATGTACTGGTATCGACAAGACCCTGGTCTGGGGCTACAGCTGATTTATATCTCATACGATGTTGATAGTAACAGCGAAGGAGACATCCCTAAAGGATACAGGGTCTCACGGAAGAAGCGGGAGCATTTCTCCCTGATTCTGGATTCTGCTAAAACAAACCAGACATCTGTGTACTTCTGTGCTAGCAGTTCAACAAACACAGAAGTCTTCTTT(SEQ ID NO:187)

ATGGGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTTCTGGTATGTGCAACATCTCAATGAATCCCCTCGGCTACTCCTGAAGAGCTTCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTTTCGACACAAATGCTTACAAAGTCATCTTTGGAAAAGGGACACATCTTCATGTTCTCCCTAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACACCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO:188)

ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCCTAGGAACAGGCCTTGTGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAGAATGGGAGAGAATGTTTTGCTGGAATGTGGACAGGACATGAGCCATGAAACAATGTACTGGTATCGACAAGACCCTGGTCTGGGGCTACAGCTGATTTATATCTCATACGATGTTGATAGTAACAGCGAAGGAGACATCCCTAAAGGATACAGGGTCTCACGGAAGAAGCGGGAGCATTTCTCCCTGATTCTGGATTCTGCTAAAACAAACCAGACATCTGTGTACTTCTGTGCTAGCAGTTCAACAAACACAGAAGTCTTCTTTGGTAAAGGAACCAGACTCACAGTTGTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAACTCGTGA(SEQ ID NO:189)

ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCCTGGAGCTCAGATGCAGTTATTCATACAGTGCAGCACCTTACCTCTTCTGGTACGTGCAGTATCCTGGCCAGAGCCTCCAGTTTCTCCTCAAATACATCACAGGAGACACCGTTGTTAAAGGCACCAAGGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCGGATACAGGAAACTACAAATACGTCTT(SEQ ID NO:190)ATGGGCATCCAGACCCTCTGTTGTGTGATCTTTTATGTTCTGATAGCAAATCACACAGATGCTGGAGTTACCCAGACACCCAGACATGAGGTGGCAGAGAAAGGACAAACAATAATCCTGAAGTGTGAGCCAGTTTCAGGCCACAATGACCTTTTCTGGTACAGACAGACCAAGATACAGGGACTAGAGTTGCTGAGCTACTTCCGCAGCAAGTCTCTTATGGAAGATGGTGGGGCTTTCAAGGATCGATTCAAAGCTGAGATGCTAAATTCATCCTTCTCCACTCTGAAGATTCAACCTACAGAACCCAGGGACTCAGCTGTGTATCTGTGTGCCAGCAGTTTTGGGACAGCTAGTGCAGAAACGCTGTATTTT(SEQ ID NO: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)

In some embodiments, the TCR construct comprises a MAGE-A4 specific TCR chain. In some embodiments, a TCR construct comprising a MAGE-A4 specific TCR chain comprises TCR alpha and TCR beta chains targeting epitope GVYDGREHTV (SEQ ID NO: 202). In some embodiments, a TCR construct comprising a MAGE-A4 specific TCR chain comprises TCR alpha and TCR beta chains targeting epitope FMNKFIYEI (SEQ ID NO: 203). In some embodiments, MAGE-A4 specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in international patent application publications WO 2017/174824 A1 and WO 2021/229212 A1, each of which is incorporated herein by reference for the purposes described herein. In certain embodiments, the anti-MAGE-A4 TCR alpha chain variable domain can have M4V or M4L amino acid substitutions. In certain embodiments, the anti-MAGE-A4 TCR β chain variable domain can have N10E amino acid substitutions.

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 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)

In some embodiments, the TCR construct comprises a Wilms tumor antigen (WT 1) WT 1-specific TCR chain. In some embodiments, a TCR construct comprising a WT1 specific TCR chain comprises TCR alpha and TCR beta chains targeting epitope VLDFAPPGA (SEQ ID NO: 215). In some embodiments, a TCR construct comprising a WT1 specific TCR chain comprises TCR alpha and TCR beta chains targeting the epitope RMFPNAPYL (SEQ ID NO: 216). In some embodiments, WT 1-specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in international patent application publications WO 2020/185796 A1 and WO 2021/034976A1, each of which is incorporated by reference herein for the purposes described herein. In some embodiments, the leader sequence and/or signal peptide may be removed from the TCR amino acid sequence, and the percentage of sequence identity may be calculated based on the TCR amino acid sequence without the leader sequence and/or signal peptide.

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.

ATGGAGACACTGCTGGGACTACTGATTCTGTGGCTGCAACTGCAATGGGTGAGCAGCAAACAGGAGGTTACCCAGATTCCTGCTGCTCTGTCTGTTCCTGAAGGCGAGAATCTGGTGCTGAACTGCAGCTTCACAGATAGCGCCATCTACAACCTGCAGTGGTTCAGACAGGATCCTGGAAAAGGCCTGACAAGCCTGCTGCTGATTCAGAGCTCTCAGAGAGAGCAGACATCTGGAAGACTGAATGCTAGCCTGGACAAGTCTAGCGGCAGAAGCACCCTGTATATTGCCGCCTCTCAACCTGGAGATTCTGCCACATACCTGTGTGCTGTGAAGGAGACATCTGGCTCTAGACTGACCTTTGGCGAGGGAACACAACTGACCGTGAATCCTGAC(SEQ ID NO:217)

ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAACAACTACTACCTGTTCTGGTACAAGCAGCCCCCCTCTAGGCAGATGATCCTGGTGATCAGACAGGAGGCCTATAAACAGCAGAATGCCACAGAGAACCGGTTCAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAGATCTCTGATTCTCAGCTGGGCGATACAGCCATGTACTTTTGCGCCTTCATCTACCCCAGCTACACAAGCGGCACATACAAGTACATCTTCGGCACCGGCACAAGACTGAAGGTTCTGGCCAAC(SEQ ID NO:218)

ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCAGAACAGCCCCTCTCTGTCTGTGCAGGAAGGCAGAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTGTGGTACAAGAAGTACCCCGCCGAGGGCCCTACCTTTCTGATCAGCATCTCTAGCATCAAGGACAAGAACGAAGATGGCAGATTCACCGTGTTCCTGAACAAGAGCGCCAAGCACCTGAGCCTGCACATTGTGCCTTCTCAACCTGGAGATTCTGCCGTGTACTTTTGTGCTGCCTCTGGAACAGGCGGAAGCTATATCCCCACATTTGGAAGAGGAACAAGCCTGATCGTGCACCCTTAC(SEQ ID NO:219)

ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCAGAACAGCCCCTCTCTGTCTGTGCAGGAAGGCAGAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTGTGGTACAAGAAGTACCCCGCCGAGGGCCCTACCTTTCTGATCAGCATCTCTAGCATCAAGGACAAGAACGAAGATGGCAGATTCACCGTGTTCCTGAACAAGAGCGCCAAGCACCTGAGCCTGCACATTGTGCCTTCTCAACCTGGAGATTCTGCCGTGTACTTTTGTGCTGCCTCTGGCATTGGCGACTACAAACTGAGCTTTGGAGCCGGCACAACAGTGACCGTTAGAGCCAAT(SEQ ID NO:220)

ATGGTGAAGATCCGGCAGTTCCTCCTGGCTATTCTGTGGCTGCAACTGTCTTGTGTGTCTGCTGCCAAGAATGAAGTGGAGCAGTCTCCCCAGAACCTTACAGCCCAGGAAGGCGAGTTTATCACCATCAACTGCAGCTATTCTGTGGGCATTAGCGCCCTGCATTGGCTGCAGCAACACCCTGGAGGAGGAATTGTGTCTCTGTTTATGCTGTCTTCTGGCAAGAAGAAGCACGGCCGGCTGATTGCCACCATCAACATCCAGGAGAAGCACTCTTCTCTGCACATTACAGCCTCTCATCCCAGGGATTCTGCCGTGTACATCTGTGCCGTGAGAACCAGCTACGATAAGGTGATTTTCGGACCAGGCACCTCTCTGAGCGTGATCCCCAAT(SEQ ID NO:221)

ATGAAGAGCCTGAGAGTCCTGCTGGTGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGAAGTGGAGCAGAATAGCGGCCCTCTGTCTGTTCCTGAAGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGAGGATGGCAGGTTTACAGCCCAGCTGAACAAGGCCAGCCAGTATGTTTCTCTGCTGATCAGAGATAGCCAGCCTAGCGATTCTGCCACCTACCTGTGTGCCGTGAACTTACTTGGAGCTACAGGATACTCTACACTGACCTTCGGCAAAGGCACCATGCTGCTGGTGAGCCCTGAT(SEQ ID NO:222)

ATGTGGGGCGTTTTCCTTCTGTATGTGAGCATGAAGATGGGCGGCACAACAGGCCAGAACATCGATCAGCCTACCGAGATGACAGCCACAGAAGGAGCTATTGTTCAGATCAACTGCACCTACCAGACAAGCGGCTTCAACGGCCTGTTCTGGTACCAGCAGCATGCTGGAGAAGCTCCTACATTTCTGAGCTACAATGTGCTGGATGGCCTGGAGGAGAAAGGCAGGTTTAGCAGCTTCCTGAGCAGGTCTAAGGGCTATTCTTATCTGCTGCTGAAGGAGCTGCAGATGAAGGATTCCGCCAGCTACCTGTGTGCCGTTAGGGGCATCAATGATTACAAGCTGAGCTTTGGAGCCGGAACAACAGTGACCGTGAGAGCCAAC(SEQ ID NO:223)

ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCCGTGATTACCGGCTTTCAGAAGCTGGTGTTTGGCACAGGCACCAGACTGCTGGTTTCTCCCAAT(SEQ ID NO:224)

ATGAGACTGGTGGCACGCGTAACTGTGTTTCTGACCTTTGGCACCATCATCGATGCCAAGACAACCCAGCCTACAAGCATGGACTGTGCCGAGGGAAGAGCTGCTAATCTGCCATGTAATCACAGCACAATCAGCGGCAACGAGTACGTGTACTGGTACCGGCAGATCCACTCTCAAGGACCTCAGTACATCATTCATGGCCTGAAGAACAACGAGACCAACGAGATGGCCAGCCTGATCATCACCGAGGACAGGAAGTCTTCTACCCTGATTCTGCCTCATGCTACACTGAGAGATACCGCCGTGTACTACTGCATTGCCGGAGTGGGAAGAGGCCAGAATTTCGTGTTTGGACCTGGAACAAGACTGAGCGTTCTGCCCTAT(SEQ ID NO:225)

ATGGAGAAGAACCCCTTGGCAGCACCTCTGCTTATTCTGTGGTTCCACCTGGATTGTGTGAGCAGCATCCTGAATGTGGAGCAGTCTCCTCAGAGCCTGCATGTGCAAGAAGGCGATAGCACCAATTTCACCTGCAGCTTTCCAAGCAGCAACTTCTACGCCCTGCACTGGTACAGATGGGAAACCGCCAAATCTCCTGAAGCCCTGTTTGTGATGACCCTGAATGGCGACGAGAAGAAGAAGGGCAGAATTAGCGCCACCCTGAATACCAAGGAGGGCTACAGCTACCTGTACATCAAGGGCTCTCAACCTGAGGATTCTGCCACCTACCTTTGCGCCTTTCACCCCAATTTCGGCAACGAGAAACTGACCTTTGGAACCGGAACAAGGCTGACCATCATCCCCAAC(SEQ ID NO:226)

ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCTGTTCAGCCTAGAGGAGATGGCTCTAGCAATACCGGCAAGCTGATCTTTGGCCAGGGAACAACACTGCAGGTGAAGCCTGAT(SEQ ID NO:227)

ATCCAGAATCCCGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:228)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACATCTCTTCTCTGTTGGGTGGTTCTGGGCTTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTATAAGGTGACCAAGAGGGGACAGGATGTGGCTCTGAGATGTGACCCTATTAGCGGACATGTGAGCCTGTACTGGTACAGACAAGCTCTGGGACAAGGACCCGAGTTTCTGACCTACTTCAACTATGAGGCCCAGCAGGACAAATCTGGACTGCCCAACGACAGATTCAGCGCCGAAAGACCAGAAGGCTCTATTAGCACACTGACCATCCAGAGAACAGAGCAGAGGGATTCTGCCATGTACAGATGCGCCAGCAGCTTAACAGGCTCTTACGAGCAGTACTTTGGACCTGGCACAAGACTGACAGTGACAGAG(SEQ ID NO:229)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCACACCTGAAGCCTCTAGCCCATATGAGCAGTACTTTGGCCCTGGCACCAGATTAACAGTGACAGAG(SEQ ID NO:230)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGACCTGGACTGCTTCATTGGATGGCTCTGTGTTTGCTGGGAACAGGACATGGAGATGCTATGGTGATCCAGAACCCCAGGTATCAGGTGACCCAGTTTGGCAAACCAGTGACACTGAGCTGTTCTCAGACCCTGAACCACAACGTGATGTACTGGTACCAGCAGAAGTCTTCTCAGGCCCCTAAGCTGCTGTTCCACTACTACGACAAGGACTTCAACAACGAGGCCGATACCCCTGACAATTTCCAGAGCAGGAGGCCCAATACCAGCTTCTGTTTCCTGGACATTAGAAGCCCTGGACTGGGAGATGCTGCCATGTACCTGTGTGCCACCAGCAATTTACAGGGAAGACAACCTCAGCACTTTGGCGATGGCACAAGGCTGTCTATCCTGGAG(SEQ ID NO:231)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGTGGCTGCTGGCGTGATTCAATCTCCTAGACACCTGATCAAGGAGAAGAGAGAAACAGCCACCCTGAAGTGCTACCCCATCCCCAGACACGATACAGTGTACTGGTATCAGCAAGGACCTGGACAAGATCCCCAGTTCCTGATCAGCTTCTACGAGAAGATGCAGAGCGACAAAGGCAGCATCCCAGACAGATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCAGCCTGGAACTGGGCGATTCTGCTCTGTACTTCTGTGCCTCTTCTCTGAGACTGGGAAGAGAAACCCAGTACTTTGGACCCGGCACAAGACTGCTGGTTCTTGAG(SEQ ID NO:232)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACAAGACTTCTCTGCTGGGTGGTGCTTGGATTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTACAAAGTGGCCAAGAGAGGACAGGATGTGGCTCTGAGATGTGACCCTATTAGCGGACATGTGAGCCTGTTTTGGTACCAGCAAGCTCTGGGACAAGGACCCGAGTTTCTGACCTACTTCCAGAATGAAGCCCAGCTGGATAAATCTGGACTGCCTAGCGACCGGTTCTTCGCCGAAAGACCTGAAGGATCTGTTAGCACCCTGAAGATTCAGAGAACACAGCAGGAGGACTCTGCCGTGTACCTGTGTGCCTCTTCTTTAGGACAGGCCTATGAGCAGTATTTTGGACCTGGCACCAGACTGACCGTGACAGAG(SEQ ID NO:233)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACAAGACTTCTCTGCTGGGTGGCCTTTTGTCTGCTGGTGGAAGAGCTGATTGAAGCTGGAGTTGTGCAGTCTCCTAGGTACAAGATCATCGAGAAGAAGCAGCCCGTGGCCTTCTGGTGTAATCCCATTTCTGGCCACAACACCCTGTACTGGTATCTGCAGAATCTGGGACAGGGCCCTGAACTGCTGATCAGATACGAGAACGAAGAAGCCGTGGACGATTCTCAACTGCCTAAGGACCGCTTTTCTGCCGAGAGGCTGAAAGGAGTGGATTCTACCCTGAAGATCCAACCTGCTGAACTGGGCGATTCTGCTGTGTACCTGTGCGCTTCTAGCCTGACAAGAGGAGCTGAAGCCTTTTTTGGACAGGGCACAAGACTGACAGTGGTGGAG(SEQ ID NO:234)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGACCTCAGCTTCTTGGATACGTTGTGCTGTGTCTGCTTGGAGCTGGACCTCTTGAAGCTCAGGTTACCCAGAACCCCAGATACCTGATTACCGTGACAGGCAAAAAGCTGACCGTGACATGTAGCCAGAACATGAACCACGAGTACATGAGCTGGTACCGGCAGGATCCTGGATTAGGCCTGAGACAGATCTACTACAGCATGAACGTGGAGGTGACCGATAAAGGCGACGTGCCTGAGGGATACAAGGTGAGCAGAAAGGAGAAGAGGAATTTCCCCCTGATCCTGGAAAGCCCAAGCCCCAATCAGACAAGCCTGTACTTTTGTGCCAGCAGCTTTTCTGGCGGCACATATGAGCAGTACTTCGGCCCTGGCACAAGACTGACAGTTACAGAG(SEQ ID NO:235)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGTGGCTGCTGGCGTGATTCAATCTCCTAGACACCTGATCAAGGAGAAGAGAGAAACAGCCACCCTGAAGTGCTACCCCATCCCCAGACACGATACAGTGTACTGGTATCAGCAAGGACCTGGACAAGATCCCCAGTTCCTGATCAGCTTCTACGAGAAGATGCAGAGCGACAAAGGCAGCATCCCAGACAGATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCAGCCTGGAACTGGGCGATTCTGCTCTGTACTTCTGTGCCAGCAGCTATAGAGGAGGCAGCACATATGAGCAGTACTTTGGCCCTGGCACAAGACTGACAGTGACAGAG(SEQ ID NO:236)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGAGCACCAGACTCCTTTGCTGGATGGCTTTGTGTCTGCTTGGAGCTGAGCTGTCTGAAGCTGAAGTTGCCCAGTCTCCCAGATACAAGATCACCGAGAAATCTCAGGCTGTGGCCTTCTGGTGTGACCCTATTTCTGGACACGCCACCCTGTACTGGTATAGGCAAATTCTGGGACAAGGCCCTGAACTGCTGGTGCAATTTCAGGATGAGAGCGTGGTGGACGATTCTCAACTGCCTAAGGACAGGTTTTCTGCCGAGCGGCTGAAAGGAGTTGATAGCACCCTGAAGATCCAACCTGCTGAACTGGGCGATTCTGCTATGTACCTGTGCGCCTCTTCTCAGAGAGATAGCCCTAACGAGAAGCTGTTCTTTGGCTCTGGAACCCAGCTGTCTGTGCTGGAG(SEQ ID NO:237)

CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCTGTAGACTGTTGTGTTGTGCTGTGCTGTGTCTGTTGGGAGCTGTGCCTATGGAAACAGGCGTTACCCAGACACCTAGACATCTGGTTATGGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCATCTGGGCCATAACGCCATGTACTGGTATAAGCAGAGCGCCAAGAAACCACTGGAACTGATGTTCGTGTACAGCCTGGAGGAGAGGGTGGAGAATAATAGCGTGCCCAGCAGATTTAGCCCTGAGTGCCCAAATTCTTCTCACCTGTTCCTGCACCTGCACACATTACAGCCCGAGGATTCTGCCCTGTACCTGTGTGCTTCTTCTCAAGACCCTTACAAGCTGAGCGGCAATACCATCTACTTCGGCGAAGGCTCTTGGCTGACAGTGGTTGAA(SEQ ID NO:238)

GATCTGAACAAGGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCTCCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTGCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGCGGCTTTACCTCCGTGTCCTATCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACTTC(SEQ ID NO:239)

GACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:240)

ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACATAGGAAACCATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:241)

ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTCAACTGCAGTTACACAGTCAGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGCTTTCTGCACATCACAGCCCCTAAACCTGAAGACTCAGCCACTTATCTCTGTGCTGTGCAGACCATGGACGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:242)

ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAGAGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGATGCCGCGATGTATTTCTGTGCTTCCAGTCCAGGAACCTACAAATACATCTTTGGAACAGGCACCAGGCTGAAGGTTTTAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: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)

ATGGGCTGTAGACTGTTGTGTTGTGCTGTGCTGTGTCTGTTGGGAGCTGTGCCTATCGATACAGAGGTGACCCAGACCCCTAAACATCTGGTTATGGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCACATGGGCCATAGGGCCATGTATTGGTATAAGCAGAAGGCCAAGAAACCTCCTGAGCTGATGTTCGTGTACAGCTACGAGAAGCTGAGCATCAACGAGAGCGTGCCCAGCAGATTTTCTCCTGAGTGCCCTAATTCTAGCCTGCTGAATCTGCACCTGCATGCTCTGCAGCCTGAGGATTCTGCTCTGTACCTGTGTGCTTCTTCTCAGGGCACATCTGGAGCTGATACACAGTACTTCGGACCTGGCACAAGACTGACAGTGCTGGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:253)

ATGTCTATCGGTCTGCTGTGCTGTGCTGCTCTTTCTCTGCTTTGGGCTGGACCTGTGAATGCTGGAGTTACACAAACCCCCAAGTTCCAAGTGCTGAAGACAGGACAGAGCATGACCCTGCAGTGTGCTCAGGACATGAATCACGAGTACATGAGCTGGTACAGACAGGATCCTGGAATGGGCCTGAGGCTGATCCACTACTCTGTTGGAGCCGGAATTACAGATCAGGGAGAAGTGCCAAATGGCTACAACGTGAGCAGGAGCACAACCGAGGACTTCCCCTTAAGACTGTTGTCTGCTGCTCCATCTCAGACAAGCGTGTACTTTTGCGCCAGCTCCTACTCTCTGTGGGATCTGCAGGAAACCCAGTACTTTGGACCAGGCACAAGACTGTTAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:254)ATGGGCACATCTCTTCTCTGCTGGATGGCTCTTTGTCTGCTTGGAGCCGATCATGCCGATACAGGAGTTAGCCAGGATCCTAGACACAAGATCACCAAGAGAGGCCAGAATGTGACCTTCCGGTGCGATCCTATCTCTGAGCACAACAGGCTGTACTGGTACAGACAAACACTGGGACAAGGACCTGAGTTCCTGACCTACTTCCAGAACGAAGCCCAGCTGGAGAAGTCTAGACTTCTGAGCGACAGATTTAGCGCCGAGAGACCTAAAGGCAGCTTTAGCACCCTGGAGATCCAGAGAACAGAACAGGGCGATTCTGCCATGTACCTGTGTGCTAGCAGCTTTTCTGATGGAGGCGCCACCGATACACAGTATTTCGGACCTGGCACAAGACTGACAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:255)

ATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCAGACCTCACAGCCTGACCGATACACAGTACTTTGGCCCTGGCACAAGACTGACAGTGTTAGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:256)

METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVKETSGSRLTFGEGTQLTVNP(SEQ ID NO:257)

MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFIYPSYTSGTYKYIFGTGTRLKVLAN(SEQ ID NO: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)

In some embodiments, the TCR construct comprises a Human Papillomavirus (HPV) -specific TCR chain. In some embodiments, the TCR construct comprising an HPV-specific TCR chain comprises TCR a and TCR β chains that target HPV 18E6 protein and/or HPV 18E7 protein. In some embodiments, the HPV 18E6 epitope is amino acids 121-135 and/or amino acids 77-91 of the HPV 18E6 protein. In some embodiments, the TCR construct comprising an HPV-specific TCR chain comprises TCR a and TCR β chains that target HPV 18E7 protein. In some embodiments, the HPV 18E7 epitope is amino acids 11-19. In some embodiments, HPV-specific TCR sequences, TCR variable domain sequences, CDR sequences, and/or TCR constant domain sequences are described in international patent application publication WO 2015/009604A1, which is incorporated herein by reference for the purposes described herein.

NK cells

NK cells modified to express the TCR/CD3 receptor complex may be obtained from any suitable source, including fresh or frozen. In certain embodiments, the NK cells are derived from human Peripheral Blood Mononuclear Cells (PBMCs), unstimulated leukocyte isolation 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 may be isolated from umbilical Cord Blood (CB), peripheral Blood (PB), bone marrow, stem cells, NK cell lines, or mixtures thereof. In certain embodiments, NK cells are isolated from the pooled CBs. CBs may be consolidated from 2, 3,4, 5, 6, 7, 8, 9, 10 or more units. NK cells may be autologous or allogeneic to the recipient individual. The isolated NK cells may or may not match the subject haplotype to which the cell therapy is to be administered. 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 cord blood, where NK cells may be present in the cord blood in the form of a heterogeneous mixture of cells, and may deplete certain CD3 expressing cells. In other methods, umbilical cord CB is used to derive NK cells by isolating cd34+ cells.

NK cells may be pre-activated with one or more inflammatory cytokines, which may or may not be amplified. In certain cases, NK cells are pre-activated prior to modification to express the cd3±tcr or after modification to express the cd3±tcr complex. In particular embodiments, pre-activation of NK cells may include culturing the isolated NK cells in the presence of one or more cytokines. NK cells can 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 particular embodiments, the preactivated 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 pre-activation step. The pre-activation may be for a short period of time, for example 5-72 hours, for example 10-50 hours, in particular 10-20 hours, for example 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, in particular about 16 hours. The pre-activation culture may comprise IL-12 at a concentration of 0.1-150ng/mL, for example at a concentration of 0.5-50ng/mL, especially 1-20ng/mL, for example 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15ng/mL, especially about 10ng/mL. The pre-activation culture may comprise IL-18 and/or IL-15 in a concentration of 10-100ng/mL, for example in a concentration of 40-60ng/mL, in particular 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55ng/mL, in particular about 50ng/mL.

In certain instances, NK cells are expanded prior to modification to express the cd3±tcr complex or after modification to express the cd3±tcr complex. The pre-activated NK cells can be expanded in the presence of artificial antigen presenting cells (aAPCs). The pre-activated NK cells may be washed, for example 2, 3,4 or 5 times, in particular 3 times, prior to expansion. aapcs may be engineered to express CD137 ligands and/or membrane-bound cytokines. The membrane-bound cytokine may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In particular embodiments, aapcs are engineered to express CD137 ligands and mll-21. aapcs may be derived from cancer cells, such as leukemia cells. aapcs may not express endogenous HLA class I, class II or CD1d molecules. They may express ICAM-1 (CD 54) and LFA-3 (CD 58). In particular, aapcs may be K562 cells, e.g., K562 cells engineered to express CD137 ligand and mll-21. aapcs may be subjected to radiation. Engineering can be performed by any method known in the art, such as retroviral transduction. The amplification may last for about 2-30 days, such as 3-20 days, particularly 12-16 days, such as 12, 13, 14, 15, 16, 17, 18 or 19 days, particularly about 14 days. The pre-activated NK cells and aAPCs may be present in a ratio of about 3:1-1:3, e.g. 2:1, 1:1, 1:2, especially about 1:2. The amplification culture may further comprise a cytokine that promotes amplification, such as IL-2.IL-2 may be present at a concentration of about 10-500U/mL, for example 100-300U/mL, particularly about 200U/mL. IL-2 may be supplemented in the amplification culture, for example, once every 2-3 days. aapcs may be added to the culture at least a second time, for example about 7 days of expansion.

In particular 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, which are secreted by NK cells or tethered to NK cell membranes. In this case, the membrane bound cytokine may be tethered to the NK cell membrane with a specific transmembrane domain, such as the transmembrane domain of CD8, CD28, CD27, B7H3, igG1, igG4, CD4, DAP10, DAP 12.

After preparation, the modified NK cells (including an effective amount of one or more bispecific or multispecific antibodies) may be infused immediately, or the NK cells may be stored, e.g., by cryopreservation. In certain aspects, cells may be propagated ex vivo as a large population for days, weeks, or months within about 1, 2, 3,4, or 5 days.

III. heterologous proteins

In particular embodiments, the NK cells are modified not only to express one or more components of the TCR/CD3 complex, but also to express one or more other heterologous proteins. The heterologous proteins may promote the activity of NK cells in any way, including at least their activation, persistence, amplification, homing and/or cytotoxicity.

A. bispecific or multispecific antibodies

In some embodiments, NK cells are modified to express one or more bispecific or multispecific antibodies, although in other cases NK cells do not express antibodies, antibodies are used with NK cells.

In the case where NK cells are modified to express antibodies, the antibodies may be conjugation molecules that bridge specific immune effector cells with specific target cells to disrupt the target cells. The present invention allows modified NK cells to be used with standard T cell binding molecules (BiTEs) because they have been modified to express CD3, in many cases CD3 is a T cell antigen that binds to a BiTE binding molecule. In this case, the BiTE used in the present invention may also target cancer antigens or viral antigens, which may be tailored to the medical condition of the intended recipient individual. For example, biTE can be tailored to bind to cancer antigens specific to cancer cells of an individual's cancer. anti-CD 3 antibodies to BiTE may target the cd3γ chain, the cd3δ chain, the cd3ε chain, or the cd3ζ chain.

In some cases, in addition to expressing a CD3 complex (with or without TCR) that allows NK cells to be used as a treatment with BiTE, NK cells may be modified to express (or not express) one or more bispecific NK engagement molecules (bikes) but be used with one or more bispecific NK engagement molecules (bikes). The BiKE comprises antibodies that bind to NK cell surface proteins, including those naturally expressed on NK cells, and antibodies that bind to the desired target antigen. The BiKE may target NK cells by antibodies against NK surface proteins (e.g. CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR or KIR). In this case, the bipes used in the present invention may also target cancer antigens or viral antigens, which may be tailored to the medical condition of the intended recipient individual. For example, the BiKE may be tailored to bind to a cancer cell-specific cancer antigen of an individual's cancer.

In embodiments where NK cells express a CD3 complex (with or without TCR) and one or more bikes, one or more vectors may be used to transfect or transduce cells with the components of the CD3 complex (with or without TCR) and one or more bikes. In some cases, one or more of the CD3 complex component (with or without TCR) and the BiKE may or may not be on the same polycistronic vector.

B. Engineered receptors

In particular embodiments, 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 type of cancer antigen or viral antigen. Receptors can be tailored to target a desired antigen according to the medical condition of the intended recipient individual.

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, a second generation, or a third or subsequent generation. The CAR may or may not have dual specificity for two or more different antigens. The CAR may comprise one or more co-stimulatory domains. Each co-stimulatory domain may comprise, for example, a co-stimulatory domain of any one or more of the members 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 of the TNFR superfamily or a combination thereof. In a particular embodiment, the CAR comprises cd3ζ. In certain embodiments, the CAR lacks one or more specific co-stimulatory domains; for example, the CAR may lack 4-1BB and/or CD28.

In certain embodiments, the CAR polypeptide in the cell comprises an extracellular spacer domain that connects the antigen binding domain and the transmembrane domain, which may be referred to as a hinge. The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or fragment or derivative thereof, a hinge region of an antibody or fragment or derivative thereof, a CH2 region of an antibody, a CH3 region antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, CD 8-alpha hinge, CD28, artificial spacers made of polypeptides such as Gly3, or CH1, CH3 domains of IgG (e.g., human IgG1 or IgG 4). In particular cases, the extracellular spacer domain may 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 CD 8a 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 particular embodiments, the hinge is from IgG1, and in certain aspects, the CAR polypeptide comprises or is encoded by a particular IgG1 hinge amino acid sequence.

The transmembrane domain in the CAR may be from a natural or synthetic source. If the source is natural, the domain is in some way derived from any membrane-bound or transmembrane protein. The transmembrane region includes a transmembrane region derived from (i.e., comprising at least) the following transmembrane regions: an alpha, beta, or zeta chain ,CD28,CD3ζ,CD3ε,CD3γ,CD3δ,CD45,CD4,CD5,CD8,CD9,CD16,CD22,CD33,CD37,CD64,CD80,CD86,CD134,CD137,CD154,ICOS/CD278,GITR/CD357,NKG2D of a T cell receptor and a DAP molecule (e.g., DAP10 or DAP 12). Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues, such as leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine can be found at each end of the synthetic transmembrane domain.

In some embodiments, the engineered receptors utilize one or more homing receptors (which can home to the target, not necessarily because of signal release, e.g., where they utilize an adhesion molecule) 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 particular instances, the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD 62L) CXCR1, CXCR2, or CX3CR 1.

C. Cytokines and methods of use

In some embodiments, NK cell expressing cells are engineered to express one or more heterologous cytokines and/or are engineered to up-regulate normal expression of one or more heterologous cytokines. The cells may or may not transduce or transfect one or more cytokines on the same vector as the other genes.

One or more cytokines may be co-expressed from the vector, including the isolated polypeptide as any component from the TCR/CD3 complex. For example, interleukin-15 (IL-15) is tissue restricted and can only be observed at any level in serum or throughout the body under pathological conditions. IL-15 has several properties that are required for adoptive therapy. IL-15 is a homeostatic cytokine that induces natural killer cell development and cell proliferation, promotes eradication of established tumors by alleviating functional inhibition of tumor resident cells, and inhibits 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 for human use. NK cells expressing IL-15 are able to sustain cytokine signaling, which is useful for their survival after infusion.

In certain embodiments, the cells express one or more exogenously supplied cytokines. As one example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. Cytokines can be supplied exogenously to NK cells because they are expressed from intracellular expression vectors. In another case, endogenous cytokines in the cell are up-regulated upon manipulation of the regulation of expression of the endogenous cytokines, such as gene recombination at the promoter site of the cytokine. Where the cytokine is provided to the cell on an expression construct, the cytokine may be encoded by the same vector as one or more components of the CD3 complex (with or without the TCR complex).

In some embodiments, specific sequences of IL-15 are used, such as the following sequences (underlined refers to signal peptide sequences):

D. Antigens

The modified NK cells of the invention are used 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. Where NK cells are modified with one or more engineered antigen receptors, the antigen targeted by the bispecific or multispecific antibody and the antigen targeted by the one or more engineered antigen receptors may or may not be the same antigen. In some cases, the antigen targeted by the bispecific or multispecific antibody and the antigen targeted by the one or more engineered antigen receptors are different antigens, but are associated with the same type of cancer.

An antigen targeted by an antibody and/or engineered antigen receptor is an antigen expressed in the context of a disease, condition, or cell type targeted by adoptive cell therapy. These diseases and conditions are proliferative, neoplastic and malignant diseases and disorders, including cancers and tumors, including cancers of the blood, immune system, such as lymphomas, leukemias and/or myelomas, such as B, T and myelogenous leukemias, lymphomas and multiple myelomas. In some embodiments, the antigen is selectively expressed or over-expressed on cells of a disease or condition (e.g., tumor or pathogenic cells) as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or on engineered cells.

Any suitable antigen may be targeted in the present method. In some cases, the antigen may be associated with certain cancer cells, but not non-cancer cells. Exemplary antigens include, but are not limited to, antigenic molecules from infectious agents, self/self antigens, tumor/cancer associated antigens, and tumor neoantigens (LINNEMANN et al 2015). In a particular aspect, the antigen comprises: 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 (CS 1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, P53, mutated P53, ras, C-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin dependent kinase), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, 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-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, cyp-B, hTERT, hTRT, iCE, MUC, MUC2, phosphatidylinositol 3-kinase (PI 3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, wilms tumor antigen (WT 1), AFP, -cycloprotein/M, caspase-8/M, CDK-4/M, ELF-2, gv-Gv, g250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myoglobin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, annexin II, CDC27/m, TPI/mbcr-ABL, BCR-ABL, interferon regulator 4 (IRF 4), ETV6/AML, LDLR/FUT, pml/RAR, tumor-associated calcium signal transducer 1 (TACSTD 1) TACSTD2, receptor tyrosine kinases (e.g., epidermal Growth Factor Receptor (EGFR) (particularly 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 transducer and transcriptional activators STAT3, STATS and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor κB (NF-B), notch receptors (e.g., notch 1-4), NY ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T 4, SM22α, carbonic Anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, protease 3, hTERT, sarcoma translocation breakpoint, ephA2, ML-IAP, epCAM, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, rhoC, GD3, fucose GM1, mesothelium (mesothelian), PSCA, sLe, PLAC1, GM3, BORIS, tn, GLoboH, NY-BR-1, RGS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, legumain, TIE2, page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKRD2A, MAD2L1, CTA, G1B, SUNC1 and LRR 1. Examples of sequences of antigens are known in the art, e.g. inIn the 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).

Tumor-associated antigens may be derived from the following cancers: prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cancer, mesothelioma, ovarian cancer, liver cancer, brain cancer, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gall bladder cancer, thyroid cancer or melanoma cancer. Exemplary tumor-associated antigens or antigens derived from tumor cells include MAGE 1,3 and MAGE 4 (or other MAGE antigens such as those disclosed in international patent publication No. WO 99/40188); PRAME; BAGE; RAGE, lange (also known as NY ESO 1); SAGE; and HAGE or GAGE. Non-limiting examples of these tumor antigens are expressed in a variety of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. patent No. 6,544,518. Tumor-associated antigens for prostate cancer include, for example, prostate Specific Membrane Antigen (PSMA), prostate Specific Antigen (PSA), prostate phosphate, NKX3.1, and prostate hexatransmembrane epithelial antigen (STEAP).

Other tumor associated antigens include Plu-1, HASH-1, hasH-2, cripto, and Criptin. In addition, the tumor antigen may be a self-peptide hormone, such as full length gonadotropin releasing hormone (GnRH), wie a short 10 amino acid long peptide, useful in the treatment of many cancers.

Antigens may include epitope regions or peptides derived from genes mutated in tumor cells or from genes transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, bcr/abl rearrangements, her2/neu, mutated or wild-type P53, cytochrome P450 1B1 and abnormally expressed intron sequences, such as N-acetylglucosaminyl transferase-V; clonal rearrangement of immunoglobulin genes produces unique idiotypes in myeloma and B-cell lymphoma; tumor antigens including epitope regions or peptides derived from oncoviral processes, such as human papilloma virus proteins E6 and E7; epstein barr virus protein LMP2; non-mutated carcinoembryonic proteins, such as carcinoembryonic antigen and alpha fetoprotein, with tumor selective expression.

E. Suicide gene

In particular embodiments, suicide genes are used with NK cell therapies to control their use and allow termination of cell therapies at a desired event and/or time. Suicide genes are used in transduced cells in order to trigger death of the transduced cells when desired. The cells of the invention are modified to contain one or more vectors encompassed by the invention, which may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that affects the conversion of a gene product to a compound that kills its host cell upon administration of a prodrug or other agent. In other embodiments, the suicide gene encodes a gene product that is targeted, when desired, by an agent (e.g., an antibody) that targets the suicide gene product.

In some cases, when an individual who is receiving and/or has received cell therapy exhibits one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, allergic reaction/allergy, and/or toxicity to/off-target tumors (for example)), or is deemed at risk of one or more symptoms (including upcoming symptoms), the cell therapy may employ any variety of one or more suicide genes. The use of suicide genes may be part of the planned treatment regimen or may be used only under recognized use requirements. In some cases, cell therapy is terminated by using agents that target suicide genes or gene products thereof, as therapy is no longer needed.

Application of suicide genes may be initiated upon occurrence of at least one adverse event on an individual and the adverse event may be identified by any means, including routine monitoring that may or may not be continuous from the beginning of cellular therapy. Adverse events may be detected during inspection and/or testing. In the case of an individual suffering from a cytokine release syndrome (also known as a 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; heart/kidney/liver dysfunction; or a combination thereof. In cases where the individual has neurotoxicity, the individual may suffer from confusion, delirium, hypoplasia and/or epilepsy. In some cases, the individual is tested for markers associated with the onset and/or severity of cytokine release syndrome, such as C-reactive protein, IL-6, TNF- α, and/or ferritin.

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), and they may be affected by the delivery of antibodies that bind TNF-alpha mutants. Examples of suicide gene/prodrug combinations that may be used are herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductases and cycloheximides; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk:: tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Coli (e.coli) purine nucleoside phosphorylase, a so-called suicide gene, can be used to convert the prodrug 6-methylpurine deoxyribose to the toxic purine 6-methylpurine. Other suicide genes include, for example, CD20, CD52, inducible caspase 9, purine Nucleoside Phosphorylase (PNP), cytochrome p450 enzyme (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase, methionine- α, γ -lyase (MET), EGFRv3, and Thymidine Phosphorylase (TP).

Administering a therapeutic composition

Administering to an individual in need thereof a CD3 expressing NK cell and a bispecific or multispecific antibody, including in a manner that enables the anti-CD 3 antibody of the bispecific or multispecific antibody to bind to CD3 on the CD3 expressing NK cell. In some cases, the two components are administered separately to the individual, while in other cases, the two components are compounded together prior to administration, e.g., in an ex vivo manner. In another embodiment, the NK cells express the antibody. In some cases, the two components are not pre-compounded prior to administration, but are co-administered by any suitable route of administration, for example, by co-infusion to a patient.

Embodiments of the present invention relate to methods of treating or preventing a medical disease or disorder using compositions comprising NK cells and antibodies provided herein. The method comprises administering to the subject a therapeutically effective amount of CD3 (±tcr) modified NK cells and antibodies, thereby treating or preventing a 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 invention, the cancer or infection is treated by transferring a composition comprising a population of NK cells and the corresponding antibody. In at least some cases, NK cells can reverse the anti-inflammatory tumor microenvironment and increase the adaptive immune response by promoting differentiation, activation and/or recruitment of helper immune cells to the malignant tumor site as they release pro-inflammatory cytokines.

Cancers for which the present treatment methods can be used include any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors may include, but are not limited to, tumors of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include bone marrow tumors, T-cell or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Other 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, lung adenocarcinoma, and lung squamous carcinoma), peritoneal cancer, stomach cancer or gastric 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 cancer or uterine cancer, salivary gland cancer, renal cancer or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various head and neck cancers, and melanoma.

The cancer may be of the following histological type in particular, although it is not limited to these: tumors, malignant; cancer; cancer, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinomas; gastrinomas, malignant; bile duct cancer; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; liang Xianai smaller; adenoid cystic carcinoma; adenocarcinomas among adenomatous polyps; adenocarcinomas, familial polyposis; solid cancer; carcinoid, malignant; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell cancer; eosinophilic cancer; eosinophilic adenocarcinoma; basophilic granulocyte cancer; clear cell adenocarcinoma; granulosa cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-enveloped sclerotic cancers; adrenal cortex cancer; endometrial-like cancer; skin accessory cancer; apocrine adenocarcinoma; sebaceous gland cancer; cervical adenocarcinoma; epidermoid carcinoma of mucous; cystic adenocarcinoma; papillary cyst adenocarcinoma; papillary serous cystic adenocarcinoma; mucinous cystic adenocarcinoma; mucinous adenocarcinoma; printing ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease, mammary gland; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinomas are accompanied by squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignancy; follicular carcinoma, malignant; granulocytoma, malignant; androgenic blastoma, malignant; celetoly cell carcinoma; testicular stromal cell tumor, malignant; lipid cell neoplasms, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomerular sarcoma; malignant melanoma; no melanin melanoma; superficial diffuse melanoma; malignant nevus malachite melanoma; lentigo acromioclavis melanoma; nodular melanoma; malignant melanoma in giant pigmented nevi; epithelioid cell melanoma; blue nevi, malignant; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryo rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; mixed tumor, malignant; miaole tube mixed tumor; nephroblastoma; hepatoblastoma; carcinoma sarcoma; a mesenchymal neoplasm, malignancy; boehringer's tumor, malignant; phylliform tumor, malignant; synovial sarcoma; mesothelioma, malignant; a vegetative cell tumor; embryo cancer; teratoma, malignant; ovarian goiter, malignancy; choriocarcinoma; mesonephroma, malignancy; hemangiosarcoma; vascular endothelial tumor, malignant; kaposi's sarcoma; vascular epidermocytoma, malignant; lymphangiosarcoma; osteosarcoma; a cortical bone sarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; bone giant cell tumor; ewing's sarcoma; odontogenic tumors, malignancy; ameloblastic osteosarcoma; enameloblastoma, malignant; ameloblastic fibrosarcoma; pineal tumor, malignancy; chordoma; glioma, malignant; ventricular tube membranoma; astrocytoma; plasmacytoma; fibroastrocytoma; astrocytoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglion neuroblastoma; neuroblastoma; retinoblastoma; an olfactory neurogenic tumor; meningioma, malignancy; neurofibrosarcoma; schwannoma, malignancy; granulocytoma, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; granuloma parades; malignant lymphoma, small lymphocytes; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-hodgkin lymphomas; b cell lymphoma; low grade/follicular non-hodgkin lymphoma (NHL); small Lymphocytes (SL) NHL; intermediate grade/follicular NHL; medium grade diffuse NHL; advanced immunoblastic NHL; higher lymphoblastic NHL; advanced small non-nucleated cells NHL; large mass NHL; mantle cell lymphoma; AIDS-related lymphoma; primary macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestine disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic granulocytic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; hairy cell leukemia; chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); acute Myelogenous Leukemia (AML); and chronic myeloid leukemia.

The therapies provided herein can include administration of a combination of therapeutic agents, e.g., a first cancer therapy and a second cancer therapy. These therapies may be administered in any suitable manner known in the art. For example, the first and second cancer treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered as separate compositions. In some embodiments, the first and second cancer treatments are in the same composition. Embodiments of the present invention relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or more than one composition, for example in 2 compositions, 3 compositions or 4 compositions. Various combinations of agents may be used. Examples of therapies other than the present invention include surgery, chemotherapy, drug therapy, radiation therapy, hormonal therapy, immunotherapy (other than the present invention) or a combination thereof.

The therapeutic agents of the invention may be administered by the same route of administration or by different routes of administration. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implantable, inhaled, intrathecally, intraventricular, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implantable, inhaled, intrathecally, intraventricular, or intranasally. The appropriate dosage may be determined according to the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual and the response to the treatment, as well as the discretion of the attendant physician.

Treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, as well as the particular route and formulation, is within the skill of the clinical technician's determination. The unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some embodiments, the unit dose comprises a single administrable dose.

Depending on the number of treatments and the unit dose, the amount administered will depend on the desired therapeutic effect. An effective dose is understood to mean the amount necessary to achieve a particular effect. In the practice of certain embodiments, it is contemplated that dosages in the range of 10mg/kg to 200mg/kg may affect the protective capabilities of these agents. Thus, the expected dosages include dosages of 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 may be administered multiple times during the day, and/or over multiple days, weeks or months.

In certain embodiments, an effective dose of the pharmaceutical composition is a dose that can provide blood levels of about 1 μm to 150 μm. In another embodiment, an effective dose provides a blood level 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 drug blood levels resulting from the therapeutic agent administered to the 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、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 or 100 μm or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in vivo to a metabolized therapeutic agent, in which case blood levels may refer to the amount of metabolized therapeutic agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the therapeutic agent not metabolized.

The precise amount of therapeutic composition will also depend on the discretion of the practitioner and is specific to each individual. Factors that affect the dosage include the physical and clinical state of the patient, the route of administration, the intended target of the treatment (relief of symptoms and cure), and the efficacy, stability, and toxicity of the particular therapeutic substance or other treatment that the subject may be receiving.

Those skilled in the art will understand and appreciate that dosage units of μg/kg or mg/kg body weight may be converted and expressed in units of comparable concentration of μg/ml or mM (blood level), for example 4 μM to 100 μM. It is also understood that uptake is species and organ/tissue dependent. Suitable conversion factors and physiological assumptions regarding uptake and concentration measurements are well known and allow one skilled in the art to convert one concentration measurement to another and to reasonably compare and draw conclusions about the dosages, efficacy and results described herein.

V. kit

Certain aspects of the invention also relate to kits comprising the compositions of the invention or compositions for practicing the methods of the invention. In particular embodiments, the kit comprises fresh or frozen NK cells and may or may not have been pre-activated or expanded. NK cells may or may not express one or more components of the TCR/CD3 complex. In the case where the NK cells have not expressed one or more components of the TCR/CD3 complex, the kit may comprise reagents for the corresponding transfection or transduction of the NK cells, including reagents such as vectors expressing the components, primers for amplifying the components, and the like. In some cases, NK cells may or may not express one or more heterologous proteins as defined herein, and when they do not express, the kit may comprise vectors expressing the heterologous proteins, primers for amplifying the heterologous proteins, and the like.

The kit may include components that may be packaged separately or placed in containers, such as tubes, bottles, vials, syringes, or other suitable container means. The individual components may also be provided in the kit in concentrated amounts; in some embodiments, the components are provided separately at the same concentration in solution as the other components. The concentration of the components may be 1x, 2x, 5x, 10x or 20x or more.

VI. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate 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.

Example 1

Preparation and efficient use of NK cells expressing CD3

This example relates to cancer immunotherapy as a strategy to redirect NK cells' specificity for one or more target antigens by "arming" or pre-complexing them with bispecific or multispecific antibodies (e.g. prior to infusion or by co-infusing the two products separately). NK cells are transduced with one or more CD3 chains, including the cd3ζ, cd3γ, cd3δ, and cd3ε chains, 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 IL12/15/18, 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, and/or cytokine genes and/or homing/chemokine receptors.

FIGS. 1A and 1B show different embodiments of NK cells engineered for use with bispecific or multispecific antibodies. As shown in fig. 1A, in the first generation of NK cells, cells engineered to express CD3 can be activated by bispecific or multispecific antibodies including bispecific T cell engagement molecules (bites) comprising anti-CD 3 antibodies that bind to NK cell surface expressed heterologous CD3. In another embodiment, NK cells expressing CD3 are capable of binding by BiTE comprising anti-CD 3 antibodies and NK cells also express one or more specific cytokines (e.g. IL-15 and/or IL-21), resulting in increased efficacy and potency, which is particularly useful for treating solid tumors. In another embodiment, NK cells are engineered to express CD3, not only are capable of being activated by BiTE comprising an anti-CD 3 antibody, but can also be used with bispecific or multispecific antibodies (e.g., bispecific NK cell binding molecules or BiKE) comprising antibodies that bind to a surface antigen naturally occurring on NK cells, such as CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, or KIR. In this way, NK cells are responsive to both NK and T cell engagement molecules. In another embodiment, the NK cells express an engineered antigen receptor, such as a CAR or engineered TCR, in addition to CD3 for binding to a T cell engagement molecule.

FIG. 1B shows a different embodiment in which NK cells are modified to express CD3 and TCR. On the right, T cell TCRs are shown as alpha and beta chains with antigen binding sites, where the TCRs complex with cd3ζ to affect signal transduction. The T cell TCR is co-complexed with two CD3 epsilon, CD3 delta and CD3 gamma chains. In some embodiments, the NK cells express a TCR in which one or more cytoplasmic domains of any CD3 molecule are heterologous intracellular domains, e.g., one or more cytoplasmic domains from one of CD16, NKG2D, DAP, DAP12, NCR, and DNAM-1. As shown on the left side of fig. 1B, NK cells are configured to express a CD3 co-receptor component, in one embodiment, the CD3 component is CD3 epsilon. In this case, standard BiTE (upper left corner contains antibody against tumor antigen and antibody against CD 3) commonly used with T cells naturally expressing CD3 can be used with NK cells expressing CD 3. In this particular example, the NK cell expresses a polypeptide comprising the extracellular domain of CD3 epsilon (although the extracellular domain of other CD3 components may be utilized) and the extracellular domain of CD3 epsilon is linked to the transmembrane and/or cytoplasmic domain of another molecule, such as CD3 zeta, CD16, NKG2D, DAP, DAP12, NCR, or DNAM-1.

As described above, fig. 1C schematically depicts the generation of a surface expressed single chimeric CD3 construct that can be used with anti-CD 3 BiTE. For example, the CD3 epsilon extracellular domain (ECD) is fused to a CD28, CD16, or NKG2D Transmembrane (TM) domain and a CD28, CD16, or NKG2D intracellular domain (ICD), with or without a CD3 zeta and/or DAP10 intracellular domain. In one embodiment, the construct is contained within the vector backbone of an SFG retrovirus derived from Moloney murine virus, which can be used with packaging plasmids to produce the virus. In the case of CD3 BiTE used with this construct in fig. 1C, the antibody will therefore bind to the extracellular domain of CD3 epsilon.

Embodiments of the invention utilize part or all of the CD3 receptor complex. As shown in fig. 2A and 2B, NK cells can be transfected or transduced with full length cd3ζ, cd3γ, cd3δ, and cd3ε. In this case, each of the full length cd3ζ, cd3γ, cd3δ, and cd3ε includes an extracellular domain, a transmembrane domain, and an intracellular domain. When the different components of the receptor are expressed from the same vector, they may be configured to be produced as separate polypeptides, e.g., separated by IRES or 2A elements. In any case, any expression construct may be configured to express one or more cytokines, including at least IL-15.

FIG. 4 shows CD3 expression on NK cells on day 4 after CMV TCR complex transduction. The figure provides a FACS plot showing CD3 expression on NK cells 4 days after CMV TCR complex transduction. Non-transduced (NT) NK cells (CD56+CD3-) served as negative control and T cells (CD3+CD56-) served as positive control.

FIG. 5 shows TCR expression on NK cells on day 4 after transduction of NK by CMV TCR complex. In particular, FACS diagrams showing expression of TCRa/b on NK cells 4 days after CMV TCR complex transduction are provided. Non-transduced (NT) NK cells (CD56+CD3 TCRa/b-) served as negative control and T cells (CD3+ TCRa/b+CD56-) served as positive control.

FIG. 6 shows TCR/CD3 expression on NK on day 6 post CMV TCR complex transduction. Specifically, FACS plots show double CD3 and TCRa/b expression on NK cells 6 days after CMV TCR complex transduction. Non-transduced (NT) NK cells (CD56+CD3 TCRa/b-) served as negative control and T cells (CD3+ TCRa/b+CD56-) served as positive control.

In FIG. 7, the binding of CD3-CD19 BiTE to NK cells by CD3/TCR at various concentrations is shown. Specifically, various cells (non-transduced (NT) NK cells, T cells or three different NK-TCR cells) were incubated with Bonauzumab (a CD3-CD19 bispecific binding molecule (BiTe)) at 37℃for 1 hour using two different concentrations (0.5. Mu.g/. Mu.l or 4. Mu.g/. Mu.l). The biotin-labeled CD19 antigen (CD 19 CAR detection reagent from Miltenyi) was then added for 20 minutes, followed by the addition of the anti-biotin antibody at room temperature for 15 minutes. This method was used to detect any BiTE that bound to cd3+ cells. The histogram in FIG. 7 shows the binding levels of CD19 to CD3-CD19 bispecific binding molecules (BiTe) associated with CD3 expression on NK-TCR and T cells.

FIG. 8 shows NK-TCR cytokine production following stimulation with plate-bound CD3 antibody. In particular, 20. Mu.g/ml of CD3-OKT3 clones were incubated overnight at 4℃in flat bottom 96-well plates to form plate-bound antigen. The next day, T cells or NK cells (NT or TCR transduced) were added to the wells for 4 hours, and brefeldin a (which prevented cytokine release, captured in the cytoplasm, so that it could be detected by intracellular cytokine staining) was added. They were then collected for surface and intracellular staining to assess cytokine production and degranulation (tnfα and CD107 a). The FACS plot in fig. 8 shows tnfα and CD107a biscationic populations in NK cells transduced with TCRs. Non-transduced (NT) NK cells (CD56+CD3-) served as negative control and T cells (CD3+CD56-) served as positive control.

FIG. 9 shows phosphorylation of CD3 zeta after CD3 cross-linking in NK TCR/CD3 cells. 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 group was transduced with CD3ZFLGDEFL (see fig. 2A and 2B). NK cells were incubated with CD3OKT3 clones (Miltenyi, 130-093-387) at a concentration of 20. Mu.g/ml for 20 min on ice. Cells were then cross-linked with Fab2 IgG1 antibodies at different time points and stained to check CD3z phosphorylation. This analysis of cd3ζ is useful because, as an internalization signal from the surface, NK cells can only cross-link with CD3 monoclonal antibodies if they express cd3ζ. NK cells not transduced with CD3 do not show any phosphorylation or activation after stimulation.

NK cells transduced with CD3-TCR also showed basal levels of sustained signaling that increased upon CD3 OKT3 stimulation and were similar to T cells, whereas non-transduced NK cells did not show any CD3 zeta phosphorylation, neither upon basal nor upon CD3 OKT3 stimulation.

FIG. 10 shows that pre-culturing CD3-CD19 BiTE with TCR/CD3 expressing NK cells increases their killing activity against Raji cells. NK cells were either loaded with CD3-TCR#1 (CD 3ZFLGDEFL (see FIGS. 2A and 2B)) or CD3-TCR#2 (Z2, also known as CD3ZGDEFL8SP21CD 8), which includes 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 CD8 transmembrane domain) CD3/TCR construct transduced NK cells or non transduced NK cells were loaded with Blinatumumab and incubated with PBS for 1 hour and washed with PBS then co-cultured with CD19+B cell lymphoma cells at different effector cell: target cell ratios (FIG. 10A is a 1:1 ratio, FIG. 10B is a 1:5 ratio) as used herein, effector cells are CD-3-NK cells, target cells are Raji cells CD3 transduced cells loaded with TCR Blinatumumab showed both anti-emetic activity at the same rate as non transduced NK cells loaded with Blinatumumab or CD 3/transduced NK cells.

Example 2

NY-ESO TCR in NK cells

This example relates to the generation and use of NY-ESO TCRs in NK cells. In FIG. 11, there is an example of a producer cell. The schematic shows a situation where NK cells are first transduced with uTNK construct, uTNK construct comprising signaling domain from CD3 complex, NK co-stimulatory molecule and IL-15, followed by a second transduction step introducing TCR molecules, thereby generating NK cells co-expressing CD3 and NK signaling molecule, IL-15 and TCR complex. In one embodiment, NK cells are derived from cord blood and expanded with irradiated (100 Gy) universal antigen presenting cells (uAPC) feeder cells (feeder cells: NK ratio 2:1) and recombinant human IL-2 (200U/ml) in complete medium. To generate universal T cell-like NK cells (uTNK cells) that secrete IL-15, NK cells were purified and transduced 4 days after isolation with a retroviral construct containing the CD3 complex with NK costimulatory molecules and IL-15 gene. 48 hours after initial transduction, NK cells expressing uTNK were then transduced with a TCR targeting the selected antigen.

FIG. 12 shows expression of NY-ESO TCR on NK cells transduced with uTNK. NK cells were derived from cord blood and expanded in complete medium with irradiated (100 Gy) universal antigen presenting cells (uAPC) feeder cells (feeder cells: NK ratio 2:1) and recombinant human IL-2 (200U/ml). To generate universal T cell-like NK cells that secrete IL-15, NK cells were purified and transduced 4 days after isolation with a retroviral construct containing the CD3 complex with NK costimulatory molecules and IL-15 gene. 48 hours after initial transduction, uTNK cells were then transduced with TCR complexes targeting the selected antigen. After 48 hours, expression of CD3 and NY-ESO TCRs on the various uTNK constructs was assessed using flow cytometry. Non-transduced (NT) NK cells were used as negative control. CD3 and NY-ESO TCRs are highly expressed on all uTNK cells compared to NT NK cells. FIG. 13 provides the number of tumor-specific TCR molecules expressed on TCR engineered NK cells using various TCR constructs and NT NK cells as negative controls.

FIG. 14 shows the expression of NY-ESO TCR on non-transduced and transduced T cells. T cells were isolated from cord blood (same donor as NK cells as paired positive control) and activated with CD3/CD28 microbeads at a concentration of 25 μl/100 ten thousand for 48 hours in RPMI complete medium. T cells were then transduced with retroviral constructs containing NY-ESO TCR. 48 hours post transduction, flow cytometry showed that NY-ESO TCRs were highly expressed on transduced T cells compared to non-transduced T cells.

NK cells transduced with NY-ESO TCR killed target cells loaded (pulsed) with NY-ESO peptide in a dose dependent manner (FIG. 15). Chromium ⁵¹ CR killing assays were performed 7 days after TCR transduction to determine the killing capacity of TCR-engineered NK cells and T cells on LCL cells loaded with varying concentrations of NY-ESO peptide (for 2 hours). The NY-ESO TCR transduced uTNK cells showed enhanced killing of peptide-loaded LCL cells compared to non-transduced NK cells. NY-ESO TCR transduced T cells showed enhanced killing of peptide-loaded LCL cells compared to non-transduced T cells.

FIG. 16 shows that NY-ESO is expressed endogenously in myeloma, sarcoma and melanoma cell lines. Flow cytometry was used to determine the expression of NY-ESO on 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 compared to the Raji cell line used as negative control.

NY-ESO TCR transduced T cells killed tumor targets expressing NY-ESO at higher E:T ratios (FIG. 17). Chromium ⁵¹ CR killing assays were performed 7 days after TCR transduction to determine the killing capacity of NY-ESO TCR engineered T cells against NY-ESO expressing myeloma, osteosarcoma and melanoma cell lines. NY-ESO TCR transduced T cells showed enhanced killing of NY-ESO positive cell lines compared to non-transduced T cells.

FIG. 18 shows that NY-ESO TCR transduced NK cells kill NY-ESO expressing tumor targets even at low E:T ratios. Chromium ⁵¹ CR killing assays were performed 7 days after TCR transduction to determine the killing capacity of NY-ESO TCR engineered NK cells against NY-ESO expressing myeloma, osteosarcoma and melanoma cell lines. NY-ESO TCR transduced NK cells showed enhanced killing of NY-ESO positive cell lines even at very low effector: target ratios compared to non-transduced NK cells.

FIG. 19 shows that NY-ESO transduced NK cells have a phenotype similar to NT NK cells. CytoF imaging showed that the untransduced NK cells and NY-ESO TCR transduced uTNK cells had similar phenotypes. FIG. 19A shows a u-plot with similar clusters and FIG. 19B shows a heat map of various markers with similar expression on NT and NY-ESO TCR transduced uTNK cells.

FIG. 20 provides a table showing the percentage of CD3+ and CD3+ TCR+ NK cells in each uTNK 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+). The untransduced NK cells consisted of less than 1% cd3+ and cd3+ tcr+ NK cells, while the TCR transduced uTNK cell products consisted of more than 60% cd3+ and more than 25% cd3+ tcr+ NK cells.

FIG. 21A provides FACS diagrams showing successful expression of CD3 on NK cells 4 days after transduction with TCR constant α - β (TCRCab; TCR6 construct). Non-transduced (NT) NK cells (CD56+CD3-) served as negative control. In FIG. 21B, NT NK and uTNK NK cells were incubated with Blinatumumab, a CD3-CD19 bispecific binding molecule (BiTe), at 37℃for 1 hr at 10. Mu.g/. Mu.l. The biotin-labeled CD19 antigen (CD 19 CAR detection reagent from Miltenyi) was then added for 20 minutes, followed by the addition of the anti-biotin antibody at room temperature for 15 minutes. This method was used to detect any BiTE that bound to cd3+ cells. The histogram in this figure shows the level of binding of CD19 to CD3-CD19 bispecific binding molecule (BiTe) associated with CD3 expression on uTINK NK cells. In FIG. 21C, CD3/TCR transduced or non-transduced NK cells were loaded Blinatumumab and incubated for 1 hour and washed with PBS. They were then co-cultured with LCL cells of different E:T ratios (A.1:1, B.1:5) to different time points. CD3-TCR transduced NK cells loaded with Blinatumumab showed enhanced anti-tumor activity compared to either untransduced NK cells or CD3/TCR transduced NK cells loaded with Blinatumumab, but not loaded with Blinatumumab at both E: T ratios.

Example 3

NY-ESO TCR in NK cells expressing CD3 in vivo

As shown in fig. 22A-22C, NK cells comprising the constructs described herein were tested in vivo and found to strongly inhibit tumor growth. Fig. 22A is a schematic diagram showing an experimental procedure performed. Briefly, NSG mice received 300cGy radiation on day-1 and then 0.5X10 ⁶ U266-B1 cells transduced with firefly luciferase (FFluc) (myeloma cell line expressing HLA-A2 and NY-ESO antigens) on day 0, mice were infused with 5X 10 ⁶ effector cells (NY-ESO TCR NK cells with WT, # A or #BUT-NK 15-NY ESO TCR constructs, respectively; WT refers to wild-type CD3 molecules with IL-15; #A refers to CD3-CD28 with IL-15 (e.g., UT-NK 15-28); #B refers to CD3-DAP10 with IL-15 (e.g., UT-NK15-DAP 10); or NY-ESO TCR T cells) on day 3), and animals were monitored over time and sacrificed as appropriate (N=5 mice per group). Fig. 22B shows the monitoring results of the experiment described in fig. 22A as a function of time-dependent bioluminescence imaging (representative images from day 1, day 7, day 14 and day 21, respectively, are shown). FIG. 22C is a graphical quantification of the bioluminescence mean radiation shown in FIG. 22B, with the Y axis representing the mean radiation in units of p/s/cm ²/sr and the X axis representing time.

As shown in fig. 23A-B, effector cells (e.g., NK cells or T cells) comprising NY-ESO targeted TCR and UT-NK15 constructs were tested for in vitro activity. FIG. 22A is an image of a cell sphere formed by osteosarcoma tumor cell line Saos-2, which was used to test the cytotoxic activity of NK cells and T cells expressing NY-ESO1 specific TCR. Stably transducing Saos-2 cells to express GFP; 10,000 of these cells per well were seeded overnight in 96-well plates, followed by the addition of 40,000 NK cells or T cells. Images of the co-cultures were scanned over time and analyzed by the IncuCyte cell analysis system. Fig. 22B is a graph showing percent cytotoxicity (Y-axis) of effector cells captured from representative images after 3 days of co-culture. NK cells co-transduce with NY-ESO-TCR and UT-NK15 signaling complex, the UT-NK15 signaling complex co-expressing a different co-stimulatory molecule fused to the CD3 zeta signaling chain (e.g., UTNK-15-28 or UTNK-15-DAP 10). T cells were transduced with NY-ESO TCR alone. Abbreviations in the figures are as follows: 28 Cd3ζ fused to CD28 costimulatory domain; 10 Cd3ζ fused to Dap10 costimulatory domain; 8 = CD8 a/β co-receptor as part of NY ESO TCR construct; wo IL-15 = the construct contained only cd3ζ, ε, γ and δ, without co-stimulation or IL-15. The TCR NK cells expressed UTNK the DAP10 costimulatory domain fused to CD28 or to CD3ζ (e.g., UTNK-15-28 or UTNK-15-DAP10; corresponding SEQ ID NO:121 and SEQ ID NO: 119) compared to NK cells transduced with CD3 complex alone or UT-NK15 without costimulatory domain. The addition of CD8 a/β co-receptors to TCRs did not significantly improve NK cell or T cell cytotoxicity.

As shown in fig. 24A-D, effector cells (e.g., NK cells or T cells) comprising NY-ESO targeted TCR and UT-NK15 constructs were tested for in vivo activity. FIG. 24A depicts a plan for in vivo studies to test the activity of different NY ESO TCR transduced NK and T cells. The program was performed in which 10 week old NSG mice were irradiated (300 cGy) and 500,000U 266 cells (HLA-A 2 positive, NY ESO expressing myeloma cell lines) were injected by tail vein the next day. Three days later, mice received 500 ten thousand TCR-transduced T cells or TCR-transduced NK cells. Mice were then monitored for tumor control by BLI imaging. Fig. 24B shows the BLI imaging results of the test described and performed according to fig. 24A. Mice were injected with U266 tumor cells alone, or also with T cells transduced with NY ESO-specific TCR, or also with NK cells co-transduced with NY ESO TCR and UT-NK15, with CD3 zeta fused with CD28 in UT-NK15 (labeled NY ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15CD28 NK cells). Fig. 24C shows quantification of the average radiation intensity of a region of interest of an animal tested according to fig. 24A and imaged in fig. 24B. Fig. 24D shows a survival graph depicting a group of the above animals. The results indicate that NY ESO TCR T and NY-ESO TCR UTNK-15-CD28 NK cells mediate strong antitumor activity in vivo.

As shown in fig. 25, NY ESO TCRs and effector cells (e.g., NK cells) of the CD3 complex with or without IL-15 were tested for in vivo activity. NSG mice were irradiated (300 cGy) and the next day by tail vein injection of 500,000U 266 cells (HLA-A 2 positive, NY ESO expressing myeloma cell line). Three days later, mice received 500 ten thousand TCR-transduced T cells or NK cells. Tumor control in mice was monitored by BLI imaging. NK cells were transduced with NY ESO-specific TCR and co-transduced with either a CD3 complex without IL-15 or with UT-NK15 expressing CD3 ζ fused to CD28 co-stimulatory molecule (UT-NK 15-28) or UT-NK15 expressing CD3 ζ fused to DAP10 co-stimulatory molecule (UT-NK 15-DAP 10), with or without expression of CD8 α/β co-receptor. The results show that the lack of IL-15 results in a decrease in anti-tumor activity in vivo.

Together, these results demonstrate that effector cells (e.g., NK cells) comprising a construct described herein (e.g., NY-ESO TCR construct and/or CD3 construct, e.g., UT-NK15 or modified versions thereof, e.g., UT-NK-15-28 or UT-NK15-DAP 10) are sufficient to strongly inhibit tumor growth in vivo.

Example 4

PRAME TCR in NK cells expressing CD3 in vitro

As shown in fig. 26A-C, NK cells comprising constructs targeting the melanoma preferential expression antigen (PRAME) described herein were tested in vitro and found to strongly inhibit the growth of tumor cells. FIG. 26A shows the expression of UT-NK15 (x-axis, CD 3) and PRAME-specific TCR (y-axis, TCR) in NK cells (TCR clone 46, 54 or DSK3, respectively), or PRAME-specific TCR in T cells transduced with the same (TCR clone 46 or 54). PRAME-specific TCR expression on NK cells was confirmed using antibodies against TCR and CD 3. Expression of PRAME-specific TCRs in T cells was confirmed by tetramer staining using 46/54 peptide/MHC-specific tetramers. Figure 26B shows in vitro cytotoxicity of NK cells expressing PRAME-specific TCRs against the U266 myeloma cell line. Cytotoxicity of U266 myeloma cells on T cells transduced with PRAME-specific TCR and NK cells transduced with UT-NK15 and PRAME-specific TCR was measured using Incucyte live cell imaging. GFP expressing U266 cells were co-cultured with PRAME specific TCR expressing T cells or NK cells at an effector to target ratio of 1:1 (50,000 effectors and 50,000 target cells were seeded in each well of a 96 well plate). A decrease in GFP expression indicates cell death. After 26 hours, a second round of 50,000 tumor cells (referred to as "re-challenge") was added to each well for the tumor re-challenge assay. NK cells expressing UT-NK15 and PRAME specific TCR clone 46 or PRAME specific TCR clone 54 exert optimal antitumor activity upon re-challenge with U266 cells and showed excellent cytotoxicity compared to control T cells transduced with PRAME specific TCR clone 46 or 54, respectively. Figure 26C shows in vitro cytotoxicity of NK cells expressing PRAME-specific TCRs against UA375 melanoma cell lines. Cytotoxicity of UA375 melanoma cells by T cells transduced with PRAME-specific TCRs and NK cells transduced with UT-NK15 and PRAME-specific TCRs was measured using intucyte live cell imaging. GFP-expressing UA375 cells were co-cultured with PRAME-expressing T cells or NK cells at a 1:1 effector to target ratio (50,000 effectors and 50,000 target cells were seeded in each well of a 96-well plate). A decrease in GFP expression indicates cell death. After 26 hours, a second round of 50,000 tumor cells were added to each well for tumor re-challenge assays. The open symbols represent T cells, while the filled symbols represent NK cells. Nt=untransduced. NK cells expressing UT-NK15 and PRAME specific TCR clone 46 (TCR-46), PRAME specific TCR clone 54 (TCR-54) or PRAME specific TCR clone DSK3 (DSK) showed excellent anti-tumor activity when re-challenged with UA375 cells and showed excellent cytotoxicity compared to control T cells transduced with PRAME specific TCR clone 46, 54 or DSK3, respectively.

Together, these results demonstrate that effector cells (e.g., NK cells) comprising the constructs described herein (e.g., PRAME-specific TCR constructs) are sufficient to strongly inhibit tumor growth in vivo. Furthermore, NK cells comprising the CD3 construct and PRAME-specific TCR construct described herein show increased cytotoxicity compared to T cell control cells comprising the same TCR construct, especially in case of tumor cell progression and/or re-challenge.

Example 5

TCR in NK cells expressing CD3 in vivo

NK cells comprising the constructs described herein were tested in vivo and strongly inhibited tumor growth. Experiments were performed according to the schematic and experimental procedure described herein. Briefly, NSG mice are irradiated (e.g., about 300 cGy) on day-1, then on day 0, individual mice receive tail vein injected cancer cells (e.g., 0.5x10 ⁶ cells, e.g., cells expressing (native and/or transduced) antigens described herein) and transduced with appropriate markers (e.g., firefly luciferase (FFluc)), and on day 3 mice are injected with effector cells transduced with a transgenic TCR (e.g., TCR constructs comprising γ/δ TCR chains and/or α/β TCR chains, e.g., TCR constructs targeted to antigens 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., about 5x 10 ⁶ NK cells with UT-NK15 constructs with or without IL15, with or without co-stimulatory molecules and with or without additional constructs with or without co-stimulatory molecules). Animals were then monitored over time and sacrificed as appropriate. The monitoring results of the above experiments are recorded, for example, as a function of bioluminescence imaging over time (e.g., on day 1, day 7, day 14, day 21, etc.).

In vitro activity of effector cells (e.g., NK cells or T cells) comprising TCRs (e.g., TCR constructs comprising gamma/delta TCR chains and/or alpha/beta TCR chains, e.g., targeting antigens described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.) and UT-NK15 constructs is tested. The cytoballs formed from an appropriate tumor cell line (e.g., 0.5x10 ⁶ cells, e.g., cells expressing (naturally and/or with) an antigen as described herein) comprising the antigen of interest are used to test the cytotoxic activity of NK and/or T cells expressing a specific TCR. Stably transducing cancer cells to express appropriate markers (e.g., GFP, FFluc, etc.); many of these cells (e.g., about 10,000) were seeded overnight in each well of a 96-well plate, and then many effector cells (e.g., about 40,000) were added. Images of the co-cultures are scanned over time and analyzed by a suitable system (e.g., the IncuCyte cell analysis system). After several days of co-culture (e.g., 1 day, 3 days, 7 days, etc.), the percent cytotoxicity of effector cells was captured from representative images. NK cells co-transduce with antigen-targeting TCR and UT-NK15 signaling complexes, the UT-NK15 signaling complex co-expressing a different co-stimulatory molecule fused to the CD3 zeta signaling chain (e.g., UTNK-15-28 or UTNK-15-DAP 10). Appropriate control cells are transduced with the appropriate constructs described herein. Superior in vitro cytotoxicity was observed with TCR NK cells expressing UTNK with CD28 or DAP10 costimulatory domains fused to CD3 ζ (e.g., UTNK-15-28 or UTNK-15-DAP10; e.g., corresponding SEQ ID NO:121 and SEQ ID NO: 119) compared to NK cells transduced with CD3 complex alone or UT-NK15 transduced with NO costimulatory domain.

In vivo activity of effector cells (e.g., NK cells or T cells) comprising antigen-specific TCRs (e.g., TCR constructs comprising gamma/delta TCR chains and/or alpha/beta TCR chains, e.g., targeting antigens described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.) and UT-NK15 constructs is tested. Assays for in vivo analysis of effector cells (e.g., NK cells or T cells) comprising the engineered constructs were performed in an experimental plan similar to that described in fig. 24. Briefly, NSG mice of the appropriate age (e.g., 10 week old NSG mice) are irradiated (e.g., with about 300 cGy) and tumor cells containing the target antigen of interest (e.g., about 500,000 cells; e.g., naturally expressed and/or transduced with the antigens described herein) are injected by tail vein the next day. Three days later, mice received effector cell bolus injections (e.g., about 500 ten thousand TCR-transduced T cells and/or TCR-transduced NK cells). Mice are then monitored for tumor control (e.g., by BLI imaging). The mean radiation of the region of interest is measured and quantified, and animals comprising the test construct containing the TCR targeting the antigen of interest and the UT-NK15 construct with or without CD3 fusion and/or IL-15 expression show improved survival and/or reduced mean radiation relative to control animals. The results indicate that TCR UTNK-15NK cells mediate strong antitumor activity in vivo.

TCR constructs comprising TCRs (e.g., TCR constructs comprising a gamma/delta TCR chain and/or an alpha/beta TCR chain, e.g., targeted to antigens described herein, e.g., NY-ESO, tyrosinase, MAGEA3, MAGEA4, HPV E7, WT1, PRAME, gp100, MART-1, etc.) and effector cells (e.g., NK cells) with or without the CD3 complex of IL-15 are tested. NSG mice are irradiated (e.g., with about 300 cGy), and tumor cells expressing the antigen (e.g., about 500,000 cells; e.g., naturally expressed and/or transduced with the antigens described herein) are injected by tail vein the next day. Three days later, mice received effector cell bolus injections (e.g., about 500 ten thousand TCR-transduced T cells and/or TCR-transduced NK cells). Mice are monitored for tumor control (e.g., by BLI imaging). NK cells were transduced with antigen specific TCR and co-transduced with either a CD3 complex without IL-15 or UT-NK15 (UT-NK 15-28) expressing CD3 zeta fused to a CD28 co-stimulatory molecule or UT-NK15 (UT-NK 15-DAP 10) expressing CD3 zeta fused to a DAP10 co-stimulatory molecule with or without expression of a CD8 alpha/beta co-receptor. The results indicate that the lack of IL-15 results in reduced antitumor activity in vivo.

Together, these results demonstrate that effector cells (e.g., NK cells) comprising a construct described herein (e.g., a TCR construct and/or a CD3 construct, e.g., UT-NK15 or modified versions thereof, e.g., UT-NK-15-28 or UT-NK15-DAP 10) are sufficient to strongly inhibit tumor growth in vivo.

All 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 of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All 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

1. A composition comprising NK cells modified to express part or all or any combination of the individual chains of cd3δ, cd3ε, cd3γ, or cd3ζ.

2. The composition of claim 1, wherein the NK cells are modified to express one or more of a TCR a chain, a TCR β chain, a TCR γ chain, and a TCR δ chain.

3. The composition of claim 1 or 2, wherein the NK cells are modified to express a T Cell Receptor (TCR) αβ chain or a tcrγδ chain.

4. The composition of claim 1, wherein the NK cells are modified to express part or all of only constant regions of one or more of a TCR a chain, a TCR β chain, a TCR γ chain, and a TCR δ chain.

5. The composition of claim 1, wherein the NK cells are modified to express part or all of a constant region only of a T Cell Receptor (TCR) αβ chain or a tcrγδ chain.

6. The composition of any one of claims 1-5, wherein the NK cells are modified to express some or all of cd3ζ, two cd3ε, cd3δ, and cd3γ.

7. The composition of any one of claims 1-6, wherein the NK cells are modified to express full length: cd3ζ, cd3ε, cd3δ and/or cd3γ.

8. The composition of any one of claims 1-7, wherein any one or more of cd3ζ, cd3ε, cd3δ, and cd3γ is heterologously linked to one or more intracellular signaling domains.

9. The composition of claim 8, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof.

10. The composition of claim 8 or 9, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

11. The composition of any one of claims 8-10, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 115.

12. The composition of any one of claims 8-11, wherein the intracellular signaling domain comprises the amino acid sequence according to SEQ ID No. 115.

13. The composition of claim 8 or 9, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

14. The composition of any one of claims 8, 9 or 13, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID No. 116.

15. The composition of any one of claims 8, 9, 13 or 14, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 116.

16. The composition of claim 8 or 9, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

17. The composition of any one of claims 8, 9 or 16, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID No. 117.

18. The composition of any one of claims 8, 9, 16 or 17, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 117.

19. The composition of any one of claims 1-18, wherein the composition further comprises one or more bispecific or multispecific antibodies, wherein the bispecific or multispecific antibodies comprise an anti-CD 3 antibody.

20. The composition of claim 19, wherein the NK cells express the antibody.

21. The composition of claim 19 or 20, wherein the NK cells are complexed with the antibody.

22. The composition of claim 20 or 21, wherein the antibody is bolafuximab.

23. The composition of any one of claims 1-22, wherein the TCR is directed against a cancer antigen or a viral antigen.

24. The composition of any one of the preceding claims, wherein the NK cells are derived from umbilical Cord Blood (CB), peripheral Blood (PB), bone marrow, stem cells, or mixtures thereof.

25. The composition of any one of the preceding claims, wherein the NK cells are pre-activated.

26. The composition of claim 25, wherein the NK cells are pre-activated by one or more cytokines.

27. The composition of claim 26, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

28. The composition of any one of the preceding claims, wherein the NK cells are expanded.

29. The composition of claim 28, wherein the NK cells are expanded in the presence of IL-2.

30. The composition of any one of the preceding claims, wherein the NK cells are modified to express one or more heterologous proteins.

31. The composition of claim 30, wherein the heterologous protein is an engineered antigen receptor, cytokine, homing receptor, or chemokine receptor.

32. The composition of claim 31, wherein the engineered antigen receptor is a chimeric antigen receptor and/or an engineered T cell receptor.

33. The composition of claim 32, wherein the engineered antigen receptor is an engineered T cell receptor, and wherein the engineered T cell receptor targets an NY-ESO antigen.

34. The composition of claim 33, wherein the T cell receptor comprises a sequence at least 85% identical to SEQ ID No. 25 and a sequence at least 85% identical to SEQ ID No. 26.

35. The composition of claim 33, wherein the engineered antigen receptor is an engineered T cell receptor, and wherein the engineered T cell receptor targets a PRAME antigen.

36. The composition of claim 35, wherein the target PRAME epitope is SLLQHLIGL (SEQ ID NO: 131) and/or QLLALLPSL (SEQ ID NO: 132).

37. The composition of claim 35 or 36, wherein the T cell receptor comprises a sequence at least 85% identical to SEQ ID No. 135 and a sequence at least 85% identical to SEQ ID No. 136.

38. The composition of claim 35 or 36, wherein the T cell receptor comprises a sequence at least 85% identical to SEQ ID No. 139 and a sequence at least 85% identical to SEQ ID No. 140.

39. The composition of claim 35 or 36, wherein the T cell receptor comprises a sequence at least 85% identical to SEQ ID No. 143 and a sequence at least 85% identical to SEQ ID No. 144.

40. The composition of any one of claims 30-39, wherein the heterologous protein is a cytokine.

41. The composition of any one of claims 26-40, wherein the cytokine is selected from the group consisting of: IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, or combinations thereof.

42. The composition of claim 41, wherein the cytokine is membrane-bound.

43. The composition of claim 41 or 42, wherein the cytokine is IL-15.

44. The composition of claim 42 or 43, wherein the membrane-bound cytokine comprises a transmembrane domain from CD8, CD28, CD27, B7H3, igG1, igG4, CD4, DAP10, or DAP 12.

45. The composition of any one of claims 26-44, wherein said NK cells express a chimeric antigen receptor and a cytokine.

46. The composition of any one of the preceding claims, wherein the bispecific antibody comprises an antibody that targets a cancer antigen.

47. The composition of claim 46, wherein the cancer antigen is a CD19 antigen.

48. The composition of claim 46 or 47, wherein the bispecific antibody is bolafuximab.

49. A composition comprising a complex, the composition comprising:

(1) NK cells modified to express part or all of the CD3 receptor complex and optionally modified to express a T Cell Receptor (TCR) αβ chain or a tcrγδ chain; and

(2) A bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD 3 antibody which binds to CD3 on the NK cell.

50. The composition of claim 49, wherein said NK cells are modified to express a TCR αβ chain that is at least 85% identical to SEQ ID No. 25 and SEQ ID No. 26, said TCR αβ chain targets an NY-ESO antigen, and said bispecific antibody is bolaful mab.

51. The composition of claim 49 or 50, wherein the NK cells are modified to express full length: cd3ζ, cd3ε, cd3δ and/or cd3γ.

52. The composition of any one of claims 49-51, wherein any one or more of cd3ζ, cd3ε, cd3δ, and cd3γ are heterologously linked to one or more intracellular signaling domains.

53. The composition of claim 52, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

54. The composition of claim 52 or 53, wherein said intracellular signaling domain comprises a DAP10 intracellular signaling domain.

55. The composition of any one of claims 52-54, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 115.

56. The composition of any one of claims 52-55, wherein the intracellular signaling domain comprises the amino acid sequence according to SEQ ID No. 115.

57. The composition of claim 52 or 53, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

58. The composition of any one of claims 52, 53 or 57, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 116.

59. The composition of any one of claims 52, 53, 57 or 58, wherein said intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 116.

60. The composition of claim 52 or 53, wherein said intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

61. The composition of any one of claims 52, 53 or 60, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 117.

62. The composition of any one of claims 52, 53, 60, or 61, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 117.

63. The composition of any one of claims 49-62, wherein the complex is contained in a pharmaceutically acceptable excipient.

64. The composition of any one of claims 49-63, wherein the complex is contained in a delivery device.

65. A method of treating cancer in an individual, the method comprising the step of administering to the individual a therapeutically effective amount of the composition of any one of claims 1-64.

66. The method of claim 65, wherein the NK cells and the antibody are administered to the individual simultaneously.

67. The method of claim 65 or 66, wherein the NK cells and the antibody are administered in the same formulation.

68. The method of any one of claims 65-67, wherein NK cells and antibody are pre-complexed prior to administration to the individual.

69. The method of claim 65, wherein the NK cells and the antibody are administered to the individual at different times.

70. The method of any one of claims 65-69, wherein NK cells and antibody are administered by infusion.

71. The method of any one of claims 65-70, wherein the NK cells are autologous to the subject.

72. The method of any one of claims 65-71, wherein the NK cells are allogeneic with respect to the individual.

73. A method of redirecting the specificity of NK cells for a cancer antigen with a bispecific or multispecific anti-CD 3 antibody, the redirected NK cells being used to treat an individual, the method comprising the step of administering to the individual the antibody and optionally NK cells expressing part or all of the CD3 receptor complex and optionally expressing part or all of the tcrαβ chain or tcrγδ chain.

74. The method of claim 73, further comprising the step of modifying NK cells to express part or all of the CD3 receptor complex.

75. The method of claim 73 or 74, wherein the NK cells are modified to express full length: cd3ζ, cd3ε, cd3δ and/or cd3γ.

76. The method of any one of claims 73-75, wherein any one or more of cd3ζ, cd3ε, cd3δ, and cd3γ are heterologously linked to one or more intracellular signaling domains.

77. The method of claim 76, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

78. The method of claim 76 or 77, wherein said intracellular signaling domain comprises a DAP10 intracellular signaling domain.

79. The method of any one of claims 76-78, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 115.

80. The method of any one of claims 76-79, wherein the intracellular signaling domain comprises the amino acid sequence according to SEQ ID No. 115.

81. The method of claim 76 or 77, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

82. The method of any one of claims 76, 77, or 81, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 116.

83. The method of any one of claims 76, 77, 81 or 82 wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 116.

84. The method of claim 76 or 77, wherein said intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

85. The method of any one of claims 76, 77, or 84, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID No. 117.

86. The method of any one of claims 76, 77, 84, or 85, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID No. 117.

87. The method of any one of claims 73-86, further comprising the step of modifying NK cells to express part or all of a tcrαβ chain or a tcrγδ chain.

88. The method of any one of claims 73-87, wherein the tcrαβ chain or tcrγδ chain targets an NY-ESO antigen.

89. The method of any one of claims 73-88, wherein the TCR chain is a TCR αβ chain and is at least 85% identical to SEQ ID No. 25 and SEQ ID No. 26.

90. The method of any one of claims 73-86, wherein the tcra β chain or tcrγδ chain targets a PRAME antigen.

91. The method according to claim 90, wherein the target PRAME epitope is SLLQHLIGL (SEQ ID NO: 131) and/or QLLALLPSL (SEQ ID NO: 132).

92. The method of claim 90 or 91, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID No. 135 and a sequence at least 85% identical to SEQ ID No. 136.

93. The method of claim 90 or 91, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID No. 139 and a sequence at least 85% identical to SEQ ID No. 140.

94. The method of claim 90 or 91, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID No. 143 and a sequence at least 85% identical to SEQ ID No. 144.

95. The method of any one of claims 73-94, further comprising the step of modifying the NK cells to express one or more additional exogenous proteins.