Engineered natural killer cells expressing enhanced CD3 and ITCR functions for adoptive immunotherapy

Abstract

Embodiments of the present disclosure include methods and compositions in which NK cells are engineered to express an invariant NK T cell receptor and a CD3 co-receptor that are not naturally expressed on the NK cell. These engineered NK cells interact effectively with bispecific or multispecific antibodies engineered to include an anti-CD3 antibody that binds to the engineered NK cell, thereby inducing signaling, activation, and cytotoxicity of target cells that also bind the antibody. In this manner, the NK cells are specifically engineered to interact effectively with bispecific NK cell engagers (BiKEs) and bispecific T cell engagers (BiTEs).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 434,395, filed December 21, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in ST26 format and is incorporated by reference in its entirety. The ST26 copy, created on December 7, 2023, is named MDAC_P1350WO_Sequence_Listing.xml and is 487,676 bytes in size.

[0003] The present disclosure relates to at least the fields of immunology, cell biology, molecular biology, and medicine (including at least cancer medicine). [Background technology]

[0004] Natural killer (NK) cells have been investigated as potential antitumor effectors, but numerous barriers, primarily related to a lack of antigen specificity, limit their therapeutic use. One approach to overcome this is to transduce NK cells with chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) to target desired antigens. For T cells, bispecific or multispecific antibodies, such as bispecific T cell engagers (BiTEs), can be used to bind CD3 on the T cell surface and also antigens on cancer cells. CD3 is composed of four distinct chains, and in mammals, the CD3γ chain, CD3δ chain, and two CD3ε chains form a complex. These chains interact with the T cell receptor (TCR) and ζ (zeta) chain, generating activation signals in T lymphocytes. However, NK cells do not naturally express the CD3 receptor complex or TCR. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure fulfills a long-felt need in the art for improved immunotherapies, including immunotherapies that utilize NK cells. [Means for solving the problem]

[0006] overview

[0003] Embodiments of the present disclosure include methods and compositions for treating cancer patients using adoptive cell therapy. In specific embodiments, individuals are provided with a therapeutically effective amount of a two-part therapy comprising both engineered NK cells and an antibody capable of binding to the NK cells to initiate signaling, activation, and killing of target cells.

[0004] The present disclosure relates to NK cells engineered to express multiple proteins that are not naturally expressed on NK cells and work in conjunction, including heterologous proteins on the NK cell surface that are not naturally present on NK cells.

[0007] In certain embodiments, disclosed herein are engineered NK cells that have been modified to express: a) a single chain, or part or all of any combination, of CD3δ, CD3ε, CD3γ, or CD3ζ; b) a single chain, or part or all of any combination, of the invariant NK T-cell receptor (iTCR) α (iTCRα) or β (iTCRβ) chain; and c) a cytokine selected from the group including IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, and combinations thereof. In certain embodiments, the NK cells are modified to express part or all of one CD3δ, two CD3ε, one CD3γ, and / or one CD3ζ. In certain embodiments, any one or more of CD3δ, CD3ε, CD3γ, and / or CD3ζ is heterologously linked to one or more intracellular signaling domains. In certain embodiments, the intracellular signaling domain is selected from the group comprising CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof. In certain embodiments, the intracellular signaling domain comprises a DAP10 intracellular signaling domain. In certain embodiments, the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO: 115. In certain embodiments, the intracellular signaling domain comprises a CD28 intracellular signaling domain. In certain embodiments, the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO: 116. In certain embodiments, the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains. In certain embodiments, the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO: 117. In certain embodiments, the NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

[0008] In certain embodiments, the iTCR α chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 293, and the iTCR β chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315. In certain embodiments, the iTCR α chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 293, and the iTCR β chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 301, 307, 311, or 313.

[0009] In certain embodiments, the NK cells are derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a combination thereof. In certain embodiments, the NK cells are primary NK cells and are not derived from stem cells and / or induced pluripotent stem cells (iPSCs). In certain embodiments, the NK cells are complexed with one or more antibodies. In certain embodiments, the one or more antibodies are one or more bispecific or multispecific antibodies, and at least one of the bispecific or multispecific antibodies comprises a linked anti-CD3 antibody. In certain embodiments, the antibody is blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B. In certain embodiments, the antibody is blinatumomab. In certain embodiments, the NK cells express the antibody.

[0010] In certain embodiments, the cytokine is membrane-bound. In certain embodiments, the cytokine is IL-15. In certain embodiments, the NK cells are further modified to express one or more additional heterologous proteins selected from the group consisting of antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof. In certain embodiments, the NK cells have been pre-activated with one or more cytokines. In certain embodiments, the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. In certain embodiments, the NK cells further comprise one or more engineered mutations in an endogenous gene. In certain embodiments, the endogenous gene is TGFBR2, CISH, and / or CD38.

[0011] In some embodiments, compositions comprising the engineered NK cells disclosed herein are also provided.

[0012] Also provided herein is a composition comprising a complex comprising: a) engineered NK cells modified to express some or all of the CD3 receptor complex, iTCRα chain, iTCRβ, and one or more cytokines; and b) a bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody that binds to CD3 on NK cells. In some embodiments, the engineered NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122). In some embodiments, the engineered NK cells are modified to express an iTCR α chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR β chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315. In some embodiments, the engineered NK cells are modified to express an iTCR α chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR β chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 301, 307, 311, or 313. In some embodiments, the bispecific or multispecific antibody is blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B. In some embodiments, the bispecific or multispecific antibody is blinatumomab. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is comprised in a delivery device.

[0013] Also disclosed herein are methods for treating disease in an individual. In some embodiments, the method for treating disease in an individual comprises administering to the individual a therapeutically effective amount of any one of the cells or compositions of claims 1-36. In some embodiments, the disease is an autoimmune disease, an infectious disease, and / or cancer. In some embodiments, the method comprises administering to the individual engineered NK cells that have been modified to express: a) a single chain of CD3δ, CD3ε, CD3γ, or CD3ζ, or any combination thereof; b) a single chain of the invariant NK T-cell receptor (iTCR) α (iTCRα) chain or β (iTCRβ) chain, or any combination thereof; and c) a cytokine selected from the group including IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, and combinations thereof. In some embodiments, the method further comprises administering to the individual one or more bispecific or multispecific antibodies, either simultaneously or at different times. In some embodiments, the one or more bispecific or multispecific antibodies are blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B. In some embodiments, the one or more bispecific or multispecific antibodies and the engineered NK cells are administered simultaneously, and / or the one or more bispecific or multispecific antibodies and the engineered NK cells are complexed prior to administration to an individual. In some embodiments, the engineered NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122). In some embodiments, the engineered NK cells are modified to express an iTCR α chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR β chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.In some embodiments, the disease is cancer.

[0014] Particular embodiments of the present invention are characterized by the following listed aspects.

[0015] Embodiment 1 is an engineered NK cell that has been modified to express: a) some or all of a single chain of CD3δ, CD3ε, CD3γ, or CD3ζ, or any combination; b) some or all of a single chain of the invariant NK T-cell receptor (iTCR) α (iTCRα) chain or β (iTCRβ) chain, or any combination; and c) a cytokine selected from the group including IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, and combinations thereof.

[0016] Embodiment 2 is the engineered NK cell of embodiment 1, wherein the NK cell is modified to express some or all of CD3δ, CD3ε, CD3γ, and / or CD3ζ.

[0017] Embodiment 3 is the engineered NK cell of embodiment 1 or 2, wherein any one or more of CD3δ, CD3ε, CD3γ, and / or CD3ζ is heterologously linked to one or more intracellular signaling domains.

[0018] Embodiment 4 is the engineered NK cell of embodiment 3, wherein the intracellular signaling domain is selected from the group comprising CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof.

[0019] Embodiment 5 is the engineered NK cell of embodiment 3 or 4, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

[0020] Embodiment 6 is the engineered NK cell of embodiment 5, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:115.

[0021] Embodiment 7 is the engineered NK cell of embodiment 3 or 4, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

[0022] Embodiment 8 is the engineered NK cell of embodiment 7, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:116.

[0023] Embodiment 9 is the engineered NK cell of embodiment 3 or 4, wherein the intracellular signaling domain comprises a DAP10 and a CD28 intracellular signaling domain.

[0024] Embodiment 10 is the engineered NK cell of embodiment 9, wherein the intracellular signaling domain comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:117.

[0025] Embodiment 11 is the engineered NK cell of any of Embodiments 1 to 10, wherein the iTCR alpha chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 293, and the iTCR beta chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.

[0026] Embodiment 12 is the engineered NK cell of any of Embodiments 1 to 11, wherein the iTCR alpha chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 293, and the iTCR beta chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 301, 307, 311, or 313.

[0027] Embodiment 13 is the engineered NK cell of any of Embodiments 1 to 12, wherein the cell is modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

[0028] Embodiment 14 is the engineered NK cell of any of embodiments 1 to 13, wherein the NK cell is derived from cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a combination thereof.

[0029] Embodiment 15 is the engineered NK cell of any of embodiments 1 to 14, wherein the NK cell is a primary NK cell and is not derived from a stem cell and / or an engineered pluripotent stem cell (iPSC).

[0030] Embodiment 16 is the engineered NK cell of any of embodiments 1 to 15, wherein the NK cell is conjugated to one or more antibodies.

[0031] Embodiment 17 is the engineered NK cell of embodiment 16, wherein the one or more antibodies are one or more bispecific or multispecific antibodies, and at least one of the bispecific or multispecific antibodies comprises an anti-CD3 antibody linked thereto.

[0032] Embodiment 18 is the engineered NK cell of embodiment 16 or 17, wherein the antibody is blinatumomab, tebentufusp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B.

[0033] Embodiment 19 is the engineered NK cell of any one of embodiments 16 to 18, wherein the antibody is blinatumomab.

[0034] Embodiment 20 is the engineered NK cell of any one of embodiments 16 to 19, wherein the NK cell expresses an antibody.

[0035] Embodiment 21 is the engineered NK cell of any of embodiments 1 to 20, wherein the cytokine is membrane-bound.

[0036] Embodiment 22 is the engineered NK cell of any of embodiments 1 to 21, wherein the cytokine is IL-15.

[0037] Embodiment 23 is the engineered NK cell of any of embodiments 1 to 22, wherein the NK cell is further modified to express one or more additional heterologous proteins selected from the group comprising an antigen receptor, a cytokine, a homing receptor, a chemokine receptor, and combinations thereof.

[0038] Embodiment 24 is the engineered NK cell of any one of embodiments 1 to 23, wherein the NK cell is pre-activated with one or more cytokines.

[0039] Embodiment 25 is the engineered NK cell of embodiment 24, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

[0040] Embodiment 26 is the engineered NK cell of any of embodiments 1 to 25, wherein the NK cell further comprises one or more engineered mutations in an endogenous gene.

[0041] Embodiment 27 is the engineered NK cell of embodiment 26, wherein the endogenous gene is TGFBR2, CISH, and / or CD38.

[0042] Embodiment 28 is a composition comprising the engineered NK cells of any one of embodiments 1 to 27.

[0043] Embodiment 29 is a composition comprising a conjugate of: a) an engineered NK cell that has been modified to express some or all of the CD3 receptor complex, an iTCR alpha chain, an iTCR beta, and one or more cytokines; and b) a bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody that binds to CD3 on the NK cell.

[0044] Embodiment 30 is the composition of embodiment 29, wherein the engineered NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

[0045] Embodiment 30 is the composition of embodiment 29 or 30, wherein the engineered NK cell is modified to express an iTCR α chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR β chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.

[0046] Embodiment 32 is the composition of any of Embodiments 29 to 31, wherein the engineered NK cell is modified to express an iTCR α chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR β chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 301, 307, 311, or 313.

[0047] Embodiment 33 is the composition of any of embodiments 29 to 32, wherein the bispecific or multispecific antibody is blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B.

[0048] Embodiment 34 is the composition of any of embodiments 29 to 33, wherein the bispecific or multispecific antibody is blinatumomab.

[0049] Embodiment 35 is the composition of any of embodiments 29 to 34, further comprising a pharmaceutically acceptable excipient.

[0050] Embodiment 36 is the composition of any of embodiments 29 to 35, wherein the composition is contained in a delivery device.

[0051] Embodiment 37 is a method of treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of any one of the cells or compositions of embodiments 1 to 36.

[0052] Embodiment 38 is the method of embodiment 37, wherein the disease is an autoimmune disease, an infectious disease, and / or cancer.

[0053] Embodiment 39 is a method of treating a disease in an individual, comprising administering to the individual engineered NK cells that have been modified to express: a) some or all of a single chain, or any combination, of CD3δ, CD3ε, CD3γ, or CD3ζ; b) some or all of a single chain, or any combination, of the invariant NK T-cell receptor (iTCR) α (iTCRα) or β (iTCRβ) chain; and c) a cytokine selected from the group including IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, and combinations thereof.

[0054] Embodiment 40 is the method of embodiment 39, further comprising administering to the individual one or more bispecific or multispecific antibodies, either simultaneously or at different times.

[0055] Embodiment 41 is the method of embodiment 40, wherein the one or more bispecific or multispecific antibodies is blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B.

[0056] Embodiment 42 is the method of embodiment 40 or 41, wherein the one or more bispecific or multispecific antibodies and the engineered NK cells are administered simultaneously and / or the one or more bispecific or multispecific antibodies and the engineered NK cells are conjugated prior to administration to the individual.

[0057] Embodiment 43 is the method of any of embodiments 39 to 42, wherein the engineered NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

[0058] Embodiment 44 is the method of any of Embodiments 39 to 43, wherein the engineered NK cell is modified to express an iTCR α chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR β chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.

[0059] Embodiment 45 is the method of any one of embodiments 39 to 44, wherein the disease is cancer.

[0060] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this 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.

[0061] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0062] [Figure 1-1]Figures 1A-1C show various embodiments of NK cells engineered to express CD3, including their use with various heterologous proteins such as cytokines, bispecific NK cell engagers, and engineered antigen receptors (CARs and / or TCRs). Figure 1B shows CD3- and TCR-matched NK cells for optimal cancer immunotherapy. Figure 1C shows an example of a single chimeric CD3 construct. [Figure 1-2] Same as above. [Figure 1-3] Same as above.

[0063] [Figure 2-1] Figures 2A-2B show examples of expression constructs for CD3 receptor complex components for transduction or transfection of NK cells. Figure 2B shows an example of a plasmid map of a representative expression construct. [Figure 2-2] Same as above.

[0064] [Figure 3] FIG. 3 is a table of various TCR / CD3 expression construct designs for NK-TCR engineering.

[0065] [Figure 4] FIG. 4 shows CD3 expression on day 4 in engineered NK cells transfected with an example of a CMV-directed TCR complex.

[0066] [Figure 5] FIG. 5 shows TCR expression in engineered NK cells on day 4 after transfer of CMV-directed TCR complexes.

[0067] [Figure 6] FIG. 6 shows TCR / CD3 expression in engineered NK cells on day 6 after transduction of the cells with a CMV-directed TCR complex.

[0068] [Figure 7]FIG. 7 shows the binding of an exemplary CD3-CD19 BiTE on NK cells at different concentrations via the CD3 / TCR complex on NK cells.

[0069] [Figure 8] FIG. 8 shows NK-TCR cytokine production of TNFα and CD107a after stimulation with plate-bound CD3 antibody.

[0070] [Figure 9] FIG. 9 shows the phosphorylation of CD3z in NK TCR / CD3 cells after cross-linking of CD3.

[0071] [Figure 10-1] Figures 10A-10B show that pre-incubation of CD3-CD19 BiTE with TCR / CD3-expressing NK cells increases the killing activity of Raji cells. Figure 10A shows a 1:1 effector:target ratio, and Figure 10B shows a 1:5 effector:target ratio. [Figure 10-2] Same as above.

[0072] [Figure 11] FIG. 11 shows a schematic of multiple retroviral transduction to generate NK cells expressing CD3, IL-15, and the TCR complex.

[0073] [Figure 12] Figure 12 shows the expression of NY-ESO TCR on NK cells transduced with uTNK15. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15.

[0074] [Figure 13]Figure 13 shows the number of TCR molecules expressed per cell on NK cells. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15. The number of NY-ESO TCR molecules on NK cells was measured using a Phycoerythrin Fluorescence Quantitation Kit (BD Biosciences).

[0075] [Figure 14] FIG. 14 shows the expression of NY-ESO TCR on T cells.

[0076] [Figure 15] Figure 15 shows that NY-ESO TCR-transduced NK cells kill NY-ESO peptide-pulsed target cells in a dose-dependent manner. WT means wild-type CD3 molecules with IL-15; A means CD3-CD28 with IL-15; B means CD3-DAP10 with IL-15; and C means CD3-CD28-Dap10 with IL-15.

[0077] [Figure 16] FIG. 16 shows endogenous NY-ESO expression in human tumor cell lines.

[0078] [Figure 17] FIG. 17 shows that NY-ESO TCR-transduced T cells kill NY-ESO-expressing tumor targets.

[0079] [Figure 18]Figure 18 shows that NY-ESO TCR-transduced NK cells kill NY-ESO-expressing tumor targets even at low E:T ratios. WT means wild-type CD3 molecules with IL-15; A means CD3-CD28 with IL-15; B means CD3-DAP10 with IL-15; and C means CD3-CD28-Dap10 with IL-15.

[0080] [Figure 19] Figures 19A-19B show that NY-ESO-transformed NK cells have a similar phenotype (Figure 19A) and expression pattern (Figure 19B) to NT NK cells. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15.

[0081] [Figure 20] Figure 20 is a table showing the cellular composition of the expanded uTNK15 product. WT refers to wild-type CD3 molecules with IL-15; A refers to CD3-CD28 with IL-15; B refers to CD3-DAP10 with IL-15; and C refers to CD3-CD28-Dap10 with IL-15.

[0082] [Figure 21] Figures 21A-21C show that NK cells can be successfully transduced with CD3 and TCR constant α-β (TCRCab) (referred to as the TCR6 construct), and that the engineered NK cells can bind blinatumumab (Figure 21B) and selectively kill CD19+ lymphoma targets (Figure 21C).

[0083] [Figure 22-1]Figures 22A-22C show the in vivo activity of effector cells (e.g., NK cells or T cells) containing NY-ESO-targeted TCRs. Figure 22A is a schematic diagram outlining the experimental procedures performed. Figure 22B shows time-lapse bioluminescence imaging (days 1, 7, 14, and 21) of mice transplanted with U266B.1 cells transduced with FireFly luciferase (FFluc) and treated with control, NY-ESO TCR NK cells, or NY-ESO TCR T cells (NK cells containing WT, #A, or #B UT-NK15-NY ESO TCR constructs, respectively; WT refers to wild-type CD3 molecules with IL-15; #A refers to CD3-CD28 with IL-15; and #B refers to CD3-DAP10 with IL-15). Figure 22C is a graphical quantification of the mean bioluminescence intensity displayed in Figure 22B. These results demonstrated that effector cells containing the NY-ESO TCR construct described herein robustly inhibited tumor growth in vivo. [Figure 22-2] Same as above. [Figure 22-3] Same as above.

[0084] [Figure 23-1]Figures 23A-23B show the in vitro activity of effector cells (e.g., NK cells or T cells) containing NY-ESO-targeting TCR and UT-NK15 constructs. Figure 23A shows an image of a spheroid formed by the osteosarcoma tumor cell line Saos-2 stably transduced to express GFP, which was used to test the cytotoxic activity of NY-ESO1-specific TCR-expressing NK cells and T cells. Figure 23B shows a graph showing the percentage of cytotoxicity (Y-axis) of a representative image after 3 days of coculture. NK cells were cotransfected with the NY-ESO-TCR and the UT-NK15 signaling complex coexpressing different costimulatory molecules fused to the CD3ζ signaling chain or an IL-15-free TCR complex. T cells were transduced only with the NY-ESO TCR. Abbreviations in graph: 28 = CD3ζ fused to the CD28 costimulatory domain; 10 = CD3ζ fused to the Dap10 costimulatory domain; 8 = CD8α / β coreceptor as part of the NY ESO TCR construct; wo IL-15 = construct containing only the CD3 zeta, epsilon, gamma, delta TCR complex without costimulation or IL-15. [Figure 23-2] Same as above.

[0085] [Figure 24-1]Figures 24A-24D show the in vivo activity of effector cells (e.g., NK cells or T cells) containing NY-ESO-targeting TCR and UT-NK15 constructs. Figure 24A shows the in vivo study design for testing the activity of different NY-ESO TCR-transduced NK cells and T cells. Figure 24B depicts BLI imaging results from a study outlined and performed according to Figure 24A. Mice were injected with U266 tumor cells and, 3 days later, administered T cells transduced with NY-ESO-specific TCRs or NK cells cotransduced with NY-ESO TCR and UT-NK15 carrying CD3ζ fused to CD28 (labeled as NY-ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15 CD28 NK cells). A tumor-only group was used as a control. Figure 24C is a graph showing the region of interest mean luminance intensity of the animals tested according to Figure 24A and imaged in Figure 24B. Figure 24D is a graph depicting the cohort survival curves of the aforementioned animals. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above.

[0086] [Figure 25] Figure 25 shows the in vivo activity of effector cells (e.g., NK cells) engineered to express the NY-ESO TCR and CD3 complex, with or without an IL-15 transgene incorporated into the construct. NSG mice were irradiated (300 cGy) and the following day injected via the tail vein with 0.5 million U266 cells (an HLA-A2-positive, NY-ESO-expressing myeloma cell line). Three days later, mice received 5 million TCR-transduced T cells or NK cells. Mice were monitored for tumor control by BLI imaging. NK cells were transduced with the NY-ESO-specific TCR, with or without expression of the CD8α / β co-receptor, and co-transduced with the CD3 complex without IL-15 transduction, or with UT-NK15 expressing CD3ζ fused to CD28 (UT-NK15 CD28) or CD3ζ fused to DAP10 (UT-NK15 DAP10) as costimulatory molecules.

[0087] [Figure 26-1]Figures 26A-26C show the in vitro expression of PRAME (Preferentially Expressed Antigen in Melanoma) TCR in effector cells (e.g., NK cells or T cells) and the in vitro activity of these cells. Figure 26A shows the expression of both UT-NK15 (x-axis, CD3) and PRAME-specific TCR (y-axis, TCR) in NK cells (TCR clones 46, 54, or DSK3, respectively), or the expression of PRAME-specific TCR in T cells transduced with them (TCR clones 46 or 54). Figure 26B shows the in vitro cytotoxicity of NK cells expressing PRAME-specific TCR against the U266 myeloma cell line. IncuCyte® Live Cell Imaging was used to measure the cytotoxicity of PRAME-specific TCR-transduced T cells and NK cells transduced with UT-NK15 and PRAME-specific TCR against U266 myeloma cells. GFP-expressing U266 cells were cocultured with T cells or NK cells expressing PRAME-specific TCRs at an effector:target ratio of 1:1. A decrease in GFP expression indicated cell death. 26 hours later, 50,000 tumor cells (labeled "rechallenging") were added to each well for a second time for tumor rechallenge assay. Open symbols represent T cells, and filled symbols represent NK cells. NT = non-transduced. Figure 26C shows the in vitro cytotoxicity of NK cells expressing PRAME-specific TCRs against the UA375 melanoma cell line. Using IncuCyte® live cell imaging, we measured the cytotoxicity of T cells transduced with PRAME-specific TCRs and NK cells transduced with UT-NK15 and PRAME-specific TCRs (PRAME-specific TCR clone 46 (TCR-46), PRAME-specific TCR clone 54 (TCR-54), or PRAME-specific TCR clone DSK3 (DSK)) against UA375 melanoma cells. GFP-expressing UA375 cells were cocultured with PRAME-expressing T cells or NK cells at an effector:target ratio of 1:1. A decrease in GFP expression indicates cell death. After 26 hours, 50,000 tumor cells were added to each well for tumor rechallenge assay.Open symbols represent T cells, filled symbols represent NK cells. NT = non-transduced. [Figure 26-2] Same as above. [Figure 26-3] Same as above.

[0088] [Figure 27-1]Figures 27A-27D show the in vitro expression and cytotoxic function of invariant natural killer T cell receptors (iTCRs) on NK cells. Additional experimental details are provided in Example 6, below. Figure 27A depicts exemplary flow cytometry plots showing the expression of three different iTCR pairs, each consisting of iTCRα (SEQ ID NO: 295) and either iTCRβ chain 1 (SEQ ID NO: 297), iTCRβ chain 2 (SEQ ID NO: 299), or iTCRβ chain 3 (SEQ ID NO: 301), and CD3 on NK cells, using antibodies specific for the iTCR target Vα24 and Vβ11 regions (y-axis and x-axis, respectively). Figure 27B shows exemplary flow cytometry plots showing the binding of blinatumomab (y-axis) to NK cells expressing the three different CD3 / iTCR complexes described in (A), e.g., iTCR1, iTCR2, and iTCR3, respectively. NK cells were derived from three donors: cell donor 4 (CD4), cell donor 8 (CD8), or cell donor N (CDN). Blinatumomab binding to CD3 was confirmed by flow cytometry using Miltenyi Biotech's CAR19 detection kit. Figure 27C shows the results of an IncuCyte® live cell imaging assay used to measure the cytotoxicity of NK cells preloaded with blinatumomab and co-transduced with iTCR and UT-NK15 against GFP-expressing Raji tumor cells at an effector-to-target ratio of 3:1. T cells and iNKT cells served as positive and negative controls, respectively. A decrease in GFP expression indicates cell death. Three NK donor lines, CB152, CB153, and CB154, were utilized. NK cells were transduced with three different CD3 / iTCR complexes, e.g., iTCR1, iTCR2, or iTCR3, as described in (A), or left untransduced (NT) (as a negative control). Figure 27D depicts a bar graph showing the % residual tumor (Y-axis) for the data shown in Figure 27C. An unpaired t-test was used to individually compare iNKT cells bearing each of the different iTCRs. CD3 / iTCR NK cells were significantly (P=<0.01) more cytotoxic than iNKT cells (N=3 replicates). [Figure 27-2] Same as above. [Figure 27-3] Same as above. [Figure 27-4] Same as above.

[0089] [Figure 28-1]Figures 28A-28D show the cloning of iTCR sequences from NK T cells isolated from human umbilical cord blood, and the transgenic expression and efficacy of a subset of iTCR clones in transduced cord blood-derived NK cells. Additional experimental details are described in Example 6 below. Figure 28A shows exemplary flow cytometry results demonstrating the isolation of iTCR clones from iNKT cells purified from five cord blood donors and the expression of iTCR on the donor's iNKT cells. iNKT cells were isolated from five cord blood donors using Miltenyi Biotech™ iNKT isolation kits. Isolated cells were stimulated and expanded with irradiated cord blood PBMCs (40 Gry) in the presence of 100 ng / ml α-galactosylceramide and 200 U / ml IL-2. Seven days after iNKT expansion, the purity of the iNKT cultures was confirmed by iNKT-specific antibodies against Vα24 and Vβ11. Total mRNA was extracted from iNKT cells, and cDNA of the Vβ-DJ region was cloned and sequenced (100 clones). Figure 28B shows the sequence of the iTCRβ clone Vβ-DJ sequence. Figure 28C depicts the transgenic expression of Vα24 and CD3 in transgenic CD3 / iTCR-expressing NK cell populations from three umbilical cord blood donors. NK cells were co-transduced with iTCR (eight randomly selected iTCRβ clones with coding sequences represented by SEQ ID NOs: 300, 302, 304, 306, 308, 310, 312, or 314, clones 3, 18, 24, 51, 56, 76, 93, and 96, respectively, and the iTCRα coding sequence represented by SEQ ID NO: 292) and UT-NK15 on day 5. iTCR expression was confirmed by flow cytometry using an iTCR-specific antibody against Vα24 and an antibody against CD3. Figure 28D is a graph depicting the results of IncuCyte® live cell imaging-mediated measurement of the cytotoxicity of NK cells co-transduced with iTCR (as shown in Figure 28B) and UT-NK15 and pre-loaded with blinatumomab (1 hour at 37°C) against GFP-expressing Raji cells at a target-effector ratio of 3:1. T cells served as a positive control, and non-transformed (NT) cells served as a negative control. A decrease in GFP expression indicated tumor cell death. [Figure 28-2] Same as above. [Figure 28-3] Same as above. [Figure 28-4] Same as above. [Figure 28-5] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0090] Detailed Description Following long-standing patent law practice, the words "a" and "an" herein, when used in conjunction with the word "comprising," including within the scope of the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments may be combined.

[0091] Throughout this specification, unless the context dictates otherwise, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of the recited step or element or steps or elements, but not the exclusion of other steps or elements or steps or elements. "Comprising" means including and limited to what follows the phrase "comprises." Thus, the phrase "comprising" indicates that the recited elements are required or essential, and that other elements may not be present. "Essentially comprising" means including the elements recited after the phrase, limited to other elements that do not interfere with or contribute to the activity or function specified in the disclosure for the recited elements. Thus, the phrase "essentially comprising" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0092] Throughout this specification, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0093] As used herein, the terms "or" and "and / or" are used to describe multiple components in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0094] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0095] As used herein, the term "CD3 receptor complex" or "CD3 co-receptor complex" refers to the protein complex that acts as a T cell co-receptor in nature and is composed of the CD3ζ chain, the CD3γ chain, the CD3δ chain, and two CD3ε chains (although alternatively only one CD3ε chain is used).

[0096] As used herein, the term "engineered" refers to an entity created by the hand of man, including cells, nucleic acids, polypeptides, vectors, etc. In at least some cases, an engineered entity is synthetic and comprises elements that do not occur in nature or are not constructed by the methods utilized in this disclosure. In specific embodiments, the vector is engineered by recombinant nucleic acid techniques and the cell is engineered by transfection or transduction of the engineered vector. The cell can be engineered to express a heterologous protein that is not naturally expressed by the cell, either because the heterologous protein is a recombinant or synthetic protein, or because the cell does not naturally express the protein.

[0097] The phrases "pharmaceutically or pharmacologically acceptable" refer, as appropriate, to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human. The preparation of pharmaceutical compositions containing an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Furthermore, it will be understood that when administered to an animal (e.g., a human), preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biological Standards.

[0098] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., parenteral vehicles such as water, alcoholic / aqueous solutions, saline, sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluid and nutrient replenishers, and such materials and combinations thereof will be known to those skilled in the art. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0099] The term "subject" as used herein generally refers to an individual who has or is suspected of having cancer. A subject can be any living organism or animal subject that is the subject of a method or material, including mammals, e.g., humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cows, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals. A subject can be, for example, a patient who has or is suspected of having a disease (sometimes called a pathology), such as a benign or malignant neoplasm or cancer. A subject may be undergoing or have undergone treatment. A subject may be asymptomatic. A subject may be a healthy individual but wishing to prevent cancer. The terms "individual" and "subject" are used interchangeably, at least in some cases. As used herein, a "subject" or "individual" may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive "one or more medical compositions" via the internet. Individuals may include humans or non-human animals of any age, and thus include both adults and juveniles (i.e., children) and infants, as well as individuals in utero. The term does not imply the need for medical treatment, and therefore individuals may participate in experiments, whether clinical or in support of basic scientific research, whether voluntary or involuntary.

[0100] As used herein, "therapy" 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 one or more measurable markers of the disease or condition being treated, such as cancer. Treatment can optionally include either a reduction or amelioration of one or more symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily indicate a complete eradication or cure of a disease or condition or its associated symptoms. Treatment can also refer to the alleviation of at least one symptom of a disease or condition.

[0101] As used herein, the term "TCR / CD3 complex" refers to a protein complex that is naturally present on the surface of T cells and contains the CD3ζ, CD3γ, CD3δ, and CD3ε chains, as well as the T cell receptor (TCR) α and β chains, the invariant natural killer T cell receptor (iTCR) α and β chains, and / or the T cell receptor γ and δ chains.

[0102] Disclosed Embodiments Natural killer (NK) cells are an emerging cellular immunotherapy for patients with hematological malignancies as well as solid cancers. The present disclosure relates, inter alia, to NK cells that have been modified to enhance their immunotherapeutic function compared to unmodified NK cells. This modification allows the NK cells to have greater versatility when used with other therapeutic agents and, at least in some embodiments, T-cell-like activity by utilizing the CD3 / TCR receptor complex. In specific embodiments, NK cells are engineered to express either (i) a single CD3 chain (CD3-zeta, CD3-epsilon, CD3-delta, or CD3-gamma), or part or all of the human CD3 receptor complex (including any combination of CD3-delta, epsilon (one or two copies of epsilon), gamma, and zeta); or (ii) a single CD3 chain or the human CD3 receptor complex (including any combination of CD3-delta, epsilon (one or two copies), gamma, and zeta), either as full-length proteins or as partial proteins heterologously linked to one or more intracellular signaling domains); and (iii) the CD3 complex may or may not include a T cell receptor (αβ or γδ) and / or an iTCR receptor (αβ). The present disclosure relates to the use of CD3-expressing NK cells in the diagnosis and treatment of disease, including the use of cells in combination with bispecific or multispecific antibodies in which one epitope of the antibody binds to CD3 on the CD3-expressing NK cells. CD3-expressing NK cells can be pre-complexed with bi / multispecific antibodies ex vivo or complexed in vivo to direct their specificity to the target antigen. In a diagnostic embodiment, labeled NK cells can be loaded with any type of bispecific or multispecific antibody, including at least an anti-CD3 antibody, and the loaded labeled NK cells can be monitored for trafficking to the site of the target antigen to which another antibody on the bispecific or multispecific antibody binds.

[0103] In certain embodiments of the present disclosure, the TCR recognizes an antigen and / or epitope presented by a major histocompatibility complex (MHC). In certain embodiments, the antigen and / or epitope is a peptide, lipid, and / or glycolipid. In certain embodiments, the MHC is class I MHC. In certain embodiments, the MHC is class II MHC. In certain embodiments, the MHC is a non-classical MHC. In certain embodiments, the MHC is class I-like MHC. In certain embodiments, the MHC is CD1d.

[0104] In certain embodiments, the TCR target antigen is not primarily what confers target antigen specificity to the transduced effector cell, hi certain embodiments, the TCR acts primarily as a stabilizer of the CD3 co-receptor complex, while the antibody provides the primary target antigen specificity to the transduced effector cell.

[0105] Disclosed Compositions The present disclosure relates to compositions comprising at least engineered NK cells that express at least a portion of a TCR / CD3 complex. In some cases, the compositions also include bispecific or multispecific antibodies, including in the same formulation, although in alternative embodiments, the NK cells and antibodies are utilized as physically separate compositions.

[0106] A. TCR / CD3 modification of NK cells In certain embodiments, provided herein are compositions comprising NK cells engineered to express part or all of the TCR receptor complex, iTCR receptor complex, and / or CD3 co-receptor complex. In specific embodiments, the NK cells are engineered to contain all components of the CD3 complex, including CD3ζ, CD3ε, CD3γ, and CD3δ. While certain aspects utilize the full length of CD3ζ, CD3ε, CD3γ, and CD3δ, including their extracellular, transmembrane, and intracellular domains, alternative embodiments utilize only a portion of one or more of CD3ζ, CD3ε, CD3γ, and CD3δ, each of which may or may not be combined with one or more intracellular signaling domains, such as CD16, NKG2D, DAP10, DAP12, CD28, 41BB, 2B4, CD27, OX40, or any combination thereof. NK cells can also be engineered to express a TCR receptor complex and / or an iTCR receptor complex, although in alternative embodiments, neither the TCR receptor complex nor the iTCR receptor complex components are utilized.

[0107] In certain embodiments, an amino acid sequence (eg, a polypeptide) may include amino acids represented by the single letter "X" or the three letter code "Xaa." In some embodiments, the amino acid represented by "X" or "Xaa" is any naturally occurring amino acid, including, but not limited to, arginine (Arg, R), histidine (His, H), lysine (Lys, K), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), glycine (Gly, G), proline (Pro, P), cysteine ​​(Cys, C), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), or tryptophan (Trp, W).

[0108] In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is arginine (Arg, R). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is histidine (His, H). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is lysine (Lys, K). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is aspartic acid (Asp, D). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is glutamic acid (Glu, E). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is serine (Ser, S). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is threonine (Thr, T). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is asparagine (Asn, N). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is glutamine (Gln, Q). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is glycine (Gly, G). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is proline (Pro, P). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is cysteine ​​(Cys, C). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is alanine (Ala, A). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is valine (Val, V).In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is isoleucine (Ile, I). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is leucine (Leu, L). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is methionine (Met, M). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is phenylalanine (Phe, F). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is tyrosine (Tyr, Y). In some embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is tryptophan (Trp, W).

[0109] In certain embodiments, any particular sequence of a CD3 receptor component, including wild-type or mutant components, is utilized, as the case may be, for the modulation of CD3ε, CD3δ, CD3γ, CD3ζ, or NK cells, so long as the mutant CD3 receptor allows signaling through the CD3 complex leading to target activation and killing.

[0110] CD3 epsilon (UniProtKB-P07766(CD3E_HUMAN))

[0111] signal peptide MQSGTHWRVLGLCLLSVGVW (SEQ ID NO: 1)

[0112] Extracellular domain sp|P07766|23-126 DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 2)

[0113] Transmembrane domain sp|P07766|127-152 VMSVATIVDITGLLLVYYWS (SEQ ID NO: 3)

[0114] Intracellular domain sp|P07766|153-207 KNRKAKPVTRGAGGRQRGQNKRPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 4)

[0115] An example of the Homo sapiens CD3e molecule (CD3E), mRNA, can be found in the NCBI reference sequence: GENBANK® Accession No. NM_000733.4 (SEQ ID NO: 5)

[0116] An example showing the entire CD3 epsilon sequence in its respective nucleic acid and amino acid form is shown below (the underlined portion indicates the signal peptide sequence): ATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAG (SEQ ID NO: 37) MQSGTHWRVLGLCLLSVGVWGQ DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 38)

[0117] CD3 delta (UniProtKB-P04234(CD3D_HUMAN))

[0118] signal peptide MEHSTFLSGLVLATLLSQVS (SEQ ID NO: 6)

[0119] Extracellular domain sp|P04234|22-105 FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA (SEQ ID NO: 7)

[0120] Transmembrane domain sp|P04234|106-126 GIIVTDVIATLLLALGVFCFA (SEQ ID NO: 8)

[0121] Intracellular domain sp|P04234|127-171 GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 9)

[0122] Homo sapiens CD3d molecule, Delta (CD3-TCR complex), mRNA (cDNA clone MGC:88324 IMAGE:30412345), complete cd GENBANK®:BC070321.1 ATGGAACATAGCACGTTTCTCTCTGGCCTGGTACTGGCTACCCTTCTCTCGCAAGTGAGCCCCTTCAAGATACCTATAGAGGAACTTGAGGACAGAGTGTTTGTGAATTGCAATACCAGCATCACATGGGT AGAGGGAACGGTGGGAACACTGCTCTCAGACATTACAAGACTGGACCTGGGAAAACGCATCCTGGACCCACGAGGAATATATAGGTGTAATGGGACAGATATATACAAGGACAAAGAATCTACCGTGCAAG TTCATTATCGAATGTGCCAGAGCTGTGTGGAGCTGGATCCAGCCACCGTGGCTGGCATCATTGTCACTGATGTCATTGCCACTCTGCTCCTTGCTTTGGGAGTCTTCTGCTTTGCTGGACATGAGACTGGA AGGCTGTCTGGGGCTGCCGACACACAAGCTCTGTTGAGGAATGACCAGGTCTATCAGCCCCTCCGAGATCGAGATGATGCTCAGTACAGCCACCTTGGAGGAAACTGGGCTCGGAACAAGTGA (SEQ ID NO: 10)

[0123] An example of the complete CD3 delta sequence, in nucleic acid and amino acid form, is shown below (the underlined portion indicates the signal peptide sequence): ATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGG AGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAG (SEQ ID NO: 35) MEHSTFLSGLVLATLLSQVSP FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 36)

[0124] CD3 gamma (T cell surface glycoprotein CD3 gamma chain gene CD3G P09693) signal peptide MEQGKGLAVLILAIILLQGTLA (SEQ ID NO: 11)

[0125] Extracellular domain sp|P09693|23-116 QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO: 12)

[0126] Transmembrane domain sp|P09693|117-137 GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 13)

[0127] Intracellular domain sp|P09693|138-182 GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 14)

[0128] Homo sapiens CD3g molecule (CD3G), mRNA;NM_000073.3:81-629 Homo sapiens CD3g molecule (CD3G), mRNA (SEQ ID NO: 15)

[0129] An example showing the entire CD3 gamma sequence, in its respective nucleic acid and amino acid form, is shown below (the underlined portion indicates the signal peptide sequence): ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCC CAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGT GTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAAT (SEQ ID NO: 33) MEQGKGLAVLILAIILLQGTLA QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 34)

[0130] CD3 Zeta

[0131] signal peptide sp|P20963| SP MKWKALFTAAILQAQLPITEA (SEQ ID NO: 16)

[0132] Extracellular domain sp|P20963|22-30 ECD QSFGLLDPK (SEQ ID NO: 17)

[0133] Transmembrane domain sp|P20963|31-51 tmd LCYLLDGILFIYGVILTALFL (SEQ ID NO: 18)

[0134] Intracellular domain sp|P20963|52-164 ICD RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 19)

[0135] An example showing the entire CD3 zeta sequence, in its respective nucleic acid and amino acid form, is shown below (the underlined portion indicates the signal peptide sequence): ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA CAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCC TGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 31) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 32)

[0136] Homo sapiens CD247 molecule (CD247; also known as CD3 zeta), transcript variant 1, mRNA NCBI reference sequence NM_198053.3 NM_198053.3:65-559 Homo sapiens CD247 molecule (CD247), transcript variant 1, mRNA ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTA TGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGAT TGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 20)

[0137] In specific embodiments, NK cells are engineered to express one or more of the TCR α chain, TCR β chain, TCR γ chain, and TCR δ chain, and any combination thereof may be utilized. In certain embodiments, the TCR may be an invariant natural killer cell TCR (iTCR). In certain embodiments, NK cells are engineered to express a T cell receptor (TCR) α β chain, iTCR α β chain, or TCR γ δ chain. In certain embodiments, NK cells are engineered to express only a portion or all of the constant region of one or more of the TCR α chain, iTCR α chain, TCR β chain, iTCR β chain, TCR γ chain, and TCR δ chain. NK cells can be engineered to express only a portion or all of the constant region of the T cell receptor (TCR) α β chain, TCR γ δ chain, or iTCR α β chain. When a portion of a constant region is utilized, 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 any contiguous amino acids of the constant region. The portion of the constant region can include 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 any contiguous amino acids of the constant region.

[0138] In specific embodiments, any of the sequences encompassed herein are utilized to modify NK cells, while in other embodiments, sequences related to these in identity are utilized, for example, related sequences that are at least 80, 85, 90, 95, 96, 97, 98, 99% identical to any of the sequences encompassed herein may be utilized in the present disclosure.

[0139] Specific constructs for the expression of various TCR / CD3 proteins in NK cells can be utilized in a variety of configurations. In particular, NK cells can be transduced or transfected with one or more vectors for expressing any of the various proteins encompassed herein, including at least one or more components of the TCR / CD3 complex. In particular, the one or more vectors themselves may or may not be multicistronic, ultimately capable of producing two or more distinct polypeptides. When one or more multicistronic vectors are employed, they may utilize one or more internal ribosome entry sites (IRES) and / or one or more 2A self-cleaving peptide sites. When one or more 2A sequences are utilized, GSG is an optional linker:

[0140] T2A (GSG) EGRGSLLTCGDVEENPGP (SEQ ID NO: 21)

[0141] P2A (GSG) ATNFSLLKQAGDVEENPGP (SEQ ID NO: 22)

[0142] E2A (GSG) QCTNYALLKLAGDVESNPGP (SEQ ID NO: 23)

[0143] F2A (GSG) VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 24)

[0144] In situations where multiple protein components are expressed from a multicistronic vector, the order from 5' to 3' on the polynucleotide vector can be any order, although in other aspects, they are present on the vector in a specific order. The multicistronic vector expresses multiple components of the CD3 receptor complex and no other heterologous proteins, or the multicistronic vector expresses multiple components of the CD3 receptor complex and one or more other heterologous proteins. The multicistronic vector expresses multiple components of the TCR receptor complex and no other heterologous proteins, or the multicistronic vector expresses multiple components of the TCR receptor complex and one or more other heterologous proteins. The multicistronic vector may or may not express one or more components of the TCR receptor complex and one or more components of the CD3 complex. In specific embodiments, the multicistronic vector contains one or more components of the CD3 receptor complex and one or more heterologous proteins, such as a cytokine or an engineered antigen receptor such as a CAR.

[0145] Figure 2A shows an example of a multicistronic vector in which full-length CD3ε, CD3δ, CD3γ, and CD3ζ are present, separated by the same or different 2A self-cleaving peptide sites. As further illustrated in the plasmid map in Figure 2B, the multicistronic vector may contain the signal peptide, extracellular domain, transmembrane domain, and intracellular domain of each of CD3ε, CD3δ, CD3γ, and CD3ζ.

[0146] Figure 3 is a table showing various examples of TCR expression constructs for engineering TCR-expressing NK cells. In certain embodiments of the present disclosure, the CD3 receptor component and the TCR receptor component are expressed in NK cells from different vectors. In either case, the vector can express a TCR directed against a specific antigen, such as a cancer antigen or a viral antigen. The TCR may or may not include at least a portion of CD3ζ, including the intracellular domain of CD3ζ, and NK cells also express CD3ζ as both a molecule separate from the TCR and as part of the CD3 receptor complex. Similarly, the CAR may or may not include at least a portion of CD3ζ, including the intracellular domain of CD3ζ, in addition to NK cells expressing CD3ζ as both a molecule separate from the TCR and as part of the CD3 receptor complex.

[0147] In a specific embodiment, the TCR of the modified NK cell is utilized as a structural support or scaffold to promote the function or enhanced function of the CD3 receptor complex, not necessarily as a therapeutic aspect of the cell. That is, the TCR can be any TCR, and is not utilized for its ability to specifically target a desired antigen. In such a case, for example, a TCR targeting a viral antigen can be employed in NK cells used for cancers that are not necessarily related to that particular virus. Alternatively, a TCR targeting a specific cancer antigen may be selected. Examples of antigens targeted by TCRs are described elsewhere herein.

[0148] Figure 3 shows the following example configuration:

[0149] TCR1 : refers to TCRpp65 (TCR against HLA-A2 restricted CMVpp65) linked to the intracellular CD3 zeta domain and full length CD3 gamma, full length CD3 delta, and full length CD3 epsilon, the construct may also be referred to as TCRpp65ZicdGDEFL, which may consist of the following sequence:

[0150] In TCRpp65ZicdGDEFL, the corresponding component sequences are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0151] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 40) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 41)

[0152] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO: 42) (where the P2A sequence is at the C-terminus) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO: 43) (where the lowercase sequence is the P2A sequence)

[0153] TCRa extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 44) ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO: 45)

[0154] CD3 gamma, delta, epsilon (CD3GDE): MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLP NDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGI IVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP (SEQ ID NO: 46) (where the E2A sequence is at the C-terminus)

[0155] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0156] TCR2 : refers to TCRpp65 linked to full length CD3 zeta, full length CD3 gamma, full length CD3 delta, full length CD3 epsilon, lacking IL-15. Representative sequences are as follows:

[0157] TCR3 : This refers to TCRpp65 bound to the intracellular CD3z domain and IL-15, and is sometimes called TCRpp65Zicd15. A representative sequence is as follows: *(SEQ ID NO: 52)

[0158] In TCRpp65Zicd15, the corresponding component sequences are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0159] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 40) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 41)

[0160] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO: 42) (where the P2A sequence is at the C-terminus) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO: 43) (where the lowercase sequence is the P2A sequence)

[0161] TCRa extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 44) (SEQ ID NO: 45)

[0162] CD3 zeta intracellular domain (Z-ICD) (in certain embodiments, two or more Z-ICD sequences may be utilized): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP (SEQ ID NO: 53) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 54)

[0163] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0164] TCR4 : Refers to TCRpp65, also known as TCRpp65βα, and a representative sequence is as follows: *(SEQ ID NO: 55)

[0165] For TCRpp65βα, the corresponding component sequences are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0166] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 40) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 41)

[0167] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO: 42) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC (SEQ ID NO: 54)

[0168] TCRa extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 44) ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO: 45)

[0169] CD3 zeta intracellular domain (Z-ICD): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP(SEQ ID NO: 53) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 54)

[0170] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0171] A representative sequence of TCRpp65βα is as follows: *(SEQ ID NO: 57)

[0172] Z1 : Refers to full-length CD3zeta, full-length CD3γ, full-length CD3δ, and full-length CD3ε linked to IL15 (see Figures 2A and 2B), and may also be referred to as CD3ZFLGDEFL15, with representative sequences as follows: (SEQ ID NO: 58)

[0173] Z2 This refers to the full-length CD3zeta, full-length CD3γ, full-length CD3δ, or full-length CD3ε bound to membrane-bound IL21 (membrane-bound IL21 has the CD8 transmembrane domain), also known as CD3ZGDEFLSP821CD28. The representative sequence is as follows:

[0174] For CD3ZGDEFLSP821CD28, the corresponding component sequences are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0175] CD3: (SEQ ID NO: 61)

[0176] SP CD8: MRICLTSDRLAPAAGLAAPRRQAV (SEQ ID NO: 63) atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg (SEQ ID NO: 64)

[0177] IL-21: HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 65) CATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAACGGA TAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT (SEQ ID NO: 66)

[0178] CD8 Hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 67) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 68)

[0179] CD28 transmembrane domain: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 70)

[0180] Z3 : Refers to full-length CD3zeta, full-length CD3γ, full-length CD3δ, and full-length CD3ε bound to membrane-bound IL21 (membrane-bound IL21 has the CD28 transmembrane domain), and the representative sequence is as follows, also known as CD3ZGDEFL8SP21CD8:

[0181] For CD3ZGDEFL8SP21CD8, the corresponding component sequences are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0182] CD3: (SEQ ID NO: 61)

[0183] SP CD8: MRICLTSDRLAPAAGLAAPRRQAV (SEQ ID NO: 63) atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg (SEQ ID NO: 64)

[0184] IL-21: HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 65) cataaatcttcctctcaaggtcaggaccgccatatgattcgaatgcggcagctgattgacatagtcgatcaactgaagaactatgtgaatgatcttgtgcccgag tttttgccagcccctgaagacgtagaaactaattgtgagtggagtgccttttcctgctttcaaaaggcacagctgaaatccgccaacacgggcaataacgaacgga taattaacgtatccattaagaagctgaagcggaagccgccctcaaccaatgcgggacggcggcaaaagcatcgcttgacctgtccgtcatgcgacagctacgaga aaaagcccccgaaggagttcttggaacgcttcaagagtctccttcagaaaatgattcaccagcacctgtcctcacggacgcacggaagcgaggacagt (SEQ ID NO: 66)

[0185] CD8 Hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 67) ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 68)

[0186] CD8 transmembrane domain: IYIWAPLAGTCGVLLLSLVIT* (SEQ ID NO: 72) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC (SEQ ID NO: 73)

[0187] In certain embodiments, provided herein are CD3 constructs comprising a fusion with an intracellular costimulatory domain derived from CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, or any combination thereof. In certain embodiments, the intracellular costimulatory domain is fused to CD3δ, CD3ε, CD3γ, and / or CD3ζ. In certain embodiments, such CD3 fusion constructs comprise CD3ζ fused to the DAP10 intracellular costimulatory domain. In certain embodiments, such CD3 fusion constructs comprise CD3ζ fused to the CD28 intracellular costimulatory domain. In certain embodiments, such CD3 fusion constructs comprise CD3ζ fused to the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain. In certain embodiments, CD3ζ fused to a DAP10 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:106. In certain embodiments, CD3ζ fused to a CD28 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:107. In certain embodiments, CD3ζ fused to a DAP10 intracellular costimulatory domain and a CD28 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:108.In certain embodiments, CD3ζ fused to a DAP10 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:109. In certain embodiments, CD3ζ fused to a CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:110. In certain embodiments, CD3ζ fused to the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 111. In certain embodiments, CD3ζ fused to the intracellular domain may not include a C-terminal 2A domain. In certain embodiments, CD3ζ fused to the intracellular domain may not include an N-terminal signal peptide domain. ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 106) ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 107) ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO: 108) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP(SEQ ID NO: 109) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (SEQ ID NO: 110) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP (SEQ ID NO: 111)

[0188] In certain embodiments, the DAP10 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 112. In certain embodiments, the CD28 intracellular costimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 113. In certain embodiments, the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain are represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 114. In certain embodiments, the DAP10 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 115. In certain embodiments, the CD28 intracellular costimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 116. In certain embodiments, the DAP10 intracellular costimulatory domain and the CD28 intracellular costimulatory domain are represented by amino acid sequences that are at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 117. CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 112) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCA (SEQ ID NO: 113) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 114) LCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 115) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 116) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRG (SEQ ID NO: 117)

[0189] UTNK15-DAP10: refers to full length CD3 zeta, full length CD3 gamma linked to IL15, full length CD3 delta, and full length CD3 epsilon comprising a fusion with an intracellular costimulatory domain from DAP10, which may be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 118. In certain embodiments, the UTNK15-DAP10 amino acid sequence may be represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:119.

[0190] UTNK15-28 : refers to full length CD3 zeta comprising a fusion with an intracellular costimulatory domain from CD28, full length CD3 gamma linked to IL15, full length CD3 delta, and full length CD3 epsilon, which may be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 120. In certain embodiments, the UTNK15-28 amino acid sequence may be represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 121.

[0191] UTNK15-28-DAP10 : refers to full length CD3 zeta comprising a fusion of the intracellular costimulatory domain from DAP10 and the intracellular costimulatory domain from CD28, and full length CD3 gamma, full length CD3 delta, and full length CD3 epsilon 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: 122. In certain embodiments, the UTNK15-28-DAP10 amino acid sequence may be represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 123.

[0192] As depicted in Figure 3 and described above, the term "linked" refers to the presence of two polypeptides on the same polynucleotide vector, and does not necessarily mean that the two polypeptides are expressed as a single polypeptide. For example, a cytokine produced from a vector of the present disclosure may ultimately be produced as a molecule separate from any one or more TCR / CD3 receptor complex components. In contrast, the term "fused" or "fusion" refers to two polypeptides that contain a peptide bond linking the two molecules, i.e., the two polypeptides are covalently linked by an amide bond and are not separated by a dividing element, such as a 2A element.

[0193] One specific example of a TCR that can be utilized intracellularly is the NY-ESO TCR, and specific examples of sequences include at least the following:

[0194] TCRα: XQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 25)

[0195] TCRβ: GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 26)

[0196] In certain embodiments, the TCR may comprise a TCR alpha chain variable region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:85. aaacaggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaactgcagtttcact gatagcgctatttacaac ctccagtggtttaggcaggaccctgggaaaggtctcacatctctgttgctt attcagtcaagtcagagagag caaacaagtggaagacttaatgcctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgactcagccacctacctc tgtgctgtgaggcccctttatggaggaagctacatacctacattt ggaagaggaaccagccttattgttcatccgtat (SEQ ID NO: 85)

[0197] In certain embodiments, the TCR may comprise a TCR alpha chain constant region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:86. atccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaag taaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcat gtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacg aacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc (SEQ ID NO: 86)

[0198] In certain embodiments, the TCR may comprise a TCR alpha chain encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:87. atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagcaaacaggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaactgcagtttcact gatagcgctatttacaacctccagtggtttaggcaggaccctgggaaaggtctcacatctctgttgctt attcagtcaagtcagagagag caaacaagtggaagacttaatgcctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgactcagccacctacctc tgtgctgtgaggcccctttatggaggaagctacatacctacattt ggaagaggaaccagccttattgttcatccgtatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttg attctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaa caaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaa acagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc (SEQ ID NO: 87)

[0199] In certain embodiments, the TCR may comprise a TCR alpha 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. XQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVRPLYGGSYIPTF GRGTSLIVHPY (SEQ ID NO: 88)

[0200] In certain embodiments, the TCR may comprise a TCR alpha 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:89. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 89)

[0201] In certain embodiments, the TCR may comprise an alpha chain CDR1 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:90. DSAIYN (SEQ ID NO: 90)

[0202] In certain embodiments, the TCR may comprise an alpha chain CDR2 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:91. IQSSQRE (SEQ ID NO: 91)

[0203] In certain embodiments, the TCR may comprise an alpha chain CDR3 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:92. CAVRPLYGGSYIPTF (SEQ ID NO: 92)

[0204] In certain embodiments, the TCR may comprise a TCR beta 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. ggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactgcagtgtgcccaggat atgaaccatgaatac atgtcctggtatcgacaagacccaggcatggggctgaggctgattcattac tcagttggtgctggtatc actgaccaaggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctcccagacatctgtgtacttc tgtgccagcagttacgtcgggaacaccggggagctgtttttt ggagaaggctctaggctgaccgtactggag (SEQ ID NO: 93)

[0205] In certain embodiments, the TCR may comprise a TCR beta chain constant region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:94. (SEQ ID NO: 94)

[0206] In certain embodiments, the TCR may comprise a TCR beta chain encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:95. Atgagcatcggcctcctgtgctgtgcagccttgtctctcctgtgggcaggtccagtgaatgctggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactgcagtgtgcccaggat atgaaccatgaatac atgtcctggtatcgacaagacccaggcatggggctgaggctgattcattac tcagttggtgctggtatcactgaccaaggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctcccagacatctgtgtacttc tgtgccagcagttacgtcgggaacaccggggagctgtttttt (SEQ ID NO: 95)

[0207] In certain embodiments, the TCR may comprise a TCR beta 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. GVTQTPKFQVLKTGQSMTLQCAQD MNHEY MSWYRQDPGMGLRLIHY SVGAGI TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF CASSYVGNTGELFF GEGSRLTVLE (SEQ ID NO: 96)

[0208] In certain embodiments, the TCR may comprise a TCR beta 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. DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 97)

[0209] In certain embodiments, the TCR may comprise a β chain CDR1 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:98. MNHEY (SEQ ID NO: 98)

[0210] In certain embodiments, the TCR may comprise a β chain CDR2 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:99. SVGAGI (SEQ ID NO: 99)

[0211] In certain embodiments, the TCR may comprise a β chain CDR3 amino acid sequence that is at least, or exactly, 80% or 100% identical to SEQ ID NO:100. CASSYVGNTGELFF (SEQ ID NO: 100)

[0212] In certain embodiments, a TCR (e.g., TCR α, β, δ, and / or γ) chain may 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. atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagc (SEQ ID NO: 101) atgagcatcggcctcctgtgctgtgcagccttgtctctcctgtgggcaggtccagtgaatgct (SEQ ID NO: 102) METLLGLLILWLQLQWVSS (SEQ ID NO: 103) MSIGLLCCAALSLLWAGPVNA (SEQ ID NO: 104)

[0213] In certain embodiments, the TCR recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis Ag NY-ESO-1 in the context of the HLA-A*02 class I allele. In certain embodiments, the TCR may target an epitope characterized by the amino acid sequence according to SEQ ID NO: 105. SLLMWITQC (SEQ ID NO: 105)

[0214] One specific example of a TCR that can be utilized intracellularly is TCRpp65α, and specific examples of sequences include at least the following (underlined signal peptide sequence): ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGA CAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCCAGTGATGTAGGCATCTACTTCTGTGCTGGACCCATGAAAACCTCCTACGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ ID NO: 27) MDSWTFCCVSLCILVAKHTDAG QQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGPMKTSYDKVIFGPGTSLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 28)

[0215] One specific example of a TCR that can be utilized intracellularly is TCRpp65β, and specific examples of sequences include at least the following (underlined signal peptide sequence): ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGA GTTATCCAGTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGTTCGGCAAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO: 29) MDSWTFCCVSLCILVAKHTDAG VIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSSANYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 30)

[0216] TCRpp65ZFLGDEFL15

[0217] In certain embodiments, a construct can be utilized in which TCRpp65 is linked to full-length CD3ζ, full-length CD3γ, full-length CD3δ, or full-length CD3ε, which is further linked to IL-15 (which can be referred to as TCRpp65ZFLGDEFL15). A representative sequence of such a construct is as follows:

[0218] In TCRpp65ZFLGDEFL15, the corresponding component sequences are as follows, although these particular sequences or other sequences may be utilized in this and / or other constructs:

[0219] TCRb-extracellular domain: MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADATNFSLLKQAGDVEENPGP (SEQ ID NO: 75) (and includes the P2A sequence at its C-terminus) ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO: 76)

[0220] TCRa extracellular domain: MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSEGRGSLLTCGDVEENPGP (SEQ ID NO: 77) (and including the T2A sequence at its C-terminus) ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO: 78)

[0221] TCR5: Designated TCRCgdZFLGDEFL15, it is the constant region of TCR gamma and delta linked to full-length CD3 zeta, full-length CD3 gamma, full-length CD3 delta, full-length CD3 epsilon; and IL-15. Representative sequences are as follows:

[0222] TCR constant gamma delta (TCRCgd) MRWALLVLLAFLSPASQDKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKSGSGATNFSLLKQAGDVEENPGPMILTVGFSFLFFYRGTLCSQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO: 82)

[0223] CD3: (SEQ ID NO: 79)

[0224] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS* (SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCC GGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCT GCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCA GCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49)

[0225] TCR6: Also known as TCRCabZFLGDEFL15, this is the constant region of TCR α and β linked to full-length CD3ζ, full-length CD3γ, full-length CD3δ, full-length CD3ε; and IL-15. Representative sequences are as follows:

[0226] TCR constant αβ (TCRCab) METLLGLLILWLQLQWVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGATNFSLLKQAGDVEENPGPMSIGLLCCAALSLLWAGPVNADLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 83)

[0227] CD3: (SEQ ID NO: 79)

[0228] IL-15: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO: 48) ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO: 49)

[0229] In some embodiments, a TCR construct comprises a NY-ESO-specific TCR and a CD8α / β co-receptor molecule. In some embodiments, such a construct can include a TCR α chain variable region signal peptide, a TCR α chain variable region, a TCR α 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 CD8β polypeptide, a 2A element (e.g., a T2A element), and a CD8α polypeptide. In some embodiments, a TCR construct comprising a NY-ESO-specific TCR and a CD8α / β co-receptor molecule nucleotide coding sequence 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: 124. In some embodiments, a TCR construct comprising a NY-ESO-specific TCR and a CD8α / β co-receptor molecule amino acid sequence 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: 125.

[0230] In some embodiments, the CD8α co-receptor molecule is transcriptionally linked to any TCR molecule disclosed herein. In some embodiments, the CD8α co-receptor molecule nucleotide coding sequence 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: 126. In some embodiments, the CD8 beta co-receptor molecule nucleotide coding sequence 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: 127. In some embodiments, the CD8 alpha co-receptor amino acid sequence 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: 128. In some embodiments, the CD8β co-receptor amino acid sequence 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: 129. ATGAGGCCACGACTTTGGCTGCTGCTCGCTGCACAGTTGACTGTACTGCATGGCAATAGTGTGTTGCAGCAGACACCTGCATACATCAAGGTTCAGACAAATAAGATGGTTATGCTGAGTTGCGAGGCAAAAATTAGTTTGAGCAATATGCGGATCTACTGGTTGCGACAGAGACAGGCTCCCAGTAGTGATAGTCACCACGAATTCCTGGCTCTTTGGGATTCCGCAAAAGGAACGATTCATGGGGAAGAAGTAGAGCAGGAGAAGATTGCGGTTTTCCGCGATGCATCTCGCTTTATCCTTAATCTTACATCCGTTAAGCCTGAGGACAGTGGGATCTATTTTTGTATGATTGTAGGGTCCCCCGAATTGACATTTGGGAAGGGTACGCAGCTCTCCGTAGTTGACTTTCTGCCCACAACGGCACAACCCACTAAGAAGTCCACCCTGAAGAAGCGCGTCTGTCGCTTGCCCAGACCTGAAACCCAAAAGGGTCCACTCTGTTCCCCTATAACCCTGGGGTTGTTGGTGGCGGGCGTCTTGGTCCTGCTTGTTAGCTTGGGCGTAGCCATTCATCTGTGTTGCCGAAGACGCAGAGCCCGACTTAGATTTATGAAGCAATTCTATAAGTGA(SEQ ID NO: 126) ATGGCCTTGCCCGTCACTGCGCTTTTGCTCCCGCTCGCTCTTCTCCTGCATGCAGCCCGACCATCTCAATTTAGAGTTTCTCCACTCGACAGGACGTGGAACCTCGGCGAAACCGTCGAACTTAAATGTCAAGTACTTCTCTCAAATCCGACTTCTGGTTGCTCATGGCTCTTTCAGCCGAGAGGAGCAGCTGCCAGCCCCACCTTCCTGCTGTATCTCTCCCAGAACAAGCCGAAGGCCGCCGAAGGGCTCGATACTCAACGATTTAGCGGGAAGCGACTCGGGGACACGTTCGTTCTTACTCTCAGCGATTTTAGAAGAGAGAACGAGGGATATTATTTTTGTTCCGCACTCTCTAACAGCATCATGTACTTCAGTCATTTTGTACCAGTCTTTCTCCCTGCAAAACCAACGACTACTCCAGCACCAAGACCGCCCACTCCCGCACCTACTATTGCAAGCCAACCTTTGAGTCTCCGACCAGAGGCATGCAGACCTGCTGCTGGAGGTGCAGTACATACGCGAGGGTTGGATTTTGCCTGCGATATCTATATCTGGGCCCCCTTGGCCGGCACGTGCGGGGTGCTCCTGCTGAGTCTCGTAATTACTCTTTATTGTAATCATAGAAACCGCAGAAGGGTGTGTAAGTGTCCCCGGCCTGTCGTGAAAAGCGGGGATAAGCCCAGTTTGTCTGCTCGGTACGTC(SEQ ID NO: 127) MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK(SEQ ID NO: 128) MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 129)

[0231] 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 the TCR alpha chain and TCR beta chain found in PRAME-specific TCR clone 46, clone 54, and / or clone DSK3. In some embodiments, the TCR construct comprising a PRAME-specific TCR chain comprises the TCR alpha chain and TCR beta chain that target the PRAME epitope SLLQHLIGL (SEQ ID NO: 131) and / or QLLALLPSL (SEQ ID NO: 132).

[0232] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain 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: 133 (e.g., TCR clone 46TCRα) and / or 134 (e.g., TCR clone 46TCRβ). In some embodiments, a TCR construct comprising a PRAME-specific TCR chain 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: 135 (e.g., TCR clone 46TCRα) and / or 136 (e.g., TCR clone 46TCRβ). (query number 133) (query number 134) MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGIPRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 135) MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTPWLAGGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 136)

[0233] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain 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: 137 (e.g., TCR clone 54TCR alpha) and / or 138 (e.g., TCR clone 54TCR beta). In some embodiments, a TCR construct comprising a PRAME-specific TCR chain 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: 139 (e.g., TCR clone 54TCRα) and / or 140 (TCR clone 54TCRβ). (SEQ ID NO: 137) ATGGGCTTCCGGCTGCTGTGCTGCGTGGCCTTTTGTCTGCTGGGAGCCGGACCTGTGGATAGCGGCGTGACCCAGACCCCCAAGCACCTGATCACCGCCACCGGCCAGAGAGTGACCCTGCGCTGCAGCCCTAGAAGCGGCGACCTGAGCGTGTACTGGTATCAGCAGAGCCTCGACCAGGGCCTGCAGTTCCTGATCCAGTACTACAACGGCGAGGAACGGGCCAAGGGCAACATCCTGGAACGGTTCAGCGCCCAGCAGTTCCCCGATCTGCACAGCGAGCTGAACCTGAGCAGCCTGGAACTGGGCGACAGCGCCCTGTACTTCTGCGCCAGCGCCAGATGGGATAGAGGCGGCGAGCAGTACTTCGGCCCTGGCACCAGACTGACCGTGACCGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO: 138) MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSERALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSGQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 139) MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASARWDRGGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 140)

[0234] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain 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: 141 (e.g., TCR clone DSK3 TCR alpha) and / or 142 (e.g., TCR clone DSK3 TCR beta). In some embodiments, TCR constructs comprising PRAME-specific TCR chains 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: 143 (e.g., TCR clone DSK3 TCR alpha) and / or 144 (TCR clone DSK3 TCR beta). (query number 141) MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKDNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 142) (query number 143) MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSDGGGVYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 144)

[0235] In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a nucleotide coding 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 one or more of SEQ ID NOs: 145-152. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises the TCR alpha and TCR beta chains found in the PRAME-specific TCR clones T116-49 and / or T402-93 and / or modified versions thereof. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises a TCR alpha and TCR beta chains that target the PRAME epitope LYVDSLFFL (SEQ ID NO: 167). In some embodiments, the PRAME-specific TCR sequences, TCR variable domain sequences, CDR sequences, and / or TCR constant domain sequences are described in International Patent Application Publication No. WO 2022 / 063966 A1, which is incorporated herein by reference for purposes described herein. In some embodiments, a TCR construct comprising a PRAME-specific TCR chain comprises an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 153-166. ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTTGGGCAGCTGGACCTGGGTCCGATCTCAGCCAGCCTTTCAGTCTCCTCCAGGCCGTGATCCTGAGAGAAGGCGAGCCGCGGTATCAACTGCAGCTTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCCGAGCCCCTGTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGCTTCAACGAGAAAGCAGCAGTCCAGCCCGTGACCTGGACCCAGCTGGAGCTCAGCGCCACCTACTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGCACCCTGCTGACCGTGAATCCCAATCCAATCCAATC ATGCTGCTGATCACCTCATGCTGGTGCTGGATGCAGCTGAGCCAAGTGATCAGATCCCTCAGTCAGCAGCACGTGCAAGAAGGCGAGGACTTCACCACCCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTTACAAGCAGCGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGTCAAGTCCGGCGCAAGTGAAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGCAAGGAAGACAGCAGCCCTGCACATCACCGCCACACAGACCACCACGATGTGGCACCTACTTTTGTGTGCGCCCTGCCCTGCCTAGAGCCGGCAGCTATCAACTAGCATTCGGCAAGGCACCAAGCTGAGCGGTGATCCCCAAC (sequence number 146) ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTGTGGCAGCTGACCTGGGTCCGATCTCAGCAGCCTGTTCAGTCTCCTCAGGCCGTGATCCTGAGAGAAGGCGAGGACGCCGTGATCAACTGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCGAGGCCCCTGTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGCTTCAACGAGAAGAAGCAGCAGTCCAGCCTGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGCACCTACTTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGGCACCCTGCTGACCGTGAATCCCAATATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO: 147) ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGAGCCAAGTGAACGGCCAGCAAGTGATGCAGATCCCTCAGTACCAGCACGTGCAAGAAGGCGAGGACTTCACCACCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTACAAGCAGCGGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGGTCAAGTCCGGCGAAGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAACAGCAGCCTGCACATCACCGCCACACAGACCACCGATGTGGGCACCTACTTTTGTGCTGGCGCCCTGCCTAGAGCCGGCAGCTATCAACTGACATTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAACATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO: ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGACACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGGTTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCCAGCCAGAGCCGTGTCTCGCCATCAGCGACTACGAGGGGCACCAGGGCCTTTTTTGGCCAAGCACAAGACTGACCGTGGTG (sequence number 149) ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAAGTGTACCGTGGAAGGGCACCAGCAATCCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGGACTGCAGCTGCTGTTTTTACAGCCGTCGGCATCGCCAGATCAGCGAGGTTTCCACAGAATCTGAGCCGCAGACCCCAGGACAGACAGTTTATCCTGGAGCAGCAAAGCTGCTGCTGAGCGACACAGCGGCTTCTACCTGTGTGGCTTGGAGCCTCGGGAGCCGGCTACACCGACACAGACTATTTTGGCCCTGGCACCAGACTGACCGTGCTG(sequence number 150) ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGGACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGTGTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCAGCCAGACCAGCGTGTACTTCTGCGCCATCAGCGACTACGAGGGCACCGAGGCCTTTTTTGGCCAAGGCACAAGACTGACCGTGGTGGAAGATCTCCGGAACGTGACCCCCCCTAAAGTGACCCTGTTCGAACCCAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGAGTGTCCACCGACCCTCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAAGGCTCCCCCAAGCCCGTGACCCAGAATATCTCTGCCGAGGCCTGGGGCAGAGCCGACTGTGGAATTACCAGCGCCAGCTACCACCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAAGAAGAACAGC(SEQ ID NO: 151) ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAATGTACCGTGGAAGGCACCAGCAATCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGACTGCAGCTGCTGTTTTACAGCGTCGGCATCGGCCAGATCAGCAGCGAGGTTCCACAGAATCTGAGCGCCAGCAGACCCCAGGACAGACAGTTTATCCTGAGCAGCAAGAAGCTGCTGCTGAGCGACAGCGGCTTCTACCTGTGTGCTTGGAGCCTCGGAGCCGGCTACACCGACACACAGTATTTTGGCCCTGGCACCAGACTGACCGTGCTGGAAGATCTCCGGAACGTGACCCCCCCTAAAGTGACCCTGTTCGAACCCAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGAGTGTCCACCGACCCTCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAAGGCTCCCCCAAGCCCGTGACCCAGAATATCTCTGCCGAGGCCTGGGGCAGAGCCGACTGTGGAATTACCAGCGCCAGCTACCACCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAAGAAGAACAGC(SEQ ID NO: 152) METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTANSGGSNYKLTFGKGTLLTVNPN (SEQ ID NO: 153) MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGALPRAGSYQLTFGKGTKLSVIPN (SEQ ID NO: 154) IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 155) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 156) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 157) METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTANSGGSNYKLTFGKGTLLTVNPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 158) MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGALPRAGSYQLTFGKGTKLSVIPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 159) MGTRLFFYVALCLLWTGHVDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISDYEGTEAFFGQGTRLTVV (SEQ ID NO: 160) MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLGAGYTDTQYFGPGTRLTVL (SEQ ID NO: 161) EDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 162) DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 163) EDLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 164) MGTRLFFYVALCLLWTGHVDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISDYEGTEAFFGQGTRLTVVEDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 165) MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLGAGYTDTQYFGPGTRLTVLEDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 166)

[0236] In some embodiments, the TCR construct comprises a gp100-specific TCR chain. In some embodiments, the TCR construct comprising a gp100-specific TCR chain comprises a TCR alpha chain and a TCR beta chain found in the gp100-specific TCR clone Sp(0.01)A and / or modified versions thereof. In some embodiments, the TCR construct comprising a gp100-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the gp100 epitope KTWGQYWQV (SEQ ID NO: 168). In some embodiments, the gp100-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in Patent Publication US8,216,565B2, which is incorporated herein by reference for purposes described herein.

[0237] In some embodiments, a TCR construct comprising a gp100-specific TCR chain comprises a nucleotide coding 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 one or more of SEQ ID NOs: 169 and / or 170. In some embodiments, a TCR construct comprising a gp100-specific TCR chain comprises an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 171-174. ATGAAATCCTTGAGTGTTTCCCTAGTGGTCCTGTGGCTCCAGTTAAACTGGGTGAACAGCCAGCAGAAGGTGCAGCAGAGCCCAGAATCCCTCATTGTCCCAGAGGGAGCCATGACCTCTCTCAACTGCACTTTCAGCGACAGTGCTTCTCAGTATTTTGCATGGTACAGACAGCATTCTGGGAAAGCCCCCAAGGCACTGATGTCCATCTTCTCCAATGGTGAAAAAGAAGAAGGCAGATTCACAATTCACCTCAATAAAGCCAGTCTGCATTTCTCGCTACACATCAGAGACTCCCAGCCCAGTGACTCTGCTCTCTACCTCTGTGCAGCCAATAACTATGCCCAGGGATTAACCTTCGGTCTTGGCACCAGAGTATCTGTGTTTCCCTACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO: 169) ATGGGCTCCAGACTCTTCTTTGTGGTTTTGATTCTCCTGTGTGCAAAACACATGGAGGCTGCAGTCACCCAAAGTCCAAGAAGCAAGGTGGCAGTAACAGGAGGAAAGGTGACATTGAGCTGTCACCAGACTAATAACCATGACTATATGTACTGGTATCGGCAGGACACGGGGCATGGGCTGAGGCTGATCCATTACTCATATGTCGCTGACAGCACGGAGAAAGGAGATATCCCTGATGGGTACAAGGCCTCCAGACCAAGCCAAGAGAATTTCTCTCTCATTCTGGAGTTGGCTTCCCTTTCTCAGACAGCTGTATATTTCTGTGCCAGCAGCCCTGGGGGGGGGGGGGAACAGTACTTCGGTCCCGGCACCAGGCTCACGGTTTTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACGAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGAAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCATGA (SEQ ID NO: 170) MKSLSVSLVVLWLQLNWVNSQQKVQQSPESLIVPEGAMTSLNCTFSDSASQYFAWYRQHSGKAPKALMSIFSNGEKEEGRFTIHLNKASLHFSLHIRDSQPSDSALYLCAANNYAQGLTFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 171) MGSRLFFVVLILLCAKHMEAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSPGGGGEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKRKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 172) QQKVQQSPESLIVPEGAMTSLNCTFSDSASQYFAWYRQHSGKAPKALMSIFSNGEKEEGRFTIHLNKASLHFSLHIRDSQPSDSALYLCAANNYAQGLTFGLGTRVSVFPY (SEQ ID NO: 173) EAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSPGGGGEQYFGPGTRLTVL (SEQ ID NO: 174)

[0238] In some embodiments, the TCR construct comprises a MART-1-specific TCR chain. In some embodiments, the TCR construct comprising the MART-1-specific TCR chain comprises the TCR alpha chain and TCR beta chain found in the MART-1-specific TCR clone F4 and / or F5 and / or modified versions thereof. In some embodiments, the TCR construct comprising the MART-1-specific TCR chain comprises the TCR alpha chain and TCR beta chain that target the MART-1 epitope AAGIGILTV (SEQ ID NO: 175). In some embodiments, the MART-1-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in patent publication US9,128,080B2, which is incorporated herein by reference for purposes described herein.

[0239] In some embodiments, a TCR construct comprising a MART-1 specific TCR chain comprises a nucleotide coding 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 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 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 180-183. ATGTTGCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTACTTCTGTGCTGGTGGGACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAGGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ ID NO: 176) ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCCTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTTCTGTGCCATCAGTGAGGTAGGGGTTGGGCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO: 177) ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACTTCGGAGGAGGAAAGCTTATCTTCGGACAGGGAACGGAGTTATCTGTGAAACCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 178) ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTGTGCCAGCAGCCTAAGTTTCGGCACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO: 179) GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGGTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 180) DAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISEVGVGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 181) QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFGGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 182) IAGITQAPTSQILAAGRRMTLRCTQDMRHNAMYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDGYSVSRANTDDFPLTLASAVPSQTSVYFCASSLSFGTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 183)

[0240] In some embodiments, the TCR construct comprises a tyrosinase-specific TCR chain. In some embodiments, the TCR construct comprising the tyrosinase-specific TCR chain comprises the TCR alpha chain and TCR beta chain found in the tyrosinase-specific TCR clone TIL 1383I and / or modified versions thereof. In some embodiments, the TCR construct comprising the tyrosinase-specific TCR chain comprises a TCR alpha chain and a TCR beta chain that target the tyrosinase epitope represented by amino acids 368-376 of tyrosinase (reactive with the class I MHC (HLA-A2)-restricted epitope (368-376) of tyrosinase). In some embodiments, the tyrosinase-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in the publication Roszkowski et al., Cancer Res. 65(4):1570-6 (2005), which is incorporated herein by reference for purposes described herein.

[0241] In some embodiments, the TCR construct comprises a MAGE-A3-specific TCR chain. In some embodiments, the TCR construct comprising a MAGE-A3-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting amino acids 271-279 of MAGE-A3, e.g., epitope FLWGPRALV (SEQ ID NO: 184). In some embodiments, the TCR construct comprising a MAGE-A3-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting amino acids 112-120 of MAGE-A3, e.g., epitope KVAELVHFL (SEQ ID NO: 185). In some embodiments, the MAGE-A3-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publication No. WO 2012 / 054825 A1, which is incorporated herein by reference for the purposes described herein. In certain embodiments, the anti-MAGE-A3 112-120 TCR comprises an A118T substitution relative to wild type (where position 118 of the alpha chain is threonine). In certain embodiments, the anti-MAGE-A3 112-120 TCR comprises an A118V substitution relative to wild type (where position 118 of the alpha chain is valine).

[0242] In some embodiments, a TCR construct comprising a MAGE-A3 specific TCR chain comprises a nucleotide coding 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 one or more of SEQ ID NOs: 186-193. In some embodiments, a TCR construct comprising a MAGE-A3 specific TCR chain comprises an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 194-201. ATGGGTCCTGTCACCTGCTCAGTTCTTGTGTCTCTCCTCAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTCTGGTATGTGTGCAACATCTCAATGAATCCCTCGGCTACTCCTGGAAGAGCTTCACAGAACAAAGGACCGAGCACCAAGGTTCCACGCCACTCTCCATAAGAGCAGCCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCTGTCAGACTCTGCCCGTACTACTGTGCTTTCGACAAATGCTTACAAAGTCATCTTT(sequence number 186) ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCTAGGAAACAGGCCTTGTGGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAAGATGGGAGAGAAGTTTTGCTGGAATGTGGGACAGGACATGAGCCATGAAACAATGTACTGTGATCGACAAGACCCTGGTCTGGGCTACAGCTGATTTATATCCATACGATGTTGATAGTAACAGCGAAGGACATCCCTAAAGGATACAGGGTCTCACGGAAGAGCGGGAGCATTTTCCCCTATTCTGGATTCTGCTAAAAACAAACCAGACATCTGTGTACTTCTGTGTAGCAGTTCAACAAACACAGAAGTCTTCTTT (sequence number 187) ATGGGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTTCTGGTATGTGCAACATCTCAATGAATCCCCTCGGCTACTCCTGAAGAGCTTCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTTTCGACACAAATGCTTACAAAGTCATCTTTGGAAAAGGGACACATCTTCATGTTCTCCCTAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACACCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO: 188) ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCCTAGGAACAGGCCTTGTGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAGAATGGGAGAGAATGTTTTGCTGGAATGTGGACAGGACATGAGCCATGAAACAATGTACTGGTATCGACAAGACCCTGGTCTGGGGCTACAGCTGATTTATATCTCATACGATGTTGATAGTAACAGCGAAGGAGACATCCCTAAAGGATACAGGGTCTCACGGAAGAAGCGGGAGCATTTCTCCCTGATTCTGGATTCTGCTAAAACAAACCAGACATCTGTGTACTTCTGTGCTAGCAGTTCAACAAACACAGAAGTCTTCTTTGGTAAAGGAACCAGACTCACAGTTGTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAACTCGTGA (SEQ ID NO: 189) ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCTGGAGCTCCAGATGCAGTTTATTCATACAGTGCAGCCACCTTACCTCTTTCTGTACGTGCAGTATCCTGGCCAGAGCCTCCAGATTTCTCCTCTCAAATACATCACAGAGACACACCGTTGTTAAAGGCACCAAGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCCGGATACAGGAAACTACAAATACGTCTT (sequence number 190) ATGGGCATCCAGACCCCTGTTGTGTGATCTTTTATGTTCTGATAGCAAATCACACAGATGCTGGAGTTACCCAGACCACACATGAGGTGGCAGAGAAAGGACAAACAATAATCCTGAAGTGTGAGCCAGTTTCAGGCCACAATGACCTTTTCTGGTACAGACAGCAAGATACAGGGACTAGAGTTGCTGAGCTACTTCCGCAGCAAGTCTCTTATGGGAAGATGGGTGGGCTTTTCAAGGATCGATTCAAAGCTGATGCTAAATTCATCCTTCTCCACTCTGAAGATTCAACCTACAGAACCCAGGGACTCAGCTGTGTATCTGTGGCCAGCGATTTTGGGACAGCTAGTGCAGAAACGCTGTATTTT(sequence number: 191) ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCCTGGAGCTCAGATGCAGTTATTCATACAGTGCAGCACCTTACCTCTTCTGGTACGTGCAGTATCCTGGCCAGAGCCTCCAGTTTCTCCTCAAATACATCACAGGAGACACCGTTGTTAAAGGCACCAAGGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCGGATACAGGAAACTACAAATACGTCTTTGGAGCAGGTACCAGACTGAAGGTTATAGCACACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTCCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO: 192) (query number 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)

[0243] In some embodiments, the TCR construct comprises a MAGE-A4-specific TCR chain. In some embodiments, the TCR construct comprising the MAGE-A4-specific TCR chain comprises a TCR α chain and a TCR β chain targeting the epitope GVYDGREHTV (SEQ ID NO: 202). In some embodiments, the TCR construct comprising the MAGE-A4-specific TCR chain comprises a TCR α chain and a TCR β chain targeting the epitope FMNKFIYEI (SEQ ID NO: 203). In some embodiments, the MAGE-A4-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publications WO2017 / 174824A1 and WO2021 / 229212A1, each of which is incorporated by reference herein for purposes described herein. In certain embodiments, the anti-MAGE-A4 TCR α chain variable domain may have an M4V or M4L amino acid substitution. In certain embodiments, the anti-MAGE-A4 TCR β chain variable domain may have an N10E amino acid substitution.

[0244] In some embodiments, a TCR construct comprising a MAGE-A4 specific TCR chain comprises a nucleotide coding 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 one or more of SEQ ID NOs: 204-205. In some embodiments, a TCR construct comprising a MAGE-A4 specific TCR chain comprises an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 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. (query number 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)

[0245] In some embodiments, the TCR construct comprises a Wilms tumor antigen (WT1) WT1-specific TCR chain. In some embodiments, the TCR construct comprising the WT1-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the epitope VLDFAPPGA (SEQ ID NO: 215). In some embodiments, the TCR construct comprising the WT1-specific TCR chain comprises a TCR alpha chain and a TCR beta chain targeting the epitope RMFPNAPYL (SEQ ID NO: 216). In some embodiments, the WT1-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publications WO2020 / 185796A1 and WO2021 / 034976A1, each of which is incorporated by reference herein for purposes described herein. In some embodiments, the leader sequence and / or signal peptide may be removed from the TCR amino acid sequence, and the percent sequence identity may be calculated based on the TCR amino acid sequence without the leader sequence and / or signal peptide.

[0246] In some embodiments, a TCR construct comprising a WT1-specific TCR chain comprises a nucleotide coding 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 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 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 257-291. ATGGAGACACTGCTGGGACTACTGATTCTGTGGCTGCAACTGCAATGGGTGAGCAGCAAACAGGAGGTTACCCAGATTCCTGCTGCTCTGTCTGTCTGAAGGCGGAGAATCTGGTGCTGAACTGCAGCTTCACAGATAGCGCCATCTACAACCTGCAGTGGTTCAGACAGGATCCTGGAAAAGGCCTGACAAGCCTGCTGCTGATTCAGAGCTCTCAGAGAGCAGACATCTGGAAGACTGAATGCTAGCCTGGACAAGTCTAGCGGCAGAAGCACCCTGTATATTGCCGCCTCTCCAACCTGGAGATTCTGCCACATACCTGTTGTGCTGTGAAGGAGACATCTGGCTCTAGACTGACCTTTGGCGAGGGAACACAACTGACCGTGAATCCTGAC(sequence number 217) ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCCTGAAATGTCTGTGCAGGAAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAACAACTACTACCTGTTTCTGGTACAAGCAGCCCCCCTCTAGGCAGATGATCCTGGTGATCAGACAGGAGCCCTATAAAACAGCAGAATGCCACAGAGAACCGTTCAGCGTGAACTTCCAGAAAGCGCCAAGAGCTTCAGCCCTGAAGATCTCTGATTCTCAGCTGGGCGATACAGCCATGTACTTTGCCCTTCATCTACCCCAGCTACACAAGCGGCACATACAAGGTCACATACAAGGTCAGATACAAGTACATCTTCGGCACCGGCACAAACTGAAGGTTCTGGCCAAC (sequence number 218) ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCAGAAACACGCCCCTCTCTGTCTGTGCAGGAAGGCAGAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTTGTGTACAAGAAGTACCCCGCGAGGCCCTACCTTCTGGATCAGCATCTAGCATCAAGGACAAGAACGAAGATGGCAGATTCACGAGTCACGTGTTCCTGAAACAAGAGCGCCAAGCCAGCTGAGCCTGCACATTGGCTTCTCAACCTGGAGATTCTGCCGGTACTTTTGTGCTGCCTTGGAACAGGCGAAGCTATATCCCCACATTTGGGAAGAGGAAACAAGCCTGATGTGTCACCCTTAC (sequence number 219) ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCCAGAACACGCCCTCTCTGTCTGTGTCAGGAAGGCGAAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTTGTGGTACAAGAAGTACCCCGCGAGGCCCTACCTTCTGGATCAGCATCTCTAGCATCAAGGACAAGAACGAAGGCAGATTACCCGTGTTCCTGAAACAAGAGCGCCAAGCCACCTGAGCCTGCACATTGTGCCTTCTCAACCTGGAGATTCTGCGTGTACTTTTGTGCTGCCTTGGCATTGGCGACTACAAACTGAGCTTTTGGAGCCGGCACAACAGTGACCGTTAGACCCAAT (sequence number 220) ATGGTGAAGATCCGGCAGTTCCTCCTGGCTATTCTGTGGCTGCAACTGTCTTGTGTGTCTGCTGCCAAGAATGAAGTGGAGCAGTCTCCCCAGAACCTTACAGCCCAGGAAGGCGAGTTTTATCACCATCAACTGCAGCTATTCTGTGGCATTAGCGCCCTGCATTGGCTGCAGCAACACCCTGGGAGGGAATTGTGTCTCTGTTTATGCTGTCTTCTGCCAAGAAGAAGCACGGCCGGCTGATTGCCACCATCAACATCCAGGAAGCACTCTTCTCTGCACATTACAGCCCTCATCCAGGGATTCTGCCGTGTACATCTGTGCCGTGGAACCAGCTACGATAAGGTGATTTTTCGGACCAGGCACCTCTCTGAGCGGTGATCCCCAAT (sequence number 221) ATGAAGAGCCTGAGAGTCCTGCTGGTGTGGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGTGGAGCGAATAGCGGCCCTCTGTTCCTGAAGGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGGAGTGGCAGGTTTACAGCCCAGCTGAAACAAGGCCAGCCAGTAGTTTTCTCTGCTGATCAGAGATAGCCAGCTAGCGATTCTGCCACCTACCTGTGTCGTGAACTTACTTGGAGCTACAGGATACTCTACACTACACTTCGGCAAAGGCACCATGCTGCTGGTGAGCCCTGAT(sequence number 222) ATGTGGGGCGTTTTCCTTCTTGTATGAGCATGAAGATGGGCGGCACAACAGGCCAGAACATCGATCAGCCCTACCGAGATGACAGCCACAGAAGGAGCTATTGTTCAGATCAACTGCACCTACCAGACAAGCGGCTTTCAACGGCCTGTTCTGGTACCAGCAGCATGCTGGAGAAGCTCCTACATTTCTGAGCTACAATGTGCTGGATGGCCTGGAGAGAAAAGCAGGTTTAGCAGCTTTCCTGAGCGAGTCAAGGGCTATTCTTATCTGCTGCTGAGGAGCTGCAGATGAAGGATTCCGCCAGCTACCTGTGTGCCGTTAGGGGCATCAATGATTACAAGCTGAGCTTTGGAGCCGGAACAACAGTGACCGTGAGAGCCAAC (sequence number 223) ATGGAGAAGATGCTGGAGTTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAAAGCTCTAGCCTGAACTGCAGCTACACAGGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGCGAGGAGAAGGAAGGAAGAGACTGAAAGCTACCCTGACCAAGAAGGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCCGTGATTACCGGCCTTTCAGAAGCTGGTGTTTCTCCCAAAT (sequence number 224) ATGAGACTGGTGGCACGCGTACTGTTTTCTGACCTTTGGCACCATCATCGATGCCAAGACAACCCAGCCTTACAAGCATGGACTGTGCCGAGGGAAGAGCTGCTAATCCTGCCATGTAATCACAGCACAATCAGCGGCAACGAGTACGGTACTACTGGTACCGGCAGATCTCACTCTCAAGGACCTCAGTACATCATTCATGGCCTGAAGAAACAACGAGACCAACGAGATGGCCAGCCGTGTCATCCTGATTCTGCCTCATGCTACACTGAGAGATACCGCCGTGTACTACTGCATTGCCGGAGTGGGAAGAGGCCAGAATTTCGTTTGGACCTTGGAACACTGAGCGTTCTGCCCTAT(sequence number 225) ATGGAGAAGAACCCCTTGGCAGCACCTCTGCTTATTCTGTGGTTCCACCTGGATTGTGTGAGCAGCATCCTGAATGTGGAGCAGTCTCCTCAGAGCTGCATGTGCAAGAAGGCGATAGCACCAATTTCACCTGCAGCTTTTCCAAGCAGCAACTTCTACGCCCTGCACTGGTACAGATGGGAAACCGCCAAATTCTCCTGAAGCCCTGTTTGTGATCACCTGAATGGCGACGAAGAAAGAGGACAATTAGCGCCACCCCTGAATACCAAGGAGGGCTACAGCTACCTGTACATCAAGGCTCTCAACCTGAGATTCTGCCACCCTTTGCGCCTTTACCCCAATTTCGGCAACGAGAAACTGACCTTTGGAACCCGGAACAAGGCTGACCATCATCCCCAAC (sequence number 226) ATGGAGAAGATGCTGGAGTTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAAAGCTCTAGCCTGAACTGCAGCTACACAGGTGTTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGCGAGGAGAAGGAAGGAAGACTGAAAGCTACCCTGACCAAGAAGGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATATCTGTGTGCTTTCAGCCTAGAGGAGATGGCTCTAGCAATACCGGCAAGCTGGATCTTGGCCAGGGAACAACACTGCAGGTGAAGCCGTGAAGCCGTGAAGCCGT(sequence number 227) ATCCAGAATCCCGATCCTGCTGTGTACCAGCTGCGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAGCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCGTGGCCTGGTCAACAAGAGCGACTTCGCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGACATTCTTCCCAAAGCCCCGAGAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCGGATCCTGCTCGGATCCTGCTGTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGCTTGGTCCAGCTGA(sequence number 228) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCACCATGGGCACATCTCTTCTCTGTTGGGTGGTGGTTCTGGGCTTTCTGGCACAGATCATACAGGAGCTGGAGTAGCCAGTCTCTAGGTATAAGGTGACCAAGAGGGACAGGATGTGGCTCTGAGAGTGTGACCCTATTAGCGGACATGTGAGCCTGTACTGTCAAGGACAAGCTCTGGGACAAGGACCCGAGTTTTCTGACCTACTTACGGCCAGCAGGACAAATCTGGACTGCCCCAACGACAGATTCAGCGCCGAAAGACCAGAAGGCCTTATAGCACACTGACCATCCAGAGAACAGAGCAGAGGGATTCTGCCATGTTACAGATGCGCGCAGCAGCTTAACAGGCTCTTACGAGCAGCTACTTTGACCTGGCACAAACTGACAGTGACAGAG(sequence number 229) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCACCATGCTGCTTCTTCTCCTCTCTCTCGGACCTGCTGGATCTGGATCTGGATTAGGAGCTGTTGTGTTCCAGCCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCAGGCCTTCTCTGACCTTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCACACCTGAAGCCTCTAGCCATATGAGCAGTACTTTGGCCCTGGCACCAGATTAACAGTGACAGAG(sequence number 230) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCCATGGGACCTGGGACTGCTTCATTGGATGGCTCTGTGTTTGCTGGGAACAGGACATGGAGATGCTATGGTTGTCCAGAACCCCAGGTATCAGGTTACGGTGACCCAGTTTGGCAAACCAGTGACACTGAGCTGTTCTCAGACCCTGAACCACAACGTGATGTACTGGTACCAGCAGAAGTCTTCTCAGGCCCTAAGCTGCTTCCACTACTACGACAAGGACTTCAACAACGAGGCCGATACCCTGACAATTTCCAGAGAGGCCCAATACCAGCTTCGTTTCCTGGACATTAGAAGCCCTGGACTGGGAGATGCTGCCATGTACCTGTGTGCCACAGGCAATTTACAGGGAAGACAACCTCAGCACTTTGGCGATGGCACAAGGCTGTCTATCCTGGAG (sequence number 231) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGGCTGCTGGTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGTCTAGCAATCTCTAGACACCTGATCAAGGAGAGAGAGAACAGCCACCCTGAAGTGCTACCCCATCAGACACGATACAGTGTACTGGTATCAGCAAGGACCTGGGACAAAGTCCCCAGTTCCTGATCTGTCTCTACGAGAAGAGATGCAGAGCGACAAAGGCCATCCCAGACATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCCCTGGAACTGGCGATTCTGCTCTGTCTCTGGCCTTCTGAGACTGGGAAGAAACCCCAGTACTTTGGACCCCGGCCAAGACTCTGTTTCTGAG (sequence number 232) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGGCACAAGACTTCTCTGCTGGGTGGTCTTGGATTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTACAAAGTGGCCAAGAGAGGACAGGACAGGATGTGGCTCTGAGAGTGTGACCCTATTAGCGGACATGTGAGCCTGTTTTGGACCAGCAAGCTGTGGGACAAGGACCCGAGTTTTCTGACCTACTTCCAGAATGAAGCCCAGCTGGATAAATCTGGACTGCCTAGCGACCCGGTTTCTCGCCGAAAGACCTGAAGGATCTGTTAGCACCCTGAAGATTCAGAGAACACAGCAGGAGGACTCTGCCGTGTACCTGTGCCTCTCTTTAGGACAGGCCTATGAGCAGTATTTTGGACCTGGCACCAGACTGACCGTGACAGAG(sequence number 233) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGGCACAAGACTTCTCTGCTGGGTGGCGCTTTTGTCTGCTGGTGGTGGAAGAGCTGATTGAAGCTGGGAGTTGCAGTCTCCTAGGTACAAGATCATCGAGAAAGCAGCCCGTGGCCTTCTGGTGTGTAATCCATTTCTGGCCACAACACCCTGTACTGGTATCTGCAGAATCTGGGACAGGCCCTGAACTGCTGGAACTGCTGGAGATACGAGAAAGCCGTGGGACGATTCTCAACTGCCTTAAGGACCGTTTCTGCCGGAGGCTGAAAGGAGTGATTCTCACCCTTGGAGAAGTCTCCAACGTGCTGAACTGGGCGATTCTCTGTGTCACCTGTGCGCTTCTAGCCTGACAAGAGGAGCTGAAGCCTTTTTGGACAGGGCACAAGACTGACAGTGGTGGAG (sequence number 234) CTCAATAAAAGCCCACAACCCCCTCCACTCGGCGCGCACCATGGGACCTCAGCTTCTTGGATACGTTGTGCTGTGTCTGCTTGGAGCTCTTGAAGCTCAGGTTACCCAGAACCCCAGATACCTGATTACCGTGACCAGAGCAAAAAGCTGACCGTGACATGTAGCCAGAACATGAGCTCGGCAGGTACATGAGCTGGTACCGGCAGGATCCTGATGGCCTGAGACAGATCTACTACAGCATGAACGTGGAGGTGACCGATAAAGGCGACGTGCCTGAGGGATACAAGGTGAGCAGAAAGGAGAAGGAAGGAATTTCCCCTGAATCCTGGAAAGCCCAATCAGACAAGCCTGTCTTTGTGCCAGCAGCTTTTCTGGCGGCACATATGAGCAGTACTTCGGCCCTGGCACAAACTAGACAGTACAGTTACAGAG(sequence number 235) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGGCTGCTGGTGGCTGGCTGGCTGGCTGGCTGGCTGGCTGGCATCAATCTCTAGACACCTGATCAAGGAGAGAGAGAACAGCCACCCTGAAGTGCTACCATCCCCCAGACACGATACAGTGTACTGGTATCACTCTGAGCTGGACAAGATCCCCAGTTCCTGATCCTCAGAGCGACAAAGGCAGCATCCCAGACAGATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCAGCCTGGAACTGGCGATTCTGCTCTGTCTCTGTGGCCAGCTATAGAGGAGGCAGCCACATATGAGCACATATTGAGCAGTACTTTGGCCCTGGCACAAGACTGACAGTGACAGAGACTGACAGTAGGACAGAGACTGACAGAGACTGACAGATGACAGAGGAGAGACTGAGTAGACAGTAGGACAGAGTAGACAGAGGAGAGACTGAGTAGACAGTAGAGAGAGGA CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGAGCACCAGACTCCTTTGCTGGATGGCTTTGTGTCTGCTTGGAGCTGAGCTGTCTGAAGCTGAAGTTGCCCAGTTCCCAGATACAAGATCACCGGAAAATCTCAGGCTTGGCCTTCTGGTGTGACCCTATTTCTGGACACACGCCACCTGTACTGGTATAGGCAAATTCTGGGACAAGGCCCTGAACTGCTGGTGCAATTTCGAGACAGGTTTTCTGCCGAGCGCGCTGAAAGGAGTTGATAGCACCCTGAAATCCAACCTGCTACTGGGCGATTCTGCTATGTACCTGTGCGCGCTTGTCTGGAAGAGATAGCCCTAACGAGAAGCTTTCTTTGGCTTGGAACCCAGCTGTTCTGTGGAAG(sequence number 237) CTCAATAAAAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCTGTAGACTGTTGTGTGTGCTGTGCTGTGTCTGTGTGGAGCTGTGCCTATGGAAACAGGCGTTACCCAGACACCTAGACATCTGGTTATGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCATCTGGGCCATAACGGCCATGTACTGGTATAAGCAGAGCGCCAAGAAACCACTGGAACTGATGTTCGTGTACAGCCTGGAGGAGAGGTGGAGAATAATAAGCGTGCCCAGCAGATTTAGCCCTGAGTGCCCCAAATTCTTTCTCACCTGTTCTCCACCTGCACACATTACAGCCCGAGGATTCTGCCCTGTACCTGTGCTTCTTCTCAAGACCCTTACAAGCTGGAGCGGCAATACCATCTACTTCGGCGGAAGGCTCTTGGCTGACAGTGGTTGAA (sequence number 238) (query number 239) GACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC (SEQ ID NO: 240) (query number 241) ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTCAACTGCAGTTACACAGTCAGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGCTTTCTGCACATCACAGCCCCTAAACCTGAAGACTCAGCCACTTATCTCTGTGCTGTGCAGACCATGGACGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 242) (query number 243) ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAGTAATTATTATTTGTTCTGGTACAAACAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCAACCCTTGGGAGAACTATGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 244) ATGAAGAGCCTGAGAGTCCTGCTGGTGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGAAGTGGAGCAGAATAGCGGCCCTCTGTCTGTTCCTGAAGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGAGGATGGCAGGTTTACAGCCCAGCTGAACAAGGCCAGCCAGTATGTTTCTCTGCTGATCAGAGATAGCCAGCCTAGCGATTCTGCCACCTACCTGTGTGCCGTGAACATCGGAAATCACGACATGAGATTTGGAGCCGGCACAAGACTGACCGTGAAGCCCAATATCCAGAACCCTGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 245) ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCTGTGCAGACCATGGATGGCAACCAGTTCTACTTCGGCACAGGCACATCTCTGACCGTTATCCCCAATATCCAGAACCCTGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 246) ATGGCTTGTCCTGGATTCTTATGGGCTCTGGTGATCAGCACCTGTCTGGAGTTCTCTATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTGACACTGTCTTGCACCTACGATACAAGCGAGAGCGACTACTACCTGTTCTGGTACAAGCAGCCTCCCTCTAGGCAGATGATCCTGGTGATTAGACAGGAGGCCTACAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGAGATGCTGCCATGTACTTTTGTGCCAGCTCTCCAGGCACCTACAAGTACATTTTTGGCACCGGCACCAGACTGAAGGTGCTGGCCAATATCCAGAATCCCGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO: 247) ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAGCAACTACTACCTGTTCTGGTACAAGCAGCCCCCTTCTAGGCAGATGATCCTGGTGATCAGACAGGAGGCCTATAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGCGATACAGCCATGTACTTTTGTGCCTTCAACCCCTGGGAGAACTATGGCCAGAATTTCGTGTTCGGCCCTGGCACCAGACTGTCTGTTCTGCCTTATATCCAGAACCCCGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA (SEQ ID NO: 248) ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGACACTGAAGTTACCCAGACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCACAGGGCTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACCTTCACCTACACGCCCTGCAGCCAGAAGACTCAGCCCTGTATCTCTGCGCCAGCAGCCAAGGGACTAGCGGGGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO: 249) ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTCTCTTTGGGACCTTCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO: 250) ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTTTCAGACGGGGGGGCTACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO: 251) ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGACCCCATTCTCTCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO: 252) (query number 253) ATGTCTATCGGTCTGCTGTGCTGTGCTGCTCTTTCTCTGCTTTGGGCTGGACCTGTGAATGCTGGAGTTACACAAACCCCCAAGTTCCAAGTGCTGAAGACAGGACAGAGCATGACCCTGCAGTGTGCTCAGGACATGAATCACGAGTACATGAGCTGGTACAGACAGGATCCTGGAATGGGCCTGAGGCTGATCCACTACTCTGTTGGAGCCGGAATTACAGATCAGGGAGAAGTGCCAAATGGCTACAACGTGAGCAGGAGCACAACCGAGGACTTCCCCTTAAGACTGTTGTCTGCTGCTCCATCTCAGACAAGCGTGTACTTTTGCGCCAGCTCCTACTCTCTGTGGGATCTGCAGGAAACCCAGTACTTTGGACCAGGCACAAGACTGTTAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 254) ATGGGCACATCTCTTCTCTGCTGGATGGCTCTTTGTCTGCTTGGAGCCGATCATGCCGATACAGGAGTTAGCCAGGATCCTAGACACAAGATCACCAAGAGAGGCCAGAATGTGACCTTCCGGTGCGATCCTATCTCTGAGCACAACAGGCTGTACTGGTACAGACAAACACTGGGACAAGGACCTGAGTTCCTGACCTACTTCCAGAACGAAGCCCAGCTGGAGAAGTCTAGACTTCTGAGCGACAGATTTAGCGCCGAGAGACCTAAAGGCAGCTTTAGCACCCTGGAGATCCAGAGAACAGAACAGGGCGATTCTGCCATGTACCTGTGTGCTAGCAGCTTTTCTGATGGAGGCGCCACCGATACACAGTATTTCGGACCTGGCACAAGACTGACAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 255) ATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCAGACCTCACAGCCTGACCGATACACAGTACTTTGGCCCTGGCACAAGACTGACAGTGTTAGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO: 256) METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVKETSGSRLTFGEGTQLTVNP (SEQ ID NO: 257) MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFIYPSYTSGTYKYIFGTGTRLKVLAN (sequence number 258) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGTGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 259) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGIGDYKLSFGAGTTVTVRAN (SEQ ID NO: 260) MVKIRQFLLAILWLQLSCVSAAKNEVEQSPQNLTAQEGEFITINCSYSVGISALHWLQQHPGGGIVSLFMLSSGKKKHGRLIATINIQEKHSSLHITASHPRDSAVYICAVRTSYDKVIFGPGTSLSVIPN (SEQ ID NO: 261) MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNLLGATGYSTLTFGKGTMLLVSP (SEQ ID NO: 262) MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVRGINDYKLSFGAGTTVTVRAN (SEQ ID NO: 263) MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVITGFQKLVFGTGTRLLVSPN (SEQ ID NO: 264) MRLVARVTVFLTFGTIIDAKTTQPTSMDCAEGRAANLPCNHSTISGNEYVYWYRQIHSQGPQYIIHGLKNNETNEMASLIITEDRKSSTLILPHATLRDTAVYYCIAGVGRGQNFVFGPGTRLSVLPY (SEQ ID NO: 265) MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFHPNFGNEKLTFGTGTRLTIIPN (SEQ ID NO: 266) MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQPRGDGSSNTGKLIFGQGTTLQVKP (SEQ ID NO: 267) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 268) MGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLTGSYEQYFGPGTRLTVTE (SEQ ID NO: 269) MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSATPEASSPYEQYFGPGTRLTVTE (SEQ ID NO: 270) MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSNLQGRQPQHFGDGTRLSILE (SEQ ID NO: 271) MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLRLGRETQYFGPGTRLLVLE (SEQ ID NO: 272) MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLGQAYEQYFGPGTRLTVTE (SEQ ID NO: 273) MGTRLLCWVAFCLLVEELIEAGVVQSPRYKIIEKKQPVAFWCNPISGHNTLYWYLQNLGQGPELLIRYENEEAVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAVYLCASSLTRGAEAFFGQGTRLTVVE (SEQ ID NO: 274) MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSRDREQESPLHFGNGTRLTVTE (SEQ ID NO: 275) MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSFSGGTYEQYFGPGTRLTVTE (SEQ ID NO: 276) MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSYRGGSTYEQYFGPGTRLTVTE (SEQ ID NO: 277) MSTRLLCWMALCLLGAELSEAEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSQRDSPNEKLFFGSGTQLSVLE (SEQ ID NO: 278) MGCRLLCCAVLCLLGAVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQDPYKLSGNTIYFGEGSWLTVVE (SEQ ID NO: 279) DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 280) DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 281) MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPN (SEQ ID NO: 282) MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPN (SEQ ID NO: 283) MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCASSPGTYKYIFGTGTRLKVLAN (SEQ ID NO: 284) MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPY (SEQ ID NO: 285) IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 286) MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFGPGTRLTVLE (SEQ ID NO: 287) MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFGPGTRLLVLE (SEQ ID NO: 288) MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSDGGATDTQYFGPGTRLTVLE (SEQ ID NO: 289) MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFGPGTRLTVLE (SEQ ID NO: 290) DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 291)

[0247] In some embodiments, the TCR construct comprises a human papillomavirus (HPV)-specific TCR chain. In some embodiments, the TCR construct comprising the HPV-specific TCR chain comprises a TCR α chain and a TCR β chain that target the HPV18 E6 protein and / or the HPV18 E7 protein. In some embodiments, the HPV18 E6 epitope is amino acids 121-135 and / or amino acids 77-91 of the HPV18 E6 protein. In some embodiments, the TCR construct comprising the HPV-specific TCR chain comprises a TCR α chain and a TCR β chain that target the HPV18 E7 protein. In some embodiments, the HPV18 E7 epitope is amino acids 11-19. In some embodiments, the HPV-specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are described in International Patent Application Publication No. WO 2015 / 009604 A1, which is incorporated herein by reference for purposes described herein.

[0248] In some embodiments, the TCR is an invariant natural killer T cell TCR (iTCR). In some embodiments, the iTCR complex comprises an alpha (α) chain iTCR (iTCRα). In some embodiments, the construct encoding the iTCRα comprises a nucleotide coding 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: 292 (e.g., iTCRα associated with human Vα24-Jα18). In some embodiments, the iTCRα comprises 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: 293 (e.g., iTCRα associated with human Vα24-Jα18). ATGAAGAAGCACCTGACCACCTTTCTGGTCATCCTGTGGCTGTACTTCTACAGAGGCAACGGCAAGAACCAGGTGGAACAGAGCCCTCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCTCCCTTCAGCAACCTGCGGTGGTACAAGCAGGATACAGGCAGAGGCCCTGTGTCTCTGACCATCATGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACACTGGATGCCGATACAAAGCAGAGCAGCCTGCACATCACAGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGATAGAGGCAGCACCCTGGGCAGACTGTACTTTGGCAGAGGCACCCAGCTGACCGTGTGGCCCGATATTCAGAACCCTGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGtgcGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCTAGC(SEQ ID NO: 292) MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSDRGSTLGRLYFGRGTQLTVWPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 293)

[0249] In some embodiments, the iTCR complex comprises a beta (β) chain iTCR (iTCRβ). In some embodiments, the construct encoding iTCRβ comprises a nucleotide coding 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: 294, 296, or 298. In some embodiments, the iTCRβ comprises 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: 295, 297, or 299. (query number 294) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSELRALGPSSYNSPLHFGNGTRLTVTDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 295) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCTCTAGTGAAGGCGGAGGACTCAAGTTGGCGAAAAACATCCAATACTTTGGTGCTGGCACCCGGCTTTCTGTCCTGGACCTGAACAAGGTGTTCCCTCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCAGCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTTTTCCCCGATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCtGCACAGATCCCCAGCCTCTGAAAGAACAGCCCGCTCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCTGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGTGGCTTTACCAGCGTGTCATACCAGCAGGGCGTGCTGTCTGCCACCATCCTGTATGAGATTCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTGTCTGCCCTTGTGCTGATGGCCATGGTCAAGAGAAAGGACTTC(SEQ ID NO: 296) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEGGGLKLAKNIQYFGAGTRLSVLDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 297) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCTCTAGTGAATTCGCCAGCTCCGTGCGAGGGAATACTATCTACTTTGGTGAGGGATCTTGGCTTACGGTAGTAGACCTGAACAAGGTGTTCCCTCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCAGCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTTTTCCCCGATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCtGCACAGATCCCCAGCCTCTGAAAGAACAGCCCGCTCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGATAGAGCCAAGCCTGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGTGGCTTTACCAGCGTGTCATACCAGCAGGGCGTGCTGTCTGCCACCATCCTGTATGAGATTCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTGTCTGCCCTTGTGCTGATGGCCATGGTCAAGAGAAAGGACTTC(SEQ ID NO: 298) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEFASSVRGNTIYFGEGSWLTVVDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 299)

[0250] In some embodiments, the NK cells comprise an iTCR complex beta (β) chain iTCR (iTCRβ). In some embodiments, the construct encoding iTCRβ comprises a nucleotide coding 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: 300, 302, 304, 306, 308, 310, 312, or 314. In some embodiments, the iTCRβ comprises 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: 301, 303, 305, 307, 309, 311, 313, or 315.

[0251] In some embodiments, the construct encoding iTCRβ comprises a nucleotide coding 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: 300, 306, 310, or 312. In some embodiments, the iTCRβ comprises 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: 301, 307, 311, or 313. ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCACTGGACAGGGGGCGCAAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO: 300; iTCRβ clone 3) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCATGQGAQDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 301; iTCR beta clone 3) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGATGGGGTGGGGAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGACTTC(SEQ ID NO: 302; iTCRβ clone 18) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSDGVGSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 303; iTCR beta clone 18) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGAGGGGGCTGGAAACACCATATATTTTGGAGAGGGAAGTTGGCTCACTGTTGTAGAGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGACTTC(SEQ ID NO: 304; iTCRβ clone 24) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSEGAGNTIYFGEGSWLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 305; iTCR beta clone 24) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGACAGGGATAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGACTTC(SEQ ID NO: 306; iTCRβ clone 51) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSDRDSNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 307; iTCR beta clone 51) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCGTGGGTCCGGTACCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO: 308; iTCRβ clone 56) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASVGPVPSYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 309; iTCR beta clone 56) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGGGGTGACTAGCGCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO: 310; iTCRβ clone 76) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSGVTSASYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 311; iTCR beta clone 76) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGGAGGAGGGGAGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO: 312; iTCRβ clone 93) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSGGGEETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 313; iTCR beta clone 93) ATGACCATCCGGCTGCTGTGCTATATGGGCTTCTACTTCCTCGGAGCCGGCCTGATGGAAGCCGACATCTACCAGACACCTAGATACCTGGTCATCGGCACCGGCAAAAAGATCACCCTGGAATGCTCCCAGACCATGGGCCACGACAAGATGTACTGGTATCAGCAGGACCCCGGCATGGAACTGCATCTGATCCACTACAGCTACGGCGTGAACAGCACCGAGAAGGGCGATCTGTCTAGCGAGAGCACCGTGTCCAGAATCCGGACCGAGCACTTCCCACTGACACTGGAAAGCGCCAGACCTAGCCACACCAGCCAGTACCTGTGTGCCAGCAGTGCGCAGGGGGTCAGCGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAGGACCTCAATAAGGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTttcCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAgtgTCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGACTTC(SEQ ID NO: 314; iTCRβ clone 96) MTIRLLCYMGFYFLGAGLMEADIYQTPRYLVIGTGKKITLECSQTMGHDKMYWYQQDPGMELHLIHYSYGVNSTEKGDLSSESTVSRIRTEHFPLTLESARPSHTSQYLCASSAQGVSEKLFFGSGTQLSVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 315; iTCR beta clone 96)

[0252] In some embodiments, the construct encoding the iTCR β chain comprises an iTCR β Vβ-DJ region 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 NOs: 316-385. ACTGGACAGGGGGCGCAAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 316; iTCR beta clone 3) AGCAGTGCCCCTGGAGGGTCTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAG (SEQ ID NO: 317; iTCR beta clone 4) AGCAGTGAACTCGACAGGGAAGGAAACACCATATATTTTGGAGAGGGAAGTTGGCTCACTGTTGTAGAG (SEQ ID NO: 318; iTCR beta clone 7) AGCAGTGATATGGGACCCGTCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 319; iTCR beta clone 9) AGCAGTGAGGAAGAGCGGGGCCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 320; iTCR beta clone 13) AGCAGTGATGGGGTGGGGAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG (SEQ ID NO: 321; iTCR beta clone 18) AGCAGTGACCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 322; iTCR beta clone 20) AGCAGTGAGGCCCCAACAGGAACCGGGGCCAACGTCCTGACTTTCGGGGCCGGCAGCAGGCTGACCGTGCTGGAG (SEQ ID NO: 323; iTCR beta clone 21) AGCAGTGATCCTCGACTAGCGGGGGGGGTCGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 324; iTCR beta clone 23) AGCAGTGAGGGGGCTGGAAACACCATATATTTTGGAGAGGGAAGTTGGCTCACTGTTGTAGAG (SEQ ID NO: 325; iTCR beta clone 24) AGCAGTTATGCTACAGGCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 326; iTCR beta clone 26) AGCAGTGAACGGCAGGGCTCCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 327; iTCR beta clone 28) AGCAGTGCTCCGACTAGCGGGAGGGACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 328; iTCR beta clone 29) AGCAGTGAATGGACTAGCGGGGGGCCCAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 329; iTCR beta clone 30) AGCAGTGAACGGGGGGCTAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 330; iTCR beta clone 32) AGCAGTGAAGGGCTAGCGGGAGAACCTCTCTTAGGCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 331; iTCR beta clone 33) AGCAGTGAGGCAGGCGGCCACACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 332; iTCR beta clone 34) AGCAGTGAATACCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (SEQ ID NO: 333; iTCR beta clone 36) AGCACCGACAGGGGATCTTTCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 334; iTCR beta clone 37) GGAGGAGGGACATCTCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (SEQ ID NO: 335; iTCR beta clone 38) AGCAGTCCGACTAGCGGGATGGGGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (SEQ ID NO: 336; iTCR beta clone 39) AGCAGTGAGTTCGGGGCCAACGTCCTGACTTTCGGGGCCGGCAGCAGGCTGACCGTGCTGGAG (SEQ ID NO: 337; iTCR beta clone 41) AGCAGTGTCCGTAGCGGGAGAGGGGACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 338; iTCR beta clone 42) AGCAGTGTCCAGGAGGAAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 339; iTCR beta clone 43) AGCAGTGATAGTAGCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 340; iTCR beta clone 44) AGCAGTGGTACTACGGGACAGGAATCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 341; iTCR beta clone 46) AGCAGTGTAAGGGGGAACCACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 342; iTCR beta clone 47) AGCAGTGAACTTCAGCGGGAGGGTTCTCCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 343; iTCR beta clone 48) AGCAGTGTCCGGGACAGGGATGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (SEQ ID NO: 344; iTCR beta clone 49) AGCAGTGAGGGTCAGGGAGGTTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 345; iTCR beta clone 50) AGCAGTGACAGGGATAGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG (SEQ ID NO: 346; iTCR beta clone 51) AGCAGTGATCGGTCTAGCGGAGCCAAAAACATTCAGTACTTCGGCGCCGGGACCCGGCTCTCAGTGCTGGAG (SEQ ID NO: 347; iTCR beta clone 52) AGCAGTGCCACGACTAGCGGGAGGACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 348; iTCR beta clone 53) AGCAGTGAATTTCGGCAGCGGGAGTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 349; iTCR beta clone 54) AGCAGTGAAATAGCGGGAGTGGCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 350; iTCR beta clone 55) AGCGTGGGTCCGGTACCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 351; iTCR beta clone 56) AGCAGTGAACGGCGCGGGAGACGGGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 352; iTCR beta clone 57) AGCAGTGGGACAGGGTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 353; iTCR beta clone 58) AGCAGTGACCGTAGCGGGAGCTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 354; iTCR beta clone 59) AGCAGTGACAGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 355; iTCR beta clone 60) AGCAGTGCTAGCGGGAGCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 356; iTCR beta clone 61) AGCAGTGACGGGACTAGCGGCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 357; iTCR beta clone 62) AGCAGTGAATATGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (SEQ ID NO: 358; iTCR beta clone 63) AGCAGTGAGTCCGGCCCCCGCAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 359; iTCR beta clone 64) AGCAGTGGCCGACTAGCGGGAGAGGAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (SEQ ID NO: 360; iTCR beta clone 66) AGCAGTGAGGGTGGCAGGGTCGATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 361; iTCR beta clone 67) AGCAGTGAGGCTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (SEQ ID NO: 362; iTCR beta clone 68) AGCAGTCAGGACGGATTGGGATATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (SEQ ID NO: 363; iTCR beta clone 69) AGCAGTGGGCGCCTCCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 364; iTCR beta clone 70) AGCAGTGAATATAACAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 365; iTCR beta clone 71) AGCAGTGAACCCGGATTGGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 366; iTCR beta clone 72) AGCATCCTGGGAGAGGGGCGGAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 367; iTCR beta clone 73) AGCAGTGCCCCGGGACAGATCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (SEQ ID NO: 368; iTCR beta clone 74) AGCAGTGACAACCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (SEQ ID NO: 369; iTCR beta clone 75) AGCAGTGGGGTGACTAGCGCCTCCTACAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 370; iTCR beta clone 76) AGCAGTCCTGAGCCCACCACCCTAGCGGGAGTCCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 371; iTCR beta clone 77) AGCAGTGGGACACAGAGGGCTGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (SEQ ID NO: 372; iTCR beta clone 78) AGCAGTGGGACTAGCGGGAGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 373; iTCR beta clone 79) AGCAGTGAGGCGGGACAGGGTTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 374; iTCR beta clone 80) AGCACCTCTAGCCGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 375; iTCR beta clone 83) AGCAGTGAACCGGGGGAGCGGAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 376; iTCR beta clone 84) AGCAGTGAAGGTCGGGTTAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAG (SEQ ID NO: 377; iTCR beta clone 85) AGCAGTGAATCAGAAGGGGGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 378; iTCR beta clone 86) AGCAGTCCCGGGGGGACTAGCGGGAGGGCACGTCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 379; iTCR beta clone 87) AGCAGTGGGAGGGAGGGGGACCCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 380; iTCR beta clone 88) AGCAGTGGACTAGCGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 381; iTCR beta clone 89) AGCAGTGGGACGACAGGGGATACACGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 382; iTCR beta clone 90) AGCAGTGAAGACCGGGACAGGGGTCACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 383; iTCR beta clone 91) AGCAGTGAACTAGCGAATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAG (SEQ ID NO: 384; iTCR beta clone 92) AGCAGTGGAGGAGGGGAGGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAG (SEQ ID NO: 385; iTCR beta clone 93) AGCAGTGAATATGCAGGGTGGGGCGGCAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAG (SEQ ID NO: 386; iTCR beta clone 94) AGCAGTGAATTGGACGGGACTAGCGCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAG (SEQ ID NO: 387; iTCR beta clone 95) AGCAGTGCGCAGGGGGTCAGCGAAAAACTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAG (SEQ ID NO: 388; iTCR beta clone 96) AGCAGTGAAGTGGCGGGAGCGGACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAG (SEQ ID NO: 389; iTCR beta clone 97) AGCAGCGGCAGGGGGCCAGGGGAAAGTGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAG (SEQ ID NO: 390; iTCR beta clone 98)

[0253] B.NK cells NK cells modified to express the TCR / CD3 receptor complex can be obtained from any suitable source, including fresh or frozen. In certain embodiments, the NK cells are not NK cells obtained from iPSC differentiation. In certain embodiments, the NK cells are obtained from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte products (PBSCs), NK cell lines (e.g., NK-92), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art. Specifically, NK cells can be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines, or mixtures thereof. In certain embodiments, the NK cells are isolated from pooled CB. CB may be pooled from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more units. The NK cells may be autologous or allogeneic with respect to the recipient individual. Isolated NK cells can be haplotype-matched or mismatched to the recipient of cell therapy. NK cells can be detected by specific surface markers, such as CD16 and CD56 in humans. In some cases, the source of NK cells is umbilical cord blood, which is a heterogeneous cell population and may be depleted of specific cells expressing CD3. In other methods, umbilical cord blood is used to derive NK cells by isolating CD34+ cells.

[0254] NK cells may be preactivated with one or more inflammatory cytokines and may or may not have been expanded. In some cases, NK cells are preactivated before being modified to express CD3±TCR or after being modified to express the CD3±TCR complex. In a specific embodiment, preactivation of NK cells may include culturing isolated NK cells in the presence of one or more cytokines. NK cells may be stimulated with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, etc.). In certain embodiments, the preactivation cytokine may be selected from the group consisting of IL-12, IL-15, IL-18, and combinations thereof. One or more additional cytokines may be used in the preactivation step. Pre-activation may be for a short period of time such as 5 to 72 hours, for example 10 to 50 hours, particularly 10 to 20 hours, for example 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, particularly about 16 hours. The pre-activation culture may comprise IL-12 at a concentration of 0.1 to 150 ng / mL, for example 0.5 to 50 ng / mL, particularly 1 to 20 ng / mL, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng / mL, particularly about 10 ng / mL. The pre-activation culture may comprise IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, such as 40-60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, particularly about 50 ng / mL.

[0255] In some cases, NK cells are expanded before or after being modified to express a CD3±TCR complex. Preactivated NK cells can be expanded in the presence of artificial antigen-presenting cells (aAPCs) and / or feeder fragments or NK activation beads. Preactivated NK cells may be washed two, three, four, or five times, particularly three times, before expansion. aAPCs can be engineered to express CD137 ligand and / or membrane-bound cytokines. The membrane-bound cytokine may be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, aAPCs are engineered to express CD137 ligand and mIL-21. aAPCs may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. The aAPCs may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, the aAPCs may be K562 cells, such as K562 cells engineered to express CD137 ligand and mIL-21. The aAPCs may be irradiated. In some embodiments, fragments of APCs can be used to expand NK cells. Engineering can be performed by any method known in the art, such as retroviral transduction. Retroviral transduction can be performed at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days after co-culture of the NK cells with antigen-presenting cells. In some embodiments, retroviral transduction comprises co-transduction of one or more constructs. In some embodiments, retroviral transduction occurs at or about day 5 after co-culture with antigen-presenting cells. In some embodiments, co-culture with antigen-presenting cells continues after transduction of the NK cells. Expansion may be for about 2 to 30 days, such as 3 to 20 days, particularly 12 to 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 to 1:3, such as 2:1, 1:1, 1:2, particularly about 1:2.The growth medium may further contain a proliferation-promoting cytokine, such as IL-2. IL-2 may be present at a concentration of about 10-500 U / mL, e.g., 100-300 U / mL, particularly about 200 U / mL. IL-2 can be replenished in the growth culture, e.g., every 2-3 days. aAPCs can be added to the culture at least a second time, e.g., on about day 7 of growth.

[0256] In certain embodiments, NK cells are transfected or transduced with one or more membrane-bound cytokines, including IL-21, IL-12, IL-18, IL-23, IL-7, or IL-15, that are secreted by the NK cells or tethered to the NK cell membrane. In such cases, the membrane-bound cytokines may be tethered to the NK cell membrane at a particular transmembrane domain, such as the transmembrane domains of CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, or DAP12.

[0257] After preparation, the modified NK cells may be immediately infused (comprising an effective amount of one or more bispecific or multispecific antibodies), or the NK cells may be preserved, such as by cryopreservation. In some embodiments, if the NK cells are sourced from cryopreservation, the NK cells were inactivated prior to cryopreservation using an inactivating agent (e.g., a kinase inhibitor, e.g., dasatinib, nilotinib, rapamycin, etc.). In certain embodiments, the cells can be expanded ex vivo as a bulk population for days, weeks, or months within about 1, 2, 3, 4, or 5 days.

[0258] C. NK cell loading In certain embodiments, NK cells are loaded with antibody prior to use. NK cells can be loaded in any particular manner, including during culture or immediately prior to infusion, to create a complex between the NK cells and the antibody. Conditions need only be sufficient to allow an effective amount of antibody to bind to the surface of the NK cells. When a monospecific antibody is used, the Fc region of the monospecific antibody binds to the NK cell, while the antigen-binding domain of the monospecific antibody is free to bind to the target antigen. In certain embodiments when a multispecific antibody is used, one or more antigen-binding domains of the antibody can bind to the surface of the NK cell, such as via an antigen on the surface of the NK cell (e.g., but not limited to, CD3, NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.), while the other antigen-binding domain is free to bind to its target antigen. In certain embodiments when a multispecific antibody is used, one or more antigen-binding domains of the antibody can bind to one or more target antigens. In certain embodiments when a multispecific antibody is used, the Fc region of the antibody binds to an NK cell, while the antigen-binding domain of the antibody is free to bind to the target antigen. In certain embodiments, the surface of an NK cell may be recognized by the Fab of the antibody and / or through recognition of the Fc region of the antibody by a cognate receptor. In certain embodiments, the Fc region of the antibody may be recognized by an endogenous NK cell receptor (e.g., CD16, CD32, CD64, etc.).

[0259] The culture conditions under which NK cells are loaded may or may not be of a specific type having one or more specific parameters. In certain embodiments, loading of NK cells occurs during culture at a specific temperature, such as 37°C, while in alternative embodiments, the temperature is 36°C or 38°C, or lower or higher. The duration of the loading step may be any suitable time, such as from 1 minute to 24 hours or more. For example, the duration may be in the range of 1 minute to 24 hours, 1 minute to 18 hours, 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 1 hour, 30 minutes to 24 hours, 30 minutes to 18 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 1 hour, 1 to 24 hours, 1 to 18 hours, 1 to 12 hours, 1 to 6 hours, 6 to 24 hours, 6 to 18 hours, 6 to 12 hours, 12 to 24 hours, 12 to 18 hours, or 18 to 24 hours. In some embodiments, the duration of the loading step can be about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours or more, or any range derivable therein. In certain embodiments, the cell culture medium is a basal medium or a complex medium. In some cases, the culture may or may not include one or more reagents utilized during the preactivation and / or expansion steps. In certain embodiments, the culture includes one or more cytokines, including, for example, one or more of IL-12, IL-15, IL-2, and IL-18. In some embodiments, the culture includes any type of APC.

[0260] In certain embodiments, an antibody of the compositions described herein is provided to an effective amount of an NK cell of the present disclosure, thereby producing a "chimeric antigen receptor-like" complex. In particular, the antigen-binding domain of the antibody binds to the NK cell, such as via an antigen that is a cell surface protein. Multiple antibodies may be directed at multiple NK cells, resulting in multiple cell / antibody complexes. Antibodies may be of any type, such as monospecific, bispecific, or multispecific, and in certain cases, the antibody engages both the NK cell and the target antigen via its antigen-binding domain (such as art-known engagers, which are fusion proteins consisting of two single-chain variable fragments (scFv) of different antibodies). In an example where an antibody is monospecific, the antigen-binding domain of the antibody binds to a target antigen, such as a cancer antigen, while another portion of the antibody, such as the Fc region of the antibody, binds to the NK cell. In an example where an antibody is multispecific, one or more antigen-binding domains of the antibody may bind to the NK cell (e.g., via a naturally occurring or genetically introduced NK cell surface antigen, e.g., CD3), and one or more antigen-binding domains of the antibody may bind to one or more target antigens. In certain embodiments, when a multispecific antibody is used, one or more antigen-binding domains of the antibody and / or the Fc region of the antibody may bind to NK cells. The multispecific antibody may be, for example, bispecific, trispecific, or tetraspecific. If the antibody is trispecific or tetraspecific, the additional antigen-binding domain may bind to other cells, such as stem cells.

[0261] In certain embodiments, the antibody can bind to any NK cell surface antigen (which may or may not be a receptor) on NK cells, such as CD16 (including CD16a or CD16b), CD32, CD56, CD64, c-type lectins such as NKG2D and NKG2C, costimulatory molecules such as CS1, DNAM, 2B4, and CD2, NCR, NKp30, NKp44, NKp46, or KIR, and can target NK cells and enhance their reactivity and specificity against different tumors. In certain embodiments, the antibody can bind to a transgenic NK cell surface antigen such as CD3.

[0262] In some embodiments, the antibody may bind to any suitable antigen (e.g., an antigen described herein, such as those described as targets of a CAR and / or a TCR). In certain embodiments, the antibody targets CD19. In certain embodiments, the antibody targets CD20. In certain embodiments, the antibody targets CD123. In certain embodiments, the antibody targets EGFR. In certain embodiments, the antibody targets EGFR2.

[0263] In certain embodiments, the generation of loaded NK cells can be performed by any suitable means that provides conditions sufficient for the appropriate region of the antibody to bind to the appropriate surface region of the NK cell. In some instances, a specific culture medium can be utilized. In certain cases, Plasma-Lyte A and / or human serum albumin are utilized, but not in others. Once the complex is formed in culture, it may or may not be washed prior to administration to a subject, such as by injection. In another embodiment, the NK cells and antibody are administered separately, and the complex is formed in vivo.

[0264] D. Preactivation In certain embodiments, NK cells are preactivated prior to administration to a recipient individual. The preactivation step may or may not occur before any expansion step. In specific embodiments, NK cells are preactivated with one or more cytokines, and in specific embodiments, NK cells are preactivated with one or more of IL-12, IL-15, IL-2, and IL-18, including two, three, or more. If all three of IL-12, IL-15, IL-2, and IL-18 are not utilized, the combination may be IL-12 and IL-15 but not IL-18; or IL-12 and IL-18 but not IL-15; or IL-15 and IL-18 but not IL-12. IL-2 may or may not be used in place of IL-15.

[0265] In certain embodiments, the preactivation cytokines may be IL-12, IL-15, and IL-18. One or more additional cytokines may be used in the preactivation step. Preactivation may be for a short period of time, such as 5 to 72 hours, e.g., 10 to 50 hours, particularly 10 to 20 hours, e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours, and may be, in some cases, approximately 16 hours. The preactivation culture may contain IL-18 and / or IL-15 at a concentration of 10 to 100 ng / mL, e.g., 40 to 60 ng / mL, particularly 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 ng / mL, particularly about 50 ng / mL. In some cases, the pre-activation culture comprises IL-12 at a concentration of 0.1 to 150 ng / mL, including a concentration of 1 to 20 ng / mL, e.g., 10 ng / mL. In alternative embodiments, NK cells may be stimulated with IL-2 or other cytokines that bind to the common gamma chain (e.g., IL-7, IL-21, and others), which may be in addition to or as a replacement for one or more of IL-12, IL-15, and IL-18. In such cases, the pre-activation culture may comprise IL-12 at a concentration of 0.1 to 150 ng / mL, e.g., 0.5 to 50 ng / mL, particularly 1 to 20 ng / mL, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, particularly about 10 ng / mL.

[0266] E. proliferation In certain embodiments, NK cells are expanded to increase their quantity before administration to an individual in need thereof. The expanded cells may or may not be derived from preactivated NK cells, such that a preactivation step may occur before the expansion step. The NK cell expansion step may be any suitable step by which an NK cell population is expanded, but in certain embodiments, the expansion step utilizes one or more specific reagents, such as in culture, to enhance their expansion. In certain embodiments, the NK cells may not be expanded. IL-2, IL-15, IL-18, or any combination of these cytokines may be added to the expansion culture before or during expansion. In specific embodiments, NK cells may be expanded ex vivo in flasks or in one of several different bioreactor configurations with continuous perfusion of medium / additives.

[0267] In specific aspects, NK cells (whether pre-activated or not) may be washed (e.g., with PBS, Plasma Lyte, human serum albumin, or culture medium, or a combination thereof) before and / or after expansion, such as once, twice, three times, four times, or five times. In certain embodiments, the cells are washed specifically three times. In certain embodiments, the NK cells are expanded in the presence of artificial antigen-presenting cells (aAPCs). In certain embodiments, the NK cells are expanded in the presence of aAPC fragments. The aAPCs can be engineered to express CD137 ligand 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 certain embodiments, the aAPCs are engineered to express CD137 ligand and mIL-21. The aAPCs may be derived from cancer cells, such as leukemia cells. aAPCs may not express endogenous HLA class I, II, or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58) or CD48. In particular, aAPCs may be K562 cells, such as K562 cells engineered to express CD137 ligand and mIL-21. Engineering can be by methods known in the art, such as retroviral transduction, but any viral or non-viral vector can be utilized. aAPCs may or may not be irradiated. Expansion may be for a specific period of time, for example, about 2 to 30 days, for example, 3 to 20 days, particularly 12 to 16 days, for example, 12, 13, 14, 15, 16, 17, 18, or 19 days, particularly about 14 days. The preactivated NK cells and aAPCs may be present in a ratio of about 3:1 to 1:3, for example, 2:1, 1:1, 1:2, particularly about 1:2. The expansion culture may further contain one or more cytokines to promote proliferation, such as IL-2. IL-2 may be present at a concentration of about 10-500 U / mL, e.g., 100-300 U / mL, particularly about 200 U / mL. IL-2 may be replenished in the expansion culture at regular intervals, e.g., every 2-3 days. aAPCs may be added to the culture at least a second time, e.g., on about day 7.The cytokines used in the preactivation and / or expansion steps may be recombinant human cytokines.

[0268] In certain embodiments, after expansion, the NK cells may be used immediately in any manner, such as by conjugation to one or more antibodies, or may be stored, such as by cryopreservation. In certain aspects, the cells may be expanded ex vivo as a bulk population within about 1, 2, 3, 4, or 5 days for days, weeks, or months.

[0269] Activated and / or expanded NK cells secrete type I cytokines, such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells, as well as other cytokines and chemokines. Measurement of these cytokines can indicate the activation state of NK cells. Additionally, other methods known in the art for determining NK cell activation can be used to characterize the NK cells of the present disclosure.

[0270] Thus, with regard to certain preactivation and expansion aspects of the present disclosure, in certain embodiments, NK cells preactivated with any combination of IL-12, IL-15, and / or IL-18 and then expanded with aAPCs, such as K562 cells, that express mIL-21 and CD137 ligand provide highly potent cell products. Accordingly, methods of using the NK cells of the present invention for the treatment of various diseases, such as immunotherapy of cancer patients, are provided. In an exemplary method, isolated NK cells may be subjected to a short period of preactivation, such as about 16 hours, with a combination of cytokines, such as interleukin-12 (IL-12), IL-15, and / or IL-18, and then expanded with artificial antigen-presenting cells (aAPCs), such as K562 feeder cells, that express membrane-bound IL-21 and CD137 ligand, and / or exogenous IL-2. IL-2, IL-15, IL-18, or a combination of these cytokines, may be added to the expansion culture at least a second time.

[0271] F. Freezing In certain cases, the NK cells and / or antibodies of the present disclosure are preserved in a cryopreservation medium composition comprising at least one cryoprotectant, serum (human or animal serum) or a non-serum substitute (not human or animal serum), and at least one cytokine and / or at least one growth factor. In some cases, the cryoprotectant is dimethyl sulfoxide (DMSO), glycerin, glycerol, hydroxyethyl starch, or a combination thereof. The non-serum substitute may be of any type, including at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments of the composition utilizing one or more (including two or more) cytokines, the cytokines may be natural, recombinant, or synthetic proteins. At least one of the cytokines may be a Food and Drug Administration (FDA)-approved cytokine. Examples of cytokines and growth factors include at least IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, interferon, tumor necrosis factor, stem cell factor, FLT3-ligand, APRIL, thrombopoietin, erythropoietin, or combinations thereof. In serum embodiments, the serum may be human serum (including human AB serum) or animal-derived serum, such as bovine serum. DMSO and other cryoprotectants, if utilized, may comprise 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10%, etc., of the composition.For embodiments in which serum is employed, the serum may be present in an amount of 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-75%, 10-70%, 10-65%, 10-60%, 10-85%, 10-8 ...85%, 10-80%, 10-85%, 10-80%, 10-75%, 10-70%, 10-65%, 10-60%, 10-65%, 1 %, 10-55%, 10-50%, 10-45%, 10-40%, 10-35%, 10-30%, 10-25%, 10-20%, 10-15%, 20-99%, 20-95%, 20-90%, 20-85%, 20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-35%, 20-30%, 20-25%, 30-99%, 30-95%, 30-90 %, 30~85%, 30~80%, 30~75%, 30~70%, 30~65%, 30~60%, 30~55%, 30~50%, 30~45%, 30~40%, 30~35%, 40~99%, 40~95%, 40~90%, 40~85%, 40~80%, 40~75%, 40~70%, 40~65%, 40~60%, 40~55%, 40~50%, 40~45%, 50~99%, 50~95%, 50~90%, 50~85%, 50~80 %, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99% of the composition. The composition may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less serum.In certain embodiments, the composition comprises platelet lysate, which may be at any concentration in the composition, however, in certain embodiments, the platelet lysate is present in an amount of 5-99%, 5-95%, 5-90%, 5-85%, 5-80%, 5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-20%, 5-15%, 5-10%, 10-99%, 10-95%, 10-90%, 10-85%, or any combination thereof. , 10~80%, 10~75%, 10~70%, 10~65%, 10~60%, 10~55%, 10~50%, 10~45%, 10~40%, 10~35%, 10~30%, 10~25%, 10~20%, 10~15%, 20~99%, 20~95%, 20~90%, 20~85%, 20~80%, 20~75%, 20~70%, 20~65%, 20~60%, 20~55%, 20~50%, 20~45%, 20~40%, 20~35%, 20~30%, 20~ 25%, 30-99%, 30-95%, 30-90%, 30-85%, 30-80%, 30-75%, 30-70%, 30-65%, 30-60%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 40-99%, 40-95%, 40-90%, 40-85%, 40-80%, 40-75%, 40-70%, 40-65%, 40-60%, 40-55%, 40-50%, 40-45%, 50-99%, 50-95%, 50-90% , 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, 50-55%, 60-99%, 60-95%, 60-90%, 60-85%, 60-80%, 60-75%, 60-70%, 60-65%, 70-99%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 80-99%, 80-95%, 80-90%, 80-85%, 90-99%, 90-95%, or 95-99% composition. The composition may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or less platelet lysate.

[0272] The composition can have specific concentrations of components, including cytokines and / or growth factors. In specific cases, any cytokine, including, for example, IL-2, IL-21, and / or IL-15, is present in the composition at a specific concentration. IL-2 can be present at a concentration of, for example, 1-5000, 1-1000, 1-500, 1-100, 100-5000, 100-500, 500-5000, 500-1000, or 1000-5000 U / mL. In specific cases, IL-2 is present in the composition at a concentration of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 U / mL, or less. In certain embodiments, IL-21 is present in the composition at a concentration of 10-3000, 10-2000, 10-1000, 10-500, 10-100, 100-3000, 100-2000, 100-1000, 500-3000, 500-2000, 500-1000, 1000-3000, 1000-2000, or 2000-3000 ng / mL. IL-21 may be present in the composition at a concentration of at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 ng / mL, or less. IL-15 may be present in the composition at a concentration of 1-2000, 1-1000, 1-500, 1-100, 100-2000, 100-1000, 100-500, 500-2000, 500-1000, or 1000-2000 ng / mL. IL-15 may be present in the composition at a concentration of at least 10, 50, 100, 500, 1000, 1500, or 2000 ng / mL, or less.

[0273] Compositions encompassed herein that contain at least one cryoprotectant, serum or a non-serum alternative, and at least one cytokine and / or at least one growth factor may each further comprise a plurality of immune cells and / or stem cells of any type. In specific embodiments, the cells are NK cells, T cells, B cells, or NKT cells derived from mature bone marrow or peripheral blood cells; cell lines such as tumor cell lines (e.g., NK92 or other NK lines) that may be derived from bone marrow, peripheral blood, skin, adipose tissue, or a combination thereof; hematopoietic stem cells; induced pluripotent stem cells; MSCs (cell populations also referred to in the literature as "mesenchymal stem cells" and "mesenchymal stromal cells"); or mixtures thereof. In embodiments in which NK cells are utilized, the NK cells may or may not be expanded NK cells. Embodiments of the present disclosure also encompass pharmaceutical compositions comprising any of the compositions of the present disclosure and a suitable pharmaceutically acceptable carrier.

[0274] In certain embodiments, the cells and / or antibodies are treated with one or more inactivating agents (eg, kinase inhibitors such as dasatinib, nilotinib, rapamycin, etc.) prior to cryopreservation.

[0275] In some embodiments, the techniques described herein include inactivating NK cells, which includes treating NK cells with an effective amount of one or more inactivating agents under conditions to generate inactivated NK cells. In some embodiments, the inactivating agent is a kinase inhibitor. In some embodiments, the inactivating agent is a mechanistic target of rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is rapamycin, everolimus, and / or temsirolimus. In some embodiments, the mTOR inhibitor is rapamycin. In some embodiments, the inactivating agent is a tyrosine kinase (TK) inhibitor. In some embodiments, the TK inhibitor is selected from the group consisting of lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, acalabrutinib, ibrutinib, capmatinib, pexidartinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdafitinib, nintedanib, gilteritinib, and midosumab. In some embodiments, the TK inhibitor is taurine, tucatinib, neratinib, baricitinib, ruxolitinib, fedratinib, tofacitinib, ripretinib, selumetinib, binimetinib, cobimetinib, trametinib, upadacitinib, avapritinib, selpercatinib, cabozantinib, fostamatinib, larotrectinib, entrectinib, axitinib, regorafenib, pazopanib, sorafenib, lenvatinib, vandetanib, and / or sunitinib. In some embodiments, the TK inhibitor is a BCR-Abl inhibitor. In some embodiments, the TK inhibitor is bosutinib, ponatinib, nilotinib, dasatinib, and / or imatinib. In some embodiments, the TK inhibitor is dasatinib and / or nilotinib. In some embodiments, the TK inhibitor is dasatinib.

[0276] In some embodiments, treatment with an inactivating agent occurs at any time during the culture of NK cells. In some embodiments, treatment is for about 24 hours to about 96 hours, about 36 hours to about 84 hours, or about 48 hours to about 72 hours. In some embodiments, treatment is for about 24 hours, about 48 hours, or about 72 hours. In some embodiments, NK cells are treated with an inactivating agent at a concentration of about 1 to about 1000 nM. In some embodiments, NK cells are treated with an inactivating agent at a concentration of about 5 to about 500 nM. In some embodiments, NK cells are treated with an inactivating agent at a concentration of about 20 to about 200 nM. In some embodiments, NK cells are treated with an inactivating agent at a concentration of about 30 to about 100 nM. In some embodiments, inactivated NK cells have increased expression of one or more of C-kit, CCR-5, CD62L, and / or CXCR4, and / or decreased expression of one or more of NKG2D, DNAM, OX-40, TRAIL, HLA-DR, CD2, CD25, ICOS, and / or CD95 compared to activated NK cells. In some embodiments, the techniques described herein include methods of maintaining the viability of a population of cells at least 50% or greater after cryopreservation of the population, the methods including subjecting the population to an effective amount of one or more inactivating agents (e.g., tyrosine kinase inhibitors) to inactivate the cells prior to cryopreservation, cryopreserving the cells, and thawing the population, wherein the viability of the population upon thawing is at least 50% or greater. In some cases, upon thawing the cells, the viability of the population of cells after cryopreservation of the population is at least 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or greater.

[0277] III. Heterologous Proteins and Mutations In a specific embodiment, NK cells are engineered to express not only one or more components of the TCR / CD3 complex, but also one or more other heterologous proteins that can enhance the activity of the NK cells in any way, including at least their activation, persistence, proliferation, homing, and / or cytotoxicity.

[0278] A. Bispecific or Multispecific Antibodies In certain embodiments, the NK cells are engineered to express one or more bispecific or multispecific antibodies, while in other cases the NK cells do not express antibodies, but antibodies are utilized in conjunction with the NK cells.

[0279] In certain embodiments in which NK cells are engineered to express antibodies and / or utilized in conjunction with NK cells, the antibodies can be any antibody or antibody-like structure known in the art, including antibody fragments, single-domain antibodies, scFvs, bispecific antibodies, bispecific diabodies, trispecific antibodies, scFv-Fc, and other antibody constructs and engagers. In particular, the antibodies can be engagers that bridge specific immune effector cells with specific target cells to destroy the target cells. The present disclosure enables the use of engineered NK cells with standard T cell engagers (BiTEs) because they are often engineered to express CD3, the T cell antigen to which BiTE engagers bind. In such cases, BiTEs used in the present invention can also target cancer antigens or viral antigens tailored to the disease state of the intended recipient individual. For example, BiTEs can be tailored to bind to cancer antigens characteristic of cancer cells of the individual's cancer. The anti-CD3 antibody in the BiTE can target the CD3γ chain, CD3δ chain, CD3ε chain, or CD3ζ chain.

[0280] In some cases, in addition to expressing a CD3 complex (with or without a TCR) that allows NK cells to be used as a therapeutic with BiTEs, NK cells can be engineered to express (or not express, but instead be used in combination with) one or more bispecific NK engagers (BiKEs). BiKEs include antibodies that bind to surface proteins on NK cells, including surface proteins naturally expressed on NK cells (e.g., but not limited to, NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.), and also include antibodies that bind to a desired target antigen. BiKEs can target NK cells using NK surface proteins, such as CD16, CS1, CD32, CD64, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, and KIR. In such cases, BiKEs used in the present invention can also target cancer antigens or viral antigens, which can be tailored to the disease state of the intended recipient. For example, BiKE may be tailored to bind to a cancer antigen characteristic of cancer cells of an individual's cancer.

[0281] In certain embodiments, the antibody is blinatumomab. In certain embodiments, the antibody is tebentafusp. In certain embodiments, the antibody is mosunetuzumab. In certain embodiments, the antibody is teclistamab. In certain embodiments, the antibody is glofitamab. In certain embodiments, the antibody is epcolitamab. In some embodiments, the antibody is flotetuzumab. In some embodiments, the antibody is APV0436. In some embodiments, the antibody is TNB383B. In certain embodiments when a multispecific antibody is used, one or more antigen-binding domains of the antibody can bind to one or more target antigens.

[0282] In embodiments in which NK cells express a CD3 complex (with or without a TCR and / or iTCR) and one or more BiKEs, one or more vectors can be used to transfect or transduce the CD3 complex components (with or without a TCR and / or iTCR) and one or more BiKEs into the cells. In some cases, the one or more CD3 complex components (with or without a TCR and / or iTCR) and BiKEs may or may not be on the same multicistronic vector.

[0283] B. Engineered receptors In a specific embodiment, NK cells are engineered to express one or more engineered receptors.In some cases, the engineered receptor is an engineered antigen receptor that targets any kind of cancer antigen or virus antigen.Receptor can be adjusted to target desired antigen based on the disease state of intended recipient individual.

[0284] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). NK cells may be engineered to encode at least one CAR, and the CAR may be, for example, a first-generation, second-generation, third-generation, or later-generation CAR. The CAR may or may not be bispecific for two or more different antigens. The CAR may contain one or more costimulatory domains. NK cells may also be engineered to express receptors for enhancing antibody binding, such as CD16, CD32, and / or CD64 receptors. Each costimulatory domain may comprise, for example, one or more costimulatory domains from a member of the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40, or a combination thereof. In certain embodiments, the CAR comprises CD3 zeta. In certain embodiments, the CAR lacks one or more specific costimulatory domains; for example, the CAR can lack 4-1BB and / or lack CD28.

[0285] In certain embodiments, the intracellular CAR polypeptide comprises an extracellular spacer domain, sometimes referred to as a hinge, that connects the antigen-binding domain and the transmembrane domain. The extracellular spacer domain includes, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Examples of extracellular spacer domains include, but are not limited to, artificial spacers made from polypeptides such as CD8α hinge, CD28, and Gly3, or the CH1 and CH3 domains of IgG (such as human IgG1 or IgG4). In certain embodiments, the extracellular spacer domain can comprise (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8α 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 certain embodiments, the hinge is derived from IgG1, and in certain embodiments, the CAR polypeptide comprises a particular IgG1 hinge amino acid sequence or is encoded by a particular IgG1 hinge nucleic acid sequence.

[0286] The transmembrane domain of the CAR may be naturally derived or synthetic. If naturally derived, in some embodiments, a domain derived from a membrane-bound or transmembrane protein is used. Transmembrane regions include those derived from (i.e., at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD30, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules (such as DAP10 or DAP12). Alternatively, the transmembrane domain in some embodiments is synthetic. In some embodiments, synthetic transmembrane domains primarily contain hydrophobic residues such as leucine and valine. In some embodiments, triplets of phenylalanine, tryptophan, and valine may be found on either side of the synthetic transmembrane domain.

[0287] In some embodiments, the engineered receptor utilizes one or more homing receptors (which can home to a target without necessarily releasing a signal, such as through adhesion molecule-based events) and / or one or more chemokine receptors. Examples of chemokine receptors include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors. In certain cases, the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD62L), CXCR1, CXCR2, or CX3CR1.

[0288] C. Cytokines In some embodiments, NK cells are engineered to express one or more heterologous cytokines and / or to upregulate the normal expression of one or more heterologous cytokines. The cells may or may not be transduced or transfected with one or more cytokines on the same vector as other genes. In certain embodiments, NK cells may be modified to express one or more cytokines, cytokine receptors, chemokines, chemokine receptors, and / or suicide genes.

[0289] One or more cytokines can be coexpressed from vectors containing polypeptides separate from any component of the TCR / CD3 complex. For example, interleukin-15 (IL-15) is tissue-restricted and is only observed at any level in serum or systemically under pathological conditions. IL-15 possesses several desirable properties for adoptive therapy. IL-15 is a homeostatic cytokine that promotes the eradication of established tumors by inducing the development and proliferation of natural killer cells and relieving the functional suppression of tumor-resident cells, thereby inhibiting activation-induced cell death (AICD). In addition to IL-15, other cytokines are also contemplated. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to the activation and proliferation of cells used in human applications. In certain embodiments, NK cells expressing IL-15 are capable of sustained supportive cytokine signaling, which is beneficial for survival after infusion. In certain embodiments, NK cells expressing IL-21 are capable of sustained supportive cytokine signaling, which is beneficial for survival after infusion. In certain embodiments, the cytokine is expressed as part of a multicistronic construct with one or more functional and / or marker proteins.

[0290] In some embodiments, the cells express one or more exogenously provided engineered receptors, wherein the engineered receptors include a chemokine receptor and / or a cytokine receptor. In some embodiments, the cytokine receptor is an IL-15 receptor. In some embodiments, the cytokine receptor is a non-naturally occurring variant of a cytokine receptor. In some embodiments, the cytokine receptor is an IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, or GMCSF receptor, or a combination thereof.

[0291] In specific embodiments, the cells express one or more exogenously supplied cytokines. By way of example, the cytokines are IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. Cytokines may be exogenously supplied to NK cells by being expressed from an expression vector within the cells. Alternatively, endogenous cytokines within cells are upregulated by manipulating the expression control of the endogenous cytokine, such as by genetic modification of the cytokine's promoter site. When cytokines are provided to cells on an expression construct, they may be encoded from the same vector as one or more components of the CD3 complex, with or without the TCR complex. In some embodiments, the present disclosure relates to the co-use of IL-15 with a CAR and, optionally, a suicide gene.

[0292] In some embodiments, a specific sequence of IL-15 is utilized, such as the one shown below (underlined indicates signal peptide sequence): ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCC GGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGT GATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC (SEQ ID NO: 49) MRISKPHLRSISIQCYLCLLLNSHFLTEA GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 48)

[0293] D. Antigen The modified NK cells of the present disclosure are used in conjunction with bispecific or multispecific antibodies that target one or more specific antigens. Additionally, NK cells may be modified with artificial antigen receptors that target one or more specific antigens. When NK cells are modified with one or more engineered antigen receptors, the antigens targeted by the bispecific or multispecific antibodies and the antigens targeted by the one or more engineered antigen receptors may or may not be the same antigens. In some cases, the antigens targeted by the bispecific or multispecific antibodies and the antigens targeted by the one or more engineered antigen receptors are different antigens but are associated with the same type of cancer.

[0294] Some antigens targeted by antibodies and / or engineered antigen receptors are expressed in the context of the disease, condition, or cell type targeted via adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including hematologic cancers, lymphomas, leukemias, and / or cancers of the immune system, such as B, T, and myeloid leukemias, lymphomas, and myelomas, such as multiple myeloma. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., tumor or pathogenic cells, compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells.

[0295] Any suitable antigen can be targeted in this method. In some cases, the antigen may be associated with a particular cancer cell but not with a non-cancerous cell. Exemplary antigens include, but are not limited to, antigenic molecules derived from infectious agents, self / autoantigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In certain embodiments, antigens include NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate-binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp 41, CD5, CD23, CD30, HERV-K, IL-11Rα, κ chain, λ chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MA GE-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, tyrosinase-related protein, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, phospho Inositide 3-kinase (PI3K), TRK receptor, PRAME, P15, RU1, RU2, SART-1, SART-3, Wilms tumor antigen (WT1), AFP, -catenin / m, caspase-8 / m, CDK-4 / m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin / m, RAGE, SART-2, TRP-2 / INT2, 707-AP, annexin II,CDC27 / m, TPI / mbcr-abl, BCR-ABL, interferon regulatory factor 4 (IRF4), ETV6 / AML, LDLR / FUT, Pml / RAR, tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., epidermal growth factor receptor (EGFR) (especially EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia-inducible factors (e.g., HIF-1 and HIF-2), nuclear factor-κB (NF-B), Notch receptors (e.g., Notch1-4), NY ESO 1, c-Met, mammalian target of rapamycin (mTOR), WNT, extracellular signal-regulated kinase (ERK) and their regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22α, carbonic anhydrase I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL / AML1, GD2, proteinase 3, hTERT, sarcoma translocation breakpoints, EphA2, ML-IAP, EpCAM, ERG (TMPRSS2) ETS fusion gene), NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, GD3, fucosyl-GM1, mesothelial, PSCA, sLe, PLAC1, GM3, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, legumain, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos-related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNC1, and LRRN1. Examples of antigen sequences can be found, for example, in GenBank, 登録商標In 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 No. NC_000023.11), TRAIL / DR4 (Accession No. NC_000003.12), and / or CEA (Accession No. NC_000019.10) are known in the art.

[0296] For example, tumor-associated antigens can be derived from prostate cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, kidney cancer, mesothelioma, ovarian cancer, liver cancer, brain tumor, bone cancer, stomach cancer, spleen cancer, testicular cancer, cervical cancer, anal cancer, gallbladder cancer, thyroid cancer, or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE1, 3, and MAGE4 (or other MAGE antigens as disclosed in International Patent Publication No. WO99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types, such as melanoma, lung cancer, sarcoma, and bladder cancer. See, for example, U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostatic acid phosphate, NKX3.1, and six-stage membrane epithelial antigen of the prostate (STEAP).

[0297] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, Criptin, etc. Furthermore, tumor antigens can be self-peptide hormones, such as gonadotrophin-releasing hormone (GnRH), a short peptide with a total length of 10 amino acids, which is useful in the treatment of many cancers.

[0298] Antigens can include genes mutated in tumor cells, such as telomerase enzyme, survivin, mesothelin, mutant ras, bcr / abl rearrangements, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and aberrantly expressed intronic sequences such as N-acetylglucosaminyltransferase-V; clonal rearrangements of immunoglobulin genes that generate unique idiotypes in myeloma and B-cell lymphoma; tumor antigens containing epitopic regions or epitopic peptides derived from oncoviral processes, such as human papillomavirus proteins E6 and E7; Epstein-Barr virus protein LMP2; and epitopic regions or epitopic peptides derived from genes transcribed at different levels in tumor cells compared to normal cells, such as unmutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0299] E. Suicide gene In certain embodiments, suicide genes are utilized with NK cell therapy to control its use and allow for termination of cell therapy at a desired event and / or time. Suicide genes are employed in transduced cells to induce their death when necessary. Cells of the present disclosure that have been modified to carry one or more vectors encompassed by the present disclosure may contain one or more suicide genes. In some embodiments, the term "suicide gene" as used herein is defined as a gene that, upon administration of a prodrug or other agent, results in the transfer of the gene product to a compound that kills the host cell. In other embodiments, the suicide gene encodes a gene product that is optionally targeted by an agent (such as an antibody) that targets the suicide gene product.

[0300] In some cases, cell therapy may be subject to the use of one or more suicide genes of any type when an individual undergoing and / or receiving cell therapy is considered to be at risk of developing or imminently developing one or more symptoms of one or more adverse events, such as cytokine release syndrome, neurotoxicity, anaphylaxis / allergy, and / or on-target / off-tumor toxicity (for example). The use of a suicide gene may be part of a planned protocol for treatment, or may be used only if its use is deemed necessary. In some cases, cell therapy may be terminated using an agent targeting the suicide gene or its gene product because treatment is no longer necessary.

[0301] The use of suicide genes can be initiated upon the onset of at least one adverse event in an individual, which can be recognized by any means, including periodic monitoring, which may or may not be continuous from the start of cell therapy. Adverse events can be detected by examinations and / or tests. If an individual develops cytokine release syndrome (sometimes referred to as cytokine storm), the individual may experience elevated levels of inflammatory cytokine(s) (by way of example only: interferon-γ, granulocyte-macrophage colony-stimulating factor, IL-10, IL-6, and TNF-α), fever, fatigue, hypotension, hypoxia, tachycardia, nausea, capillary leak, cardiac / renal / liver dysfunction, or a combination thereof. If an individual experiences neurotoxicity, the individual may experience confusion, delirium, aplasia, and / or seizures. 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.

[0302] Examples of suicide genes include engineered non-secreted (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US19 / 62009, incorporated herein by reference in its entirety), which may be affected by delivery of antibodies that bind to TNF-α mutants. Examples of suicide gene / prodrug combinations that may be used include herpes simplex virus thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase-thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside. Escherichia coli purine nucleoside phosphorylase, a so-called suicide gene that converts the prodrug 6-methylpurine deoxyriboside to the toxic purine 6-methylpurine, may also be used. Other examples of suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome p450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine ribosyltransferase (XGRTP), glycosidase enzymes, methionine-α,γ-lyase (MET), and thymidine phosphorylase (TP).

[0303] F. Endogenous gene knockout or knockdown In certain embodiments, the NK cells of the present disclosure can include gene editing of the NK cells to remove one, two, three, four, five, six, seven, eight, nine, ten, or more endogenous genes in the NK cells. In some cases, the gene editing is performed in NK cells that express one or more heterologous transgenes (e.g., CD3, TCR, etc.), while in other cases, the gene editing is performed in at least some NK cells that do not express the heterologous transgenes, but that will eventually express one or more heterologous transgenes. In certain embodiments, the NK cells that are gene-edited are expanded NK cells.

[0304] In certain embodiments, one or more endogenous genes of NK cells are modified, such as expression disruption, which reduces part or all of the expression.In certain cases, one or more genes are knocked down or knocked out using the process of the present disclosure.In certain cases, multiple genes are knocked down or knocked out in the same process of the present disclosure.The gene that is edited in NK cells can be any, but in a specific embodiment, the gene is a gene whose gene product inhibits the activity and / or proliferation of NK cells.In certain cases, the gene that is edited in NK cells allows NK cells to work more effectively in tumor microenvironment. In a specific embodiment, the gene is one or more of NKG2A, SIGLEC-7, LAG3, TIM3, CISH, FOXO1, TGFBR2, TIGIT, CD96, ADORA2, NR3C1, PD1, PDL-1, PDL-2, CD47, SIRPA, SHIP1, ADAM17, RPS6, 4EBP1, CD25, CD40, IL21R, ICAM1, CD95, CD80, CD86, IL10R, TDAG8, CD5, CD7, SLAMF7, CD38, LAG3, TCR, β2-microglobulin, HLA, CD73, CREB, CREM, ICER, and CD39. In a specific embodiment, the TGFBR2 gene is knocked out or down in NK cells. In a specific embodiment, the CISH gene is knocked out or down in NK cells. In a specific embodiment, the CD38 gene is knocked out or knocked down in NK cells.

[0305] In some embodiments, gene editing is carried out using one or more DNA-binding nucleic acids, such as modification via RNA-guided endonuclease (RGEN).For example, modification can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins.Generally, "CRISPR system" refers collectively to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including the sequence encoding the Cas gene, tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active part tracrRNA), tracr-mate sequence (including "direct repeat" and tracrRNA processing part direct repeat in the context of endogenous CRISPR system), guide sequence (also referred to as "spacer" in the context of endogenous CRISPR system), and / or other sequences and transcripts from the CRISPR locus.Methods for utilizing CRISPR systems are well known in the art.

[0306] IV. Administration of Therapeutic Compositions The CD3-expressing NK cells and the bispecific or multispecific antibody are administered to an individual in need thereof, including in a manner that brings them into close proximity so that the anti-CD3 antibody of the bispecific or multispecific antibody can bind to CD3 on the CD3-expressing NK cells. In some embodiments, the two components are administered separately to the individual, while in other embodiments, the two components are complexed prior to administration, such as by ex vivo methods. In another embodiment, the NK cells express the antibody. In some cases, the two components are not pre-complexed prior to administration, but are co-administered by any suitable route, such as by co-infusion into the patient.

[0307] Embodiments of the present disclosure relate to methods of using compositions comprising NK cells and antibodies provided herein to treat or prevent a medical disease or disorder. The methods include administering therapeutically effective amounts of CD3 (±TCR)-modified NK cells and antibodies to a subject, thereby treating or preventing disease in the subject, including reducing the risk of disease, reducing the severity of disease, and / or delaying the onset of disease. In certain embodiments of the present disclosure, cancer or infectious disease is treated by transfer of a composition comprising a population of NK cells and a corresponding antibody. In at least some cases, NK cells, due to their release of proinflammatory cytokines, may augment the adaptive immune response by reversing an anti-inflammatory tumor microenvironment and promoting the differentiation, activation, and / or recruitment of accessory immune cells to malignant tumor sites. In certain embodiments, the providing step may include culturing the NK cells with the antibody molecule for a specified time (e.g., from about 5 minutes to about 24 hours or more) and storing the NK cells and antibody molecule for a period of time (e.g., about 1, 2, 3, 4, 5, or more than 5 days) prior to infusion / administration.

[0308] The cancers for which the therapeutic method of the present invention is useful include any malignant cell type, such as those found in solid tumors or blood tumors.Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, appendix, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.Exemplary blood tumors include bone marrow tumors, malignant tumors of T cells or B cells, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), cancer of the peritoneum, stomach 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 or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0309] Cancer may be of the following histological types, particularly, but not limited to: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilonidal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial polyposis coli; solid tumor; malignant carcinoid tumor; lobular-alveolar adenocarcinoma; papillary adenocarcinoma; chromatophore carcinoma; Eosinophilic carcinoma; Eosinophilic adenocarcinoma; Basophilic carcinoma; Clear cell adenocarcinoma; Granular cell carcinoma; Follicular adenocarcinoma; Papillary and follicular adenocarcinoma; Non-encapsulated sclerosing carcinoma; Adrenal cortical carcinoma; Endometrial carcinoma; Skin adnexal carcinoma; Apocrine adenocarcinoma; Sebaceous gland carcinoma; Keratin adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma; Papillary cystadenocarcinoma; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma; Mucinous adenocarcinoma; Signet ring cell carcinoma; Invasive ductal carcinoma; Medullary carcinoma; Lobular carcinoma; Inflammatory carcinoma; Paget's disease of the breast; Acinic cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma with squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Sarcoma, malignant; Granulosa cell Tumors, malignant; Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipocytoma, malignant; Paraganglioma, malignant; Extramammary paraganglioma, malignant; Pheochromocytoma; Angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial spreading melanoma; Lentigo maligna melanoma; Lentigo acuminata melanoma; Nodular melanoma; Malignant melanoma of giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Mixed tumors, malignant; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma ;Carcinosarcoma;Mesenchymoma, malignant;Brenner tumor, malignant;Philodes tumor, malignant;Synovial sarcoma;Mesothelioma, malignant;Dysgerminoma;Embryonal carcinoma;Teratoma, malignant;Ovarian goiter, malignant;Choriocarcinoma;Mesostosis, malignant;Angiosarcoma;Hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Paddy cortical osteosarcoma;Chondrosarcoma;Chondrosarcoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastoma;Ameloblastic odontoma;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal tumor; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granular cell tumor, malignant; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Paragranuloma; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin's lymphoma; B-cell lymphoma; Low-grade / follicular non-Hodgkin's lymphoma (NHL); Small lymphocytic (SL) NHL; Intermediate-grade / follicular NHL; Intermediate-grade diffuse NHL; High-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-necrotic cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroid leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.

[0310] Therapies provided herein may include the administration of a combination of therapeutic agents, such as a first cancer therapeutic agent and a second cancer therapeutic agent. The therapeutic agents can be administered by any suitable method known in the art. For example, the first cancer treatment and the second cancer treatment can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered in separate compositions. In some embodiments, the first and second cancer treatments are in the same composition. Embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapeutic agents may be administered in one composition or in two or more compositions, such as two, three, or four compositions. Various combinations of agents may be employed. Examples of treatments other than those disclosed herein include surgery, chemotherapy, drug therapy, radiation therapy, hormone therapy, immunotherapy (other than those disclosed herein), or combinations thereof.

[0311] The therapeutic agents of the present disclosure may be administered by the same or different routes of administration. In some embodiments, the cancer therapeutic agent may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0312] Therapeutic agents may include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the discretion of those skilled in the art. A unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose includes a single administrable dose.

[0313] The dosage depends on the desired therapeutic effect, both in the number of treatments and the unit dose.It is understood that an effective amount refers to the amount required to achieve a specific effect.In practice, it is contemplated that in certain embodiments, a dosage in the range of 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, dosages include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such dosages can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0314] In certain embodiments, an effective amount of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount 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 is about, at least, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 12 The blood concentration may be 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, a therapeutic agent administered to a subject is metabolized in the body to become a metabolic therapeutic agent, in which case blood concentration can refer to the amount of that therapeutic agent. Alternatively, to the extent that a therapeutic agent is not metabolized by the subject, blood concentrations discussed herein can refer to the unmetabolized therapeutic agent.

[0315] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (palliation of symptoms versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy the subject may be undergoing.

[0316] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg of body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood concentration), such as 4 μM to 100 μM. It is also understood that uptake is species- and organ-tissue-dependent. Conversion factors and physiological assumptions applicable to uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies, and results described herein.

[0317] V. Kit Certain aspects of the present disclosure also relate to kits comprising compositions of the invention or compositions for carrying out methods of the invention. In certain embodiments, the kits comprise NK cells, fresh or frozen, which may or may not have been pre-activated or expanded. The NK cells may or may not already express one or more components of the TCR / CD3 complex. If the NK cells do not already express one or more components of the TCR / CD3 complex, the kits may include reagents for corresponding transfection or transduction of the NK cells, including vectors expressing the components, primers for amplifying the components, and other reagents. In some cases, the NK cells may or may not also express one or more heterologous proteins as defined herein; if they do not express the components, the kits may include vectors expressing the heterologous proteins, primers for amplifying the heterologous proteins, and the like.

[0318] The kits may be comprised of components individually packaged or placed in containers, such as tubes, bottles, vials, syringes, or other suitable container means. Individual components (such as, but not limited to, NK cells, modified NK cells, means for modifying NK cells, antibodies (e.g., monospecific, bispecific, and / or multispecific), cytokines, etc.) may also be provided in the kit in concentrated amounts; in some embodiments, components are provided individually at the same concentration as they would be in solution with the other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or more. In some embodiments, components are provided in separate solutions or separate containers, while in some embodiments, components are provided in a single container. [Example]

[0319] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and as such can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand 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.

[0320] Example 1 Preparation and effective utilization of CD3-expressing NK cells This example relates to cancer immunotherapeutics as a strategy to redirect the specificity of NK cells toward one or more target antigens by "arming" or pre-conjugating them with bispecific or multispecific antibodies, for example, prior to infusion or by separately co-injecting the two products. NK cells can be transduced with one or more CD3 chains, including CD3ζ, CD3γ, CD3δ, and CD3ε chains, and can be from any source. The cells may or may not be expanded, can be pre-activated with one or more proinflammatory cytokines, such as IL-12 / 15 / 18, and / or can 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 homokine / chemokine receptors.

[0321] 1A and 1B show different embodiments of NK cells engineered to be utilized with bispecific or multispecific antibodies. As shown in FIG. 1A, in first-generation NK cells, the cells are engineered to express CD3, which can be activated with bispecific or multispecific antibodies, including bispecific T cell engagers (BiTEs) comprising anti-CD3 antibodies that bind to heterologous CD3 expressed on the surface of the NK cells. In another embodiment, the CD3-expressing NK cells can be engaged by BiTEs comprising anti-CD3 antibodies, and the NK cells also express one or more specific cytokines (e.g., IL-15 and / or IL-21), resulting in increased efficacy and potency that is particularly useful for treating solid tumors. In another embodiment, NK cells are engineered to express only CD3 so that they can be activated by a BiTE comprising an anti-CD3 antibody, but are also utilized in conjunction with a bispecific or multispecific antibody (e.g., a bispecific NK cell engager, or BiKE) comprising an antibody that binds to a surface antigen naturally present on NK cells, such as CD16, CS1, CD32, CD64, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR. In this way, the NK cells respond to both the NK engager and the T cell engager. In another embodiment, the NK cells express an engineered antigen receptor, such as a CAR or engineered TCR, in addition to expressing CD3 to engage the T cell engager.

[0322] Figure 1B shows a different embodiment in which NK cells have been engineered to express both CD3 and TCR. On the right, a T cell TCR is shown with an α chain and a β chain containing the antigen-binding site, and the TCR signaling occurs in a complex with CD3ζ. The T cell TCR is co-complexed with two CD3ε chains, a CD3δ chain, and a CD3γ chain. In some embodiments, NK cells express a TCR in which one or more of the cytoplasmic domains of any of the CD3 molecules are heterologous intracellular domains, such as those derived from CD16, NKG2D, DAP10, DAP12, NCR, and DNAM-1. As shown on the left side of Figure 1B, NK cells are engineered to express a CD3 co-receptor component; in one example, the CD3 component is CD3ε. In such cases, standard BiTEs (top left, including an antibody against a tumor antigen and an antibody against CD3) typically utilized with T cells that naturally express CD3 can be utilized with CD3-expressing NK cells. In this particular example, the NK cell expresses a polypeptide comprising the extracellular domain of CD3ε (although the extracellular domains of other CD3 components may also be utilized), which is linked to the transmembrane and / or cytoplasmic domain of another molecule, such as, for example, the transmembrane and / or cytoplasmic domain of CD3ζ, CD16, NKG2D, DAP10, DAP12, NCR, or DNAM-1.

[0323] As described above, Figure 1C illustrates the generation of a surface-expressible single chimeric CD3 construct that can be used in combination with an anti-CD3 BiTE. In one example, the CD3 epsilon extracellular domain (ECD) is fused to the CD28, CD16, or NKG2D transmembrane domain (TM), with or without the CD3 zeta and / or DAP10 intracellular domain, and the CD28, CD16, or NKG2D intracellular domain (ICD). In one example, the construct is packaged within a Moloney murine virus-derived SFG retroviral vector backbone, which can be used with a packaging plasmid for virus production. When a CD3-BiTE is used with such a construct in Figure 1C, the antibody will bind to the extracellular domain ε of CD3.

[0324] Embodiments of the present disclosure utilize part or all of the CD3 receptor complex. As shown in Figures 2A and 2B, NK cells may be transfected or transduced with full-length CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon. In such cases, the full-length CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon each comprise the extracellular, transmembrane, and intracellular domains. When different receptor components are expressed from the same vector, they may be configured to be produced as separate polypeptides, for example, by using an IRES or 2A element. In any event, any expression construct may be configured to express one or more cytokines, including at least IL-15.

[0325] Figure 4 shows CD3 expression on NK cells 4 days after CMV TCR complex transduction. This figure is a FACS plot showing CD3 expression on NK cells 4 days after CMV TCR complex transduction. Non-transduced (NT) NK cells (CD56+ CD3-) served as a negative control, and T cells (CD3+ CD56-) served as a positive control.

[0326] Figure 5 shows TCR expression on NK cells 4 days after transduction of NK with the CMV TCR complex. In particular, a FACS plot showing TCRa / b expression on NK cells 4 days after transduction of the CMV TCR complex is provided. Non-transduced (NT) NK cells (CD56+ CD3- TCRa / b-) served as a negative control, and T cells (CD3+ TCRa / b+ CD56-) served as a positive control.

[0327] Figure 6 shows TCR / CD3 expression in NK cells 6 days after CMV TCR complex transduction. Specifically, the FACS plot shows dual expression of CD3 and TCRa / b in NK cells 6 days after CMV TCR complex transduction. Non-transduced (NT) NK cells (CD56+ CD3- TCRa / b-) served as a negative control, and T cells (CD3+ TCRa / b+ CD56-) served as a positive control.

[0328] Figure 7 shows the CD3 / TCR-mediated binding of CD3-CD19 BiTE to NK cells at different concentrations. Specifically, various cells (non-transduced (NT) NK cells, T cells, or three different NK-TCR cells) were incubated with the CD3-CD19 bispecific engager (BiTE), blinatumomab, at two different concentrations (0.5 μg / μl or 4 μg / μl) for 1 hour at 37°C. Subsequently, biotin-labeled CD19 antigen (CD19 CAR Detection Reagent from Miltenyi Biotech) was added. TM ) for 20 minutes, followed by the addition of anti-biotin antibody for 15 minutes at room temperature. This strategy was used to detect BiTe bound to CD3+ cells. The histogram in Figure 7 shows the level of CD19 binding to the CD3-CD19 bispecific engager (BiTe) correlating with CD3 expression on NK-TCR and T cells.

[0329] Figure 8 shows NK-TCR cytokine production after stimulation with plate-bound CD3 antibodies. Specifically, 20 μg / ml of the CD3-OKT3 clone was incubated overnight at 4°C in a flat-bottom 96-well plate to form plate-bound antigens. The following day, T cells or NK cells (NT or TCR-transduced) were added to the wells for 4 hours, followed by the addition of Brefeldin A (which inhibits cytokine release and traps them in the cytoplasm for detection by intracellular cytokine staining). Surface and intracellular staining was then performed to assess cytokine production and degranulation (TNFα and CD107a). The FACS plot in Figure 8 shows a TNFα and CD107a double-positive population in TCR-transduced NK cells. Nontransduced (NT) NK cells (CD56+ CD3-) served as a negative control, and T cells (CD3+ CD56-) served as a positive control.

[0330] Figure 9 shows the phosphorylation of CD3ζ in NK TCR / CD3 cells after crosslinking CD3. The various cells tested included non-transduced (NT) NK cells; non-transduced (NT) T cells; or three different CD3-TCR-transduced NK cells (where CD1, CD2, or CD3 represent different donors). Each NK cell population was transduced with CD3ZFLGDEFL15 (see Figures 2A and 2B). NK cells were cloned into the CD3 OKT3 clone (Miltenyi Biotech). TM The cells were incubated with CD3ζ (130-093-387) at 20 μg / ml for 20 minutes on ice. The cells were then crosslinked with Fab2 IgG1 antibody at various time points and stained to confirm CD3z phosphorylation. This analysis of CD3ζ is useful because it acts as an internalization signal from the surface, allowing crosslinking with CD3 monoclonal antibodies only if the NK cells express it. NK cells lacking CD3 transfection do not exhibit phosphorylation or activation after stimulation.

[0331] CD3-TCR-transduced NK cells also expanded upon stimulation with CD3 OKT3 and, like T cells, displayed basal levels of tonic signaling, whereas untransduced NK cells did not exhibit CD3ζ phosphorylation either basally or upon CD3 OKT3 stimulation.

[0332] Figure 10 shows that pre-incubation of CD3-CD19 BiTEs with TCR / CD3-expressing NK cells increased their killing activity against Raji cells. NK cells were transduced with CD3-TCR#1 (CD3ZFLGDEFL15 (see Figures 2A and 2B)) or CD3-TCR#2 (Z2, also known as CD3ZGDEFL8SP21CD8, which contains full-length CD3ζ, CD3γ, CD3δ, and CD3ε linked to membrane-bound IL21 (with the CD8 transmembrane domain for membrane-bound IL21). NK cells transduced with the CD3 / TCR constructs or untransduced NK cells were loaded with blinatumumab, incubated for 1 hour, and then washed with PBS. They were then co-cultured with CD19+ B-cell lymphoma cells at different effector:target cell ratios (1:1 ratio in Figure 10A and 1:5 ratio in Figure 10B) for various time points. As used herein, effector cells are CD3-TCR NK cells and target cells are Raji cells. CD3-TCR transduced NK cells loaded with blinatumomab showed enhanced antitumor activity at all E:T ratios compared to untransduced NK cells loaded with blinatumomab or CD3 / TCR transduced NK cells not loaded with blinatumomab.

[0333] Example 2 NY-ESO TCR on NK cells This example describes the generation and use of the NY-ESO TCR in NK cells. Figure 11 shows an example of cell production. This schematic illustrates an example in which NK cells are first transduced with a uTNK15 construct incorporating signaling domains from the CD3 complex, NK costimulatory molecules, and IL-15, followed by a second transduction step to introduce TCR molecules, resulting in NK cells co-expressing CD3 and NK signaling molecules, IL-15, and the TCR complex. In one embodiment, NK cells were derived from umbilical cord blood and expanded in complete medium using irradiated (100 Gy) universal antigen-presenting cell (uAPC) feeder cells (feeder cell:NK ratio 2:1) and recombinant human IL-2 (200 U / ml). To generate universal T cell-like NK cells capable of secreting IL-15 (uTNK15 cells), NK cells were purified and transduced 4 days after isolation with a retroviral construct containing the CD3 complex carrying NK costimulatory molecules and IL-15 genes. Forty-eight hours after the initial transduction, NK cells expressing uTNK15 were transduced with a TCR targeting the antigen of choice.

[0334] Figure 12 shows the expression of NY-ESO TCR on uTNK15-transduced NK cells. NK cells were derived from umbilical cord blood and expanded in complete medium using irradiated (100 Gy) universal antigen-presenting cell (uAPC) feeder cells (feeder cell:NK ratio 2:1) and recombinant human IL-2 (200 U / ml). To generate universal T cell-like NK cells capable of secreting IL-15, NK cells were purified and transduced 4 days after isolation with a retroviral construct containing a CD3 complex carrying NK costimulatory molecules and the IL-15 gene. Forty-eight hours after the initial transduction, uTNK15 cells were transduced with a TCR complex targeting the selected antigen. Forty-eight hours later, flow cytometry was used to evaluate the expression of CD3 and NY-ESO TCR on various uTNK15 constructs. Non-transduced (NT) NK cells served as a negative control. CD3 and NY-ESO TCR were highly expressed on all uTNK15 cells compared to NT NK cells. The number of tumor-specific TCR molecules expressed on TCR-engineered NK cells with various TCR constructs is shown in Figure 13, NT NK cells were used as a negative control.

[0335] Figure 14 shows NY-ESO TCR expression in untransduced and transduced T cells. T cells were isolated from umbilical cord blood (from the same donor as the NK cells to serve as a paired positive control) and activated with CD3 / CD28 microbeads at a concentration of 25 μl / million in RPMI complete medium for 48 hours. T cells were then transduced with a retroviral construct containing the NY-ESO TCR. Forty-eight hours after transduction, flow cytometry revealed that the NY-ESO TCR was highly expressed on transduced T cells compared to untransduced T cells.

[0336] NY-ESO TCR-transduced NK cells kill target cells pulsed with NY-ESO peptide in a dose-dependent manner (Figure 15). To measure the killing ability of TCR-engineered NK cells and T cells against LCL cells loaded with different concentrations of NY-ESO peptide for 2 hours, chromosome-modified NK cells and T cells were used on day 7 after TCR transduction. 51We performed a CR killing assay. NY-ESO TCR-transduced uTNK15 cells showed enhanced killing of peptide-pulsed LCL cells compared to untransduced NK cells. NY-ESO TCR-transduced T cells also showed enhanced killing of peptide-pulsed LCL cells compared to untransduced T cells.

[0337] Figure 16 shows that NY-ESO is endogenously expressed in myeloma, sarcoma, and melanoma cell lines. NY-ESO expression was measured in U266 (myeloma), Saos-2 (sarcoma), and A375 (melanoma) cell lines using flow cytometry. U266, Saos-2, and A375 cell lines showed higher levels of NY-ESO expression than the negative control Raji cell line.

[0338] NY-ESO TCR-transduced T cells kill NY-ESO-expressing tumor targets at higher E:T ratios (Figure 17). 51 CR killing assays were performed 7 days after TCR transfer to determine the killing ability of NY-ESO TCR-transduced T cells against NY-ESO-expressing myeloma, osteosarcoma, and melanoma lines. NY-ESO TCR-transduced T cells exhibited enhanced killing ability against NY-ESO-positive cell lines compared with untransduced T cells.

[0339] Figure 18 shows that NK cells transduced with the NY-ESO TCR kill tumor targets expressing NY-ESO even at low E:T ratios. 51 CR killing assays were performed 7 days after TCR transduction to determine the killing ability of NY-ESO TCR-engineered NK cells against NY-ESO-expressing myeloma, osteosarcoma, and melanoma cell lines. NY-ESO TCR-transduced NK cells exhibited enhanced killing of NY-ESO-positive cell lines, even at very low effector:target ratios, compared with untransduced NK cells.

[0340] Figure 19 shows that NY-ESO-transduced NK cells have a similar phenotype to NT NK cells. CytoF imaging revealed that untransduced NK cells and NY-ESO TCR-transduced uTNK15 cells share a similar phenotype. Figure 19A shows a u-map plot with similar clusters, and Figure 19B shows a heatmap with similar expression of various markers in NT cells and NY-ESO TCR-transduced uTNK15 cells.

[0341] Figure 20 provides a table depicting the percentage of CD3+ and CD3+TCR+ NK cells in each uTNK15 product. Flow cytometry was used to assess the composition of single-positive CD3 NK cells (CD3+) and double-positive CD3 / TCR NK cells (CD3+TCR+). Untransduced NK cells consist of less than 1% CD3+ and CD3+TCR+ NK cells, whereas TCR-transduced uTNK15 cell products consist of more than 60% CD3+ and more than 25% CD3+TCR+ NK cells.

[0342] Figure 21A provides a FACS plot showing successful CD3 expression on NK cells 4 days after transduction with TCR constant alpha-beta (TCRCab; TCR6 construct). Non-transduced (NT) NK cells (CD56+ CD3-) serve as a negative control. In Figure 21B, NT NK cells and uTNK15 NK cells were incubated with the CD3-CD19 bispecific engager (BiTe), blinatumumab, at 10 μg / μl for 1 hour at 37°C. Next, biotin-labeled CD19 antigen (CD19 CAR detection reagent from Miltenyi) was added for 20 minutes, followed by the addition of an anti-biotin antibody for 15 minutes at room temperature. This strategy was used to detect any BiTe that engaged with CD3+ cells. The histogram in this figure shows the level of CD19 binding to the CD3-CD19 bispecific engager (BiTe), which correlates with CD3 expression on uTNK15 NK cells. In Figure 21C, CD3 / TCR-transduced or non-transduced NK cells were loaded with blinatumumab, incubated for 1 hour, and washed with PBS. They were then co-cultured with LCL cells at different E:T ratios (A. 1:1, B. 1:5) for various time points. CD3-TCR-transduced NK cells loaded with blinatumumab showed enhanced antitumor activity compared with non-transduced NK cells or CD3 / TCR-transduced NK cells loaded with blinatumumab, but not with blinatumumab, at any E:T ratio.

[0343] Example 3 NY-ESO TCR on CD3-expressing NK cells in vivo As shown in Figures 22A-22C, NK cells containing the constructs described herein were tested in vivo and found to potently inhibit tumor growth. Figure 22A shows a schematic of the experimental procedure performed. Briefly, NSG mice were irradiated with 300 cGy on day -1, and then on day 0, individual mice were injected with 0.5 x 10 NK cells transduced with FireFlyluciferase (FFluc). 6Mice were injected with 5 x 10 U266-B1 cells (a myeloma cell line expressing both HLA-A2 and NY-ESO antigens) via the tail vein, and on day 3, mice received 5 x 10 6 effector cells (NY-ESO TCR NK cells carrying the UT-NK15-NY ESO TCR construct, designated WT, #A, or #B, respectively; WT refers to wild-type CD3 molecules with IL-15; #A refers to CD3-CD28 with IL-15 (e.g., UT-NK15-28); and #B refers to CD3-DAP10 with IL-15 (e.g., UT-NK15-DAP10); or NY-ESO TCR T cells), and animals were then monitored over time and sacrificed as needed (N=5 mice / group). Figure 22B shows the results of monitoring the experiment described in Figure 22A as a function of bioluminescence imaging over time (representative images from days 1, 7, 14, and 21, respectively, are shown). Figure 22C is a graphical quantification of the bioluminescence mean radiance shown in Figure 22B, with the Y-axis representing mean radiance in p / s / cm. 2 / sr, and the X axis represents time.

[0344] As shown in Figures 23A-B, the in vitro activity of effector cells (e.g., NK cells or T cells) containing the NY-ESO-targeting TCR and the UT-NK15 construct was tested. Figure 22A shows images of spheroids formed by the osteosarcoma tumor cell line Saos-2, which were used to test the cytotoxic activity of NK cells and T cells expressing the NY-ESO1-specific TCR. Saos-2 cells were stably transduced to express GFP, and 10,000 of these cells were seeded overnight in a 96-well plate. Then, 40,000 NK cells or T cells were added. Images of the co-culture were scanned over time and analyzed using an IncuCyte® cell analysis system. Figure 22B shows a graph depicting the percentage of effector cell cytotoxicity (Y axis) captured from representative images after 3 days of co-culture. NK cells were co-transduced with the NY-ESO-TCR and the UT-NK15 signaling complex co-expressing a different costimulatory molecule (e.g., UTNK-15-28 or UTNK-15-DAP10) fused to the CD3ζ signaling chain. T cells were transduced with the NY-ESO TCR alone. Abbreviations in the graph are as follows: 28 = CD3ζ fused to the CD28 costimulatory domain; 10 = CD3ζ fused to the Dap10 costimulatory domain; 8 = CD8 alpha / beta co-receptor as part of the NY-ESO TCR construct; and wo IL-15 = construct containing only CD3 zeta, epsilon, gamma, and delta without costimulation or IL-15. The best in vitro cytotoxicity was observed with TCR NK cells expressing UTNK15 with CD28 or the DAP10 costimulatory domain fused to CD3ζ (e.g., UTNK-15-28, or UTNK-15-DAP10; SEQ ID NO: 121 and SEQ ID NO: 119, respectively) when compared with NK cells transduced with the CD3 complex alone or UT-NK15 without the costimulatory domain. Addition of CD8 alpha / beta co-receptors to the TCR did not significantly improve the cytotoxicity of NK or T cells.

[0345] As shown in Figures 24A-D, the in vivo activity of effector cells (e.g., NK cells or T cells) containing NY-ESO-targeting TCRs and UT-NK15 constructs was tested. Figure 24A shows the in vivo study design for testing the activity of NK cells and T cells transduced with different NY-ESO TCRs. Ten-week-old NSG mice were irradiated (300 cGy) and injected the following day with 500,000 U266 cells (an HLA-A2-positive, NY-ESO-expressing myeloma cell line) via the tail vein. Three days later, the mice received 5 million TCR-transduced T cells or TCR-transduced NK cells. The mice were then monitored for tumor control by BLI imaging. Figure 24B shows the BLI imaging results of the study outlined and performed according to Figure 24A. Mice were injected with U266 tumor cells alone, T cells transduced with NY-ESO-specific TCR, or NK cells co-transduced with NY-ESO TCR and UT-NK15, bearing CD3ζ fused to CD28 (labeled as NY-ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15 CD28 NK cells). Figure 24C shows quantification of the...

Claims

1. a) part or all of a single chain of CD3δ, CD3ε, CD3γ, CD3ζ or any combination; b) part or all of a single chain or any combination of the invariant NK T cell receptor (iTCR) α (iTCRα) or β (iTCRβ) chain; and c) a cytokine selected from the group consisting of IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, and combinations thereof; Engineered NK cells modified to express

2. 2. The engineered NK cell of claim 1, wherein the NK cell has been modified to express part or all of one CD3δ, two CD3ε, one CD3γ, and / or one CD3ζ.

3. 2. The engineered NK cell of claim 1, wherein any one or more of CD3δ, CD3ε, CD3γ, and / or CD3ζ are heterologously linked to one or more intracellular signaling domains.

4. 4. The engineered NK cell of claim 3, wherein the intracellular signaling domain is selected from the group comprising CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof.

5. 4. The engineered NK cell of claim 3, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

6. 6. The engineered NK cell of claim 5, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

115.

7. 4. The engineered NK cell of claim 3, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

8. 8. The engineered NK cell of claim 7, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

116.

9. 4. The engineered NK cell of claim 3, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

10. 10. The engineered NK cell of claim 9, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

117.

11. 2. The engineered NK cell of claim 1, wherein the iTCR alpha chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 293, and the iTCR beta chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.

12. 2. The engineered NK cell of claim 1, wherein the iTCR alpha chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 293, and the iTCR beta chain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 301, 307, 311, or 313.

13. 2. The engineered NK cell of claim 1, wherein the cell is modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

14. 2. The engineered NK cells of claim 1, wherein the NK cells are derived from umbilical cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a combination thereof.

15. 2. The engineered NK cell of claim 1, wherein the NK cell is a primary NK cell and is not derived from a stem cell and / or an induced pluripotent stem cell (iPSC).

16. 10. The engineered NK cell of claim 1, wherein the NK cell is conjugated to one or more antibodies.

17. 17. The engineered NK cell of claim 16, wherein the one or more antibodies are one or more bispecific or multispecific antibodies, and at least one of the bispecific or multispecific antibodies comprises a linked anti-CD3 antibody.

18. 17. The engineered NK cell of claim 16, wherein the antibody is blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B.

19. 17. The engineered NK cell of claim 16, wherein the antibody is blinatumomab.

20. 17. The engineered NK cell of claim 16, wherein the NK cell expresses an antibody.

21. 2. The engineered NK cell of claim 1, wherein the cytokine is membrane-bound.

22. 2. The engineered NK cell of claim 1, wherein the cytokine is IL-15.

23. 2. The engineered NK cell of claim 1, wherein the NK cell is further modified to express one or more additional heterologous proteins selected from the group comprising antigen receptors, cytokines, homing receptors, chemokine receptors, and combinations thereof.

24. 10. The engineered NK cell of claim 1, wherein the NK cell has been pre-activated with one or more cytokines.

25. 25. The engineered NK cell of claim 24, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

26. 2. The engineered NK cell of claim 1, wherein the NK cell further comprises one or more engineered mutations in an endogenous gene.

27. 27. The engineered NK cell of claim 26, wherein the endogenous gene is TGFBR2, CISH, and / or CD38.

28. A composition comprising the engineered NK cells of any one of claims 1 to 27.

29. a) engineered NK cells that have been modified to express some or all of the CD3 receptor complex, iTCRα chain, iTCRβ, and one or more cytokines; and b) a bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody that binds to CD3 on NK cells; A composition comprising a complex comprising:

30. 30. The composition of claim 29, wherein the engineered NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

31. 30. The composition of claim 29, wherein the engineered NK cells are modified to express an iTCR alpha chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR beta chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.

32. 30. The composition of claim 29, wherein the engineered NK cells are modified to express an iTCR alpha chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR beta chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 301, 307, 311, or 313.

33. 30. The composition of claim 29, wherein the bispecific or multispecific antibody is blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B.

34. 30. The composition of claim 29, wherein the bispecific or multispecific antibody is blinatumomab.

35. 30. The composition of claim 29, further comprising a pharmaceutically acceptable excipient.

36. 30. The composition of claim 29, wherein the composition is contained in a delivery device.

37. 37. A method of treating a disease in an individual comprising administering to the individual a therapeutically effective amount of any one of the cells or compositions of any one of claims 1 to 27, 29 to 36.

38. 38. The method of claim 37, wherein the disease is an autoimmune disease, an infectious disease, and / or cancer.

39. a) part or all of a single chain of CD3δ, CD3ε, CD3γ, or CD3ζ, or any combination; b) part or all of a single chain or any combination of the invariant NK T cell receptor (iTCR) α (iTCRα) or β (iTCRβ) chain; and c) a cytokine selected from the group comprising IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, and combinations thereof; 20. A method of treating a disease in an individual comprising administering to the individual engineered NK cells that have been modified to express the

40. 40. The method of claim 39, further comprising administering one or more bispecific or multispecific antibodies to the individual simultaneously or at different times.

41. 41. The method of claim 40, wherein the one or more bispecific or multispecific antibodies are blinatumomab, tebentafsp, mosunetuzumab, teclistamab, glofitamab, epcolitamab, flotetuzumab, APV0436, and / or TNB383B.

42. 41. The method of claim 40, wherein the one or more bispecific or multispecific antibodies and the engineered NK cells are administered simultaneously and / or the one or more bispecific or multispecific antibodies and the engineered NK cells are complexed prior to administration to the individual.

43. 40. The method of claim 39, wherein the engineered NK cells are modified to express a polynucleotide sequence at least 85% identical to UT-NK15-DAP10 (SEQ ID NO: 118), UT-NK15-28 (SEQ ID NO: 120), or UTNK15-28-DAP10 (SEQ ID NO: 122).

44. 40. The method of claim 39, wherein the engineered NK cells are modified to express an iTCR alpha chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 293, and an iTCR beta chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, or 315.

45. The method of any one of claims 39 to 44, wherein the disease is cancer.

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