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

Abstract

Embodiments of the present disclosure include methods and compositions in which NK cells are artificially modified to express T cell receptors and CD3 co-receptors on NK cells that do not naturally express expression T cell receptors and CD3 co-receptors. Such modified NK cells effectively work with monospecific, bispecific, or multispecific antibodies, where the bispecific or multispecific antibodies are tailored to comprise anti-CD3 antibodies that bind to the modified NK cells, thereby triggering signaling, activation, and cytotoxicity against the target cells to which the antibodies also bind. Thus, the NK cells are specifically configured to be able to operate efficiently with the bispecific NK cell conjugates (BiKE) as well as the bispecific T cell conjugates (BiTE).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to international application serial number PCT / US2022 / 074062, filed on July 22, 2022, and U.S. Provisional Patent Application Serial Number 63 / 481,588, filed on January 25, 2023, each of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing filed in ST26 format and incorporated herein by reference in its entirety. This ST26 copy was created on July 21, 2023, named MDAC_P1347WO_Sequence_Listing.xml, and has a file size of 422,889 bytes. I. Technical field

[0005] The present disclosure relates to at least the fields of immunology, cell biology, molecular biology, and medicine including at least cancer medicine.

[0006] II. Background

[0007] Natural killer (NK) cells have been studied as potential anti-tumor effector cells, but many obstacles limit their therapeutic applications, mainly related to their lack of antigen specificity. One way to overcome this problem is to transduce NK cells with chimeric antigen receptors (CAR) or engineered T cell receptors (TCR) to target the desired antigen. In T cells, bispecific or multispecific antibodies can be used, such as bispecific T cell engagers (BiTEs) that bind CD3 on the surface of T cells and also bind antigens on the surface of cancer cells. CD3 consists of four different chains, and in mammals, the complex includes CD3γ chains, CD3δ chains, and two CD3ε chains. These chains associate with T cell receptors (TCR) and ζ chains (zeta chains) to produce activation signals in T lymphocytes. However, NK cells do not naturally express CD3 receptor complexes or TCRs.

[0008] The present disclosure satisfies a long-felt need in the art for improved immunotherapies involving the use of NK cells.

[0009] Brief Overview

[0010] Embodiments of the present disclosure include methods and compositions for treating individuals with cancer using adoptive cell therapy. In a specific embodiment, an individual is provided with a therapeutically effective dose of a two-part therapy comprising modified NK cells and antibodies capable of binding to NK cells to initiate signaling, activation, and killing of target cells. The present disclosure relates to NK cells modified to express a variety of proteins that are not naturally expressed in NK cells and that can work in concert, including heterologous proteins on the surface of NK cells that are not naturally present in NK cells.

[0011] In a specific embodiment, NK cells are engineered to express one or more proteins from the CD3 co-receptor complex and an optional TCR receptor complex, each of which is usually present on the surface of T cells. Such engineering provides greater versatility, enabling NK cells to be used with various bispecific or multispecific antibodies, including those comprising anti-CD3 antibodies (e.g., anti-CD3 scFv). In a particular embodiment, modified NK cells are administered to individuals in need together with one or more monospecific, bispecific or multispecific antibodies, wherein each bispecific or multispecific antibody has an antibody targeting CD3 and an antibody binding to a desired antigen (such as a cancer antigen). Therefore, in certain cases, NK cells expressing CD3 are able to bind to the anti-CD3 antibody portion of a bispecific or multispecific antibody, and the antibody binding to a cancer antigen binds to a cancer antigen on the surface of a cancer cell. Such coordinated binding between NK cells and antibodies results in activation of cytotoxicity against target cancer antigens.

[0012] In certain embodiments, the disclosure relates to modified NK cells that express all or part of the CD3 complex with or without a TCR and, in some cases, a single CD3 chain heterologously linked to a NK-associated signaling domain, all of which allow the modified NK cells to be used with a variety of bispecific antibodies.

[0013] Embodiments of the present disclosure include NK cells comprising a single chain or any combination of modified to express CD3δ, CD3ε, CD3γ or CD3ζ. In some cases, NK cells are modified to express T cell receptor (TCR) αβ chain or TCRγδ chain. NK cells may be modified to express CD3ζ, two CD3ε (two of CD3ε), CD3δ and CD3γ, part or all. In some cases, NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ. In certain cases, any one or more of CD3ζ, CD3ε, CD3δ and CD3γ are heterologously connected to one or more intracellular signaling domains. The intracellular signaling domain may be selected from CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28 and combinations thereof. In certain embodiments, the intracellular signaling domain is fused with CD3ζ. In certain embodiments, the intracellular signaling domain is derived from DAP10. In certain embodiments, the intracellular signaling domain is derived from CD28. In certain embodiments, the intracellular signaling domain comprises a sequence derived from DAP10 and a sequence derived from CD28. In a specific embodiment, the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO: 115. In a specific embodiment, the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO: 116. In a specific embodiment, the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO: 117. In a specific embodiment, the NK 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). In certain embodiments, the intracellular signaling domain may also include other co-stimulatory signals associated with NK cell function, such as but not limited to 2B4, DNA, 4-1BB, DAP12, NKG2D, etc. In a specific embodiment, the composition further comprises one or more monospecific, bispecific or multispecific antibodies, wherein the bispecific or multispecific antibodies comprise anti-CD3 antibodies. NK cells can express antibodies and / or complex with antibodies. In certain embodiments, the antibody is imatinib (Imgatuzumab), amivantamab (Amivantamab) and / or cetuximab (Cetuximab). In certain embodiments, TCR targets cancer antigens or viral antigens. In a specific embodiment, NK cells are derived from cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a mixture thereof.In certain embodiments, TCR is directed to KRAS antigen. For example, NK cells can be pre-activated, such as pre-activated with one or more cytokines, including IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof. In certain embodiments, NK cells are amplified, such as amplified in the presence of IL-2. In a specific embodiment, NK cells are modified to express one or more heterologous proteins, such as one or more engineered antigen receptors, one or more cytokines, one or more homing receptors, and / or one or more chemokine receptors. In certain cases, the engineered antigen receptor is a chimeric antigen receptor and / or an engineered T cell receptor. In some cases, the heterologous protein is a cytokine, such as a cytokine selected from IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokine can be a membrane-bound cytokine, and the membrane-bound cytokine can include a transmembrane domain from CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10 or DAP12. In certain cases, NK cells express chimeric antigen receptors and cytokines. In some cases, the bispecific antibody includes an antibody targeting a cancer antigen. In some cases, the bispecific antibody includes ervantumab. In some cases, the antibody includes imatinib and / or cetuximab. In some cases, the antibody targets EGFR, C-met and / or TROP-2.

[0014] Embodiments of the present disclosure include compositions comprising a complex comprising: (1) NK cells modified to express part or all of a CD3 receptor complex and optionally modified to express a T cell receptor (TCR) αβ chain or a TCRγδ chain; and (2) a monospecific, bispecific, or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody that binds to CD3 on the NK cell. In certain embodiments, the complex is stored in a pharmaceutically acceptable excipient. The complex can be stored in a delivery device.

[0015] In certain embodiments, there is a method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of any of the compositions contemplated herein. In certain embodiments, NK cells and antibodies are administered to the individual simultaneously. NK cells and antibodies may or may not be administered in the same formulation. NK cells and antibodies may be pre-compounded before being administered to the individual. In certain embodiments, NK cells and antibodies are administered to the individual at different times. NK cells and antibodies may be administered by infusion. In certain embodiments, NK cells are autologous or allogeneic relative to the individual.

[0016] Embodiments of the present disclosure include redirecting the specificity of NK cells for cancer antigens for use in treating individuals using monospecific, bispecific or multispecific antibodies, wherein the bispecific or multispecific antibodies comprise anti-CD3 antibodies, comprising administering antibodies to individuals and expressing part or all of the CD3 receptor complex and optionally expressing part or all of the TCR α β chain or TCR γ δ chain. Steps of NK cells. In specific embodiments, the method further includes modifying NK cells to express part or all of the CD3 receptor complex. In specific embodiments, the method further includes modifying NK cells to express TCR α β chain or TCR γ δ chain. In some cases, the method further includes modifying NK cells to express one or more heterologous proteins.

[0017] Certain embodiments of the invention are characterized by the following aspects.

[0018] Aspect 1 is a composition comprising engineered NK cells modified to express one or more transgenic polynucleotides encoding: a) a CD3 protein complex comprising a single chain or part or all of any combination of CD3δ, CD3ε, CD3γ or CD3ζ, b) optionally at least one cytokine, and c) at least one engineered T cell receptor (TCR) comprising one or more of a TCRα chain, a TCRβ chain, a TCRγ chain and / or a TCRδ chain, wherein the engineered TCR targets a KRAS antigen.

[0019] Aspect 2 is the composition of aspect 1, wherein the NK cells are modified to express a TCR αβ chain or a TCR γδ chain.

[0020] Aspect 3 is the composition of aspect 1 or 2, wherein the NK cells are modified to express CD3ζ, two copies of CD3ε, CD3δ and part or all of CD3γ.

[0021] Aspect 4 is the composition of any one of aspects 1-3, wherein the NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ.

[0022] Aspect 5 is the composition of any of aspects 1-4, wherein any one or more of CD3ζ, CD3ε, CD3δ and CD3γ are heterologously linked to one or more intracellular signaling domains.

[0023] Aspect 6 is the composition of aspect 5, wherein the intracellular signaling domain is selected from CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28 and combinations thereof.

[0024] Aspect 7 is the composition of aspect 5 or 6, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

[0025] Aspect 8 is the composition of aspect 7, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:115.

[0026] Aspect 9 is the composition of aspect 7, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:115.

[0027] Aspect 10 is the composition of any one of aspects 6-9, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

[0028] Aspect 11 is the composition of aspect 10, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:116.

[0029] Aspect 12 is the composition of aspect 10, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:116.

[0030] Aspect 13 is the composition of any one of aspects 6-13, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

[0031] Aspect 14 is the composition of aspect 13, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:117.

[0032] Aspect 15 is the composition of aspect 13, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:117.

[0033] Aspect 16 is the composition of any of aspects 1-15, wherein the composition further comprises one or more monospecific, bispecific or multispecific antibodies, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody.

[0034] Aspect 17 is the composition of Aspect 16, wherein the NK cells express the antibody.

[0035] Aspect 18 is the composition of aspect 16 or 17, wherein the NK cells are complexed with antibodies.

[0036] Aspect 19 is the composition of any one of aspects 16-18, wherein the antibody is imatinib, ervantumab and / or cetuximab.

[0037] Aspect 20 is the composition of any one of aspects 1-19, wherein the NK cells are derived from cord blood (CB), peripheral blood (PB), bone marrow, stem cells, or a mixture thereof.

[0038] Aspect 21 is the composition of any one of aspects 1-20, wherein the NK cells are pre-activated.

[0039] Aspect 22 is the composition of Aspect 21, wherein the NK cells are pre-activated with one or more cytokines.

[0040] Aspect 23 is the composition of aspect 22, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

[0041] Aspect 24 is the composition of any of aspects 1-23, wherein NK cells are expanded.

[0042] Aspect 25 is the composition of aspect 24, wherein the NK cells are expanded in the presence of IL-2.

[0043] Aspect 26 is the composition of any of aspects 1-25, wherein the NK cells are modified to express one or more additional heterologous proteins.

[0044] Aspect 27 is the composition of aspect 26, wherein the additional heterologous protein is an engineered antigen receptor, cytokine, homing receptor and / or chemokine receptor.

[0045] Aspect 28 is a composition of any of Aspects 1-27, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO: 299 and a sequence at least 85% identical to SEQ ID NO: 301.

[0046] Aspect 29 is a composition of any of aspects 1-28, wherein the TCR comprises one or more sequences that are at least 85% identical to SEQ ID NOs: 299-317.

[0047] Aspect 30 is the composition of any one of aspects 1-29, wherein the target KRAS antigenic epitope comprises or consists of GADGVGKSA (SEQ ID NO: 293) and / or GADGVGKSAL (SEQ ID NO: 292).

[0048] Aspect 31 is a composition of any of aspects 1-30, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO:304 and a sequence at least 85% identical to SEQ ID NO:305.

[0049] Aspect 32 is a composition of any of Aspects 1-31, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO:306 and a sequence at least 85% identical to SEQ ID NO:307.

[0050] Aspect 33 is a composition of any of aspects 1-32, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO:308 and a sequence at least 85% identical to SEQ ID NO:309.

[0051] Aspect 34 is the composition of any one of aspects 26-33, wherein the heterologous protein is a cytokine.

[0052] Aspect 35 is the composition of aspect 34, wherein the cytokine is selected from: IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF or a combination thereof.

[0053] Aspect 36 is the composition of aspect 34 or 35, wherein the cytokine is a membrane-bound cytokine.

[0054] Aspect 37 is the composition of any one of aspects 34-36, wherein the cytokine is IL-15.

[0055] Aspect 38 is the composition of aspect 36 or 37, wherein the membrane-bound cytokine comprises a transmembrane domain from CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10 or DAP12.

[0056] Aspect 39 is the composition of any of aspects 27-38, wherein the NK cells express a chimeric antigen receptor and a cytokine.

[0057] Aspect 40 is a composition of any of aspects 16-39, wherein the monospecific, bispecific or multispecific antibody comprises an antibody targeting a cancer antigen.

[0058] Aspect 41 is the composition of aspect 40, wherein the cancer antigen is an EGFR, C-met and / or TROP-2 antigen.

[0059] Aspect 42 is a composition comprising a complex comprising: a) a NK cell modified to express part or all of the CD3 receptor complex and modified to express a T cell receptor (TCR) αβ chain or a TCR γδ chain; and b) a monospecific, bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody that binds to CD3 on the NK cell, wherein the TCR targets a KRAS antigen.

[0060] Aspect 43 is the composition of aspect 42, wherein the NK cells are modified to express a TCR αβ chain that is at least 85% identical to SEQ ID NO: 299 and SEQ ID NO: 301, and the antibody is imatinib, ervantumab and / or cetuximab.

[0061] Aspect 44 is the composition of aspect 42 or 43, wherein the NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ.

[0062] Aspect 45 is a composition of any of aspects 42-44, wherein any one or more of CD3ζ, CD3ε, CD3δ and / or CD3γ are heterologously linked to one or more intracellular signaling domains.

[0063] Aspect 46 is the composition of aspect 45, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

[0064] Aspect 47 is the composition of aspect 45 or 46, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

[0065] Aspect 48 is the composition of any of aspects 45-47, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:115.

[0066] Aspect 49 is the composition of any one of aspects 45-48, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:115.

[0067] Aspect 50 is the composition of any one of aspects 45-49, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

[0068] Aspect 51 is the composition of any of aspects 45-50, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:116.

[0069] Aspect 52 is the composition of any one of aspects 45-51, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:116.

[0070] Aspect 53 is the composition of any one of aspects 45-52, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

[0071] Aspect 54 is the composition of any of aspects 45-53, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:117.

[0072] Aspect 55 is the composition of any one of aspects 45-54, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:117.

[0073] Aspect 56 is the composition of any one of aspects 1-55, wherein the composition is stored in a pharmaceutically acceptable excipient.

[0074] Aspect 57 is the composition of any one of aspects 1-56, wherein the composition is stored in a delivery device.

[0075] Aspect 58 is a method of treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of any one of the compositions of aspects 1-57.

[0076] Aspect 59 is a method of treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of any of the compositions of Aspects 16-57, wherein the NK cells and the antibody are administered to the individual simultaneously, and optionally wherein the NK cells and the antibody are administered in the same formulation and / or pre-complexed prior to administration to the individual.

[0077] Aspect 60 is the method of aspect 58, wherein the NK cells and the antibody are administered to the individual at different times.

[0078] Aspect 61 is the method of any one of aspects 58-60, wherein the NK cells and the antibody are administered by infusion.

[0079] Aspect 62 is the method of any one of aspects 58-61, wherein the NK cells are autologous to the individual.

[0080] Aspect 63 is the method of any one of aspects 58-61, wherein the NK cells are allogeneic to the individual.

[0081] Aspect 64 is a method for redirecting the specificity of NK cells against cancer antigens for treating an individual using a monospecific, bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody, comprising the steps of administering to the individual the antibody and a NK cell expressing part or all of the CD3 receptor complex and expressing part or all of the TCR αβ chain or TCR γδ chain, wherein the TCR targets a KRAS antigen.

[0082] Aspect 65 is the method of aspect 64, wherein the NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ.

[0083] Aspect 66 is the method of aspect 64 or 65, wherein any one or more of CD3ζ, CD3ε, CD3δ and / or CD3γ are heterologously linked to one or more intracellular signaling domains.

[0084] Aspect 67 is the method of any one of aspects 64-66, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

[0085] Aspect 68 is the method of aspect 66 or 67, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

[0086] Aspect 69 is the method of any one of aspects 66-68, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:115.

[0087] Aspect 70 is the method of any one of aspects 66-69, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:115.

[0088] Aspect 71 is the method of any one of aspects 66-70, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

[0089] Aspect 72 is the method of any one of aspects 66-71, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:116.

[0090] Aspect 73 is the method of any one of aspects 66-72, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:116.

[0091] Aspect 74 is the method of any one of aspects 66-73, wherein the intracellular signaling domain comprises a DAP10 and a CD28 intracellular signaling domain.

[0092] Aspect 75 is the method of any one of aspects 66-74, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:117.

[0093] Aspect 76 is the method of any one of aspects 66-75, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:117.

[0094] Aspect 77 is the method of any one of aspects 64-76, wherein the TCR chain is a TCR αβ chain and is at least 85% identical to SEQ ID NO: 299 and SEQ ID NO: 301.

[0095] Aspect 78 is the method of any one of aspects 64-77, wherein the TCR chain is a TCR αβ chain and is at least 85% identical to one or more of SEQ ID NOs: 299-317.

[0096] Aspect 79 is the method of any one of aspects 64-78, wherein the target KRAS antigenic epitope comprises or consists of GADGVGKSA (SEQ ID NO: 293) and / or GADGVGKSAL (SEQ ID NO: 292).

[0097] Aspect 80 is the method of any one of aspects 64-79, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID NO:304 and a sequence at least 85% identical to SEQ ID NO:305.

[0098] Aspect 81 is the method of any one of aspects 64-79, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID NO:306 and a sequence at least 85% identical to SEQ ID NO:307.

[0099] Aspect 82 is the method of any one of aspects 64-79, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID NO:308 and a sequence at least 85% identical to SEQ ID NO:309.

[0100] Aspect 83 is the method of any one of aspects 64-82, further comprising the step of modifying the NK cells to express one or more additional heterologous proteins.

[0101] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate specific embodiments of the present invention, they are only for illustrative purposes, because various changes and modifications within the spirit and scope of the present invention will be obvious to those skilled in the art.

[0102] BRIEF DESCRIPTION OF THE DRAWINGS

[0103] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0104] Figures 1A-1CVarious embodiments of NK cells engineered to express CD3 are exemplified, including for expression with various heterologous proteins such as cytokines, bispecific NK cell engagers, and engineered antigen receptors (CAR and / or TCR). Figure 1B NK cells adapted for CD3 and TCR for optimized cancer immunotherapy are exemplified. Figure 1C Examples of single chimeric CD3 constructs are illustrated.

[0105] Figure 2A-2B An example of an expression construct for transducing or transfecting a CD3 receptor complex component of a NK cell is illustrated. Figure 2B Examples of plasmid maps of representative expression constructs are shown.

[0106] Figure 3 A table of various TCR / CD3 expression construct designs for NK-TCR engineering is provided.

[0107] Figure 4 Shown is the expression of CD3 on engineered NK cells at day 4 after transduction with one example of a CMV-guided TCR complex.

[0108] Figure 5 Demonstrated is the expression of TCR on engineered NK cells at day 4 after transduction with CMV-primed TCR complexes.

[0109] Figure 6 Shown is the expression of TCR / CD3 on engineered NK cells at day 6 after transduction of the CMV-guided TCR complex into the cells.

[0110] Figure 7 Shown is the binding of one example of a CD3-CD19 BiTE on NK cells at different concentrations via the CD3 / TCR complex on NK cells.

[0111] Figure 8 NK-TCR cytokine production of TNFα and CD107a after stimulation with plate-bound CD3 antibody is shown.

[0112] Fig. 9 Phosphorylation of CD3z in NK TCR / CD3 cells after cross-linking of CD3 is shown.

[0113] Figures 10A-10B It was shown that the cytotoxic activity of CD3-CD19 BiTE against Raji cells was increased after pre-incubation with NK cells expressing TCR / CD3. Fig. 10A Indicates an effector:target ratio of 1:1. Fig. 10B Indicates an effector-target ratio of 1:5.

[0114] Fig.11 A general overview of multiple retroviral transductions to generate NK cells expressing CD3, IL-15, and the TCR complex is provided.

[0115] Fig.12 The expression of NY-ESO TCR on NK cells transduced with uTNK15 is shown. WT refers to wild-type CD3 molecule and IL-15; A refers to CD3-CD28 and IL-15; B refers to CD3-DAP10 and IL-15; C refers to CD3-CD28-Dap10 and IL-15.

[0116] Fig.13 The number of TCR molecules expressed per cell on NK cells is shown. WT refers to wild-type CD3 molecules and IL-15; A refers to CD3-CD28 and IL-15; B refers to CD3-DAP10 and IL-15; C refers to CD3-CD28-Dap10 and IL-15. The number of NY-ESO TCR molecules on NK cells was determined using a phycoerythrin fluorescence quantification kit (BD Biosciences).

[0117] Fig.14 The expression of NY-ESO TCR on T cells is shown.

[0118] Fig.15 It is shown that NK cells transduced with NY-ESO TCR kill target cells loaded with NY-ESO peptide in a dose-dependent manner. WT refers to wild-type CD3 molecule and IL-15; A refers to CD3-CD28 and IL-15; B refers to CD3-DAP10 and IL-15; C refers to CD3-CD28-Dap10 and IL-15.

[0119] Fig.16 The expression of endogenous NY-ESO on human tumor cell lines is shown.

[0120] Fig.17 T cells transduced with NY-ESO TCR were shown to kill NY-ESO-expressing tumor targets.

[0121] Fig.18 Results are provided showing that NK cells transduced with NY-ESO TCR can kill tumor targets expressing NY-ESO even at low E:T ratios. WT refers to wild-type CD3 molecule and IL-15; A refers to CD3-CD28 and IL-15; B refers to CD3-DAP10 and IL-15; C refers to CD3-CD28-Dap10 and IL-15.

[0122] Figures 19A-19BIt is shown that NK cells transduced with NY-ESO have similar phenotypes (19A) and expression patterns (19B) as NT NK cells. WT refers to wild-type CD3 molecules and IL-15; A refers to CD3-CD28 and IL-15; B refers to CD3-DAP10 and IL-15; C refers to CD3-CD28-Dap10 and IL-15.

[0123] Fig. 20 A table showing the cell composition of the amplified uTNK15 products is provided. WT refers to wild-type CD3 molecules and IL-15; A refers to CD3-CD28 and IL-15; B refers to CD3-DAP10 and IL-15; C refers to CD3-CD28-Dap10 and IL-15.

[0124] Figures 21A-21C It was demonstrated that NK cells could be successfully transduced with CD3 and TCR constant α-β (TCRCab) (referred to as TCR6 construct), and that the engineered NK cells were able to bind blinatumumab ( FIG. 21B ) and selectively kill CD19+ lymphoma targets ( FIG. 21C ).

[0125] Figures 22A-22C The in vivo activity of effector cells (eg, NK cells or T cells) comprising a TCR targeting NY-ESO is shown. Fig.22A is a schematic diagram summarizing the experimental procedures performed. Fig. 22B Shown are bioluminescent imaging of mice implanted with U266B.1 cells transduced with FireFlyluciferase (FFluc) and treated with control, NY-ESO TCR NK cells, or NY-ESO TCR T cells (NK cells contained WT, #A, or #B UT-NK15-NY ESO TCR constructs, respectively; WT refers to wild-type CD3 molecule and IL-15; #A refers to CD3-CD28 and IL-15; #B refers to CD3-DAP10 and IL-15) over time (day 1, day 7, day 14, and day 21). Fig. 22C yes Fig. 22B Graphical quantification of the mean radiant intensity of bioluminescence is shown in Figure 3. These results demonstrate that effector cells containing the NY-ESO TCR constructs described herein robustly inhibit tumor growth in vivo.

[0126] Figures 23A-23B The in vitro activity of effector cells (eg, NK cells or T cells) comprising a TCR targeting NY-ESO and a UT-NK15 construct is shown. Fig.23AImages of spheroids formed by the osteosarcoma tumor cell line Saos-2 stably transduced to express GFP were used to test the cytotoxic activity of NK and T cells expressing the NY-ESO1-specific TCR. Fig. 23B Graph showing percent cytotoxicity (Y axis) of representative images after 3 days of co-culture. NK cells were co-transduced with NY-ESO-TCR and UT-NK15 signaling complexes co-expressing different co-stimulatory molecules fused to the CD3ζ signaling chain or TCR complexes without IL-15. T cells were transduced with NY-ESO TCR only. Abbreviations in the figure: 28 = CD3ζ fused to CD28 co-stimulatory domain; 10 = CD3ζ fused to Dap10 co-stimulatory domain; 8 = CD8α / β co-receptor as part of NY ESO TCR construct; wo IL-15 = The construct contains only CD3ζ, ε, γ and δ TCR complexes without co-stimulation or IL-15.

[0127] Figures 24A-24D The in vivo activity of effector cells (eg, NK cells or T cells) comprising a TCR targeting NY-ESO and a UT-NK15 construct is shown. Fig.24A Depicted is a plan for in vivo studies testing the activity of NK and T cells transduced with different NY ESO TCRs. Fig. 24B Describes the basis Fig.24A BLI imaging results of tests performed and summarized. Mice were injected with U266 tumor cells and three days later received T cells transduced with NY-ESO-specific TCRs or NK cells co-transduced with NY-ESO TCRs and UT-NK15 with CD3ζ fused to CD28 (labeled NY-ESO NK UT-NK15 CD28 or NY-ESO TCR UTNK-15CD28 NK cells). Tumor-only groups served as controls. Fig.24C Describes the basis Fig.24A Tested and imaged on Fig. 24B Average radiation intensity in the region of interest of the animal. Fig.24D It is a graph depicting the cohort survival curve of the above animals.

[0128] Fig.25The in vivo activity of effector cells (e.g., NK cells) engineered to express the NY ESO TCR and CD3 complex was shown, with or without the IL-15 transgene in the construct. NSG mice were irradiated (300 cGy) and the next day injected with 500,000 U266 cells (HLA-A2 positive, NY-ESO expressing myeloma cell line) via the tail vein. Three days later, the mice received 5 million T cells or NK cells transduced with the TCR. Tumor control in the mice was monitored by BLI imaging. NK cells were transduced with or without the NY-ESO specific TCR expressing the CD8α / β co-receptor, and were co-transduced with the CD3 complex of UT-NK15 that did not contain the IL-15 transgene or expressed CD3ζ fused to CD28 (UT-NK15 CD28) or CD3ζ fused to the DAP10 (UT-NK15 DAP10) co-stimulatory molecule.

[0129] Figures 26A-26C In vitro expression of a melanoma antigen (PRAME) TCR preferentially expressed on effector cells (eg, NK cells or T cells) and in vitro activity of the cells are shown. Fig.26A Shown are the expression of UT-NK15 (X axis, CD3) and PRAME-specific TCR (Y axis, TCR) (TCR clones 46, 54, or DSK3, respectively) in NK cells or in T cells transduced with the same content (TCR clones 46 or 54). Fig.26B In vitro cytotoxicity of NK cells expressing PRAME-specific TCR against the U266 myeloma cell line was demonstrated. Live cell imaging measures the cytotoxicity of T cells transduced with PRAME-specific TCRs and NK cells transduced with UT-NK15 and PRAME-specific TCRs against U266 myeloma cells. U266 cells expressing GFP were co-cultured with T cells or NK cells expressing PRAME-specific TCRs at a 1:1 effector-target ratio. A decrease in GFP expression indicates cell death. After 26 hours, a second round of 50,000 tumor cells (denoted as "re-attack") was added to each well for tumor re-challenge assays. Open symbols represent T cells, while closed symbols represent NK cells. NT = not transduced. Fig.26C In vitro cytotoxicity of NK cells expressing PRAME-specific TCR against UA375 melanoma cell line was demonstrated. Live cell imaging measures 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. UA375 cells expressing GFP were co-cultured with T cells or NK cells expressing PRAME at a 1:1 effector-target ratio. A decrease in GFP expression indicates cell death. After 26 hours, a second round of 50,000 tumor cells was added to each well for tumor re-challenge assays. Open symbols represent T cells, while closed symbols represent NK cells. NT = not transduced.

[0130] Figures 27A-27B In vitro expression of a transgenic Kirsten rat sarcoma virus (KRAS)-specific TCR on effector cells (eg, NK cells) and the in vitro activity of said cells are shown. Fig.27A Shown is the expression of UT-NK15 (x-axis, CD3) (e.g., SEQ ID NO: 120) and KRAS-specific TCR (y-axis, TCRab) (SEQ ID NOs: 300 and 302) in transduced NK cells (3 umbilical cord blood donors, CB25, CB26, and CB27, respectively) (bottom) compared to non-transduced (NT) NK cell controls (top). Fig.27BIn vitro cytotoxicity of NK cells expressing KRAS-specific TCR and UT-NK15 against the BcPC3 cell line (a pancreatic cancer cell line BxPC-3 transduced with constructs encoding HLA-Cw08:02 and β-2-M (SEQ ID NO:320)) loaded with 1 μg / ml KRAS-G12D-10mer (GADGVGKSAL) peptide (SEQ ID NO:292, labeled "G12D-10-mer"). Cytotoxicity was measured in real time using the Xcelligence cell analysis system, and the cytotoxicity of control untransduced NK cells (cb25-NK-NT; dotted line) or NK cells transduced with KRAS-specific TCR and UT-NK15 (cb25-kras-TCR NK; solid line) against BcPC3 cells with KRAS-G12D-10-mer (HLA-Cw08:02+BxPC-3+kras-G12D-10-mer) was measured and compared. Cells were co-cultured with an effector-target ratio of 5:1. NK cells from donors CB25, CB26, and CB27 were assayed, and the figure shows data from representative NK cell lines expressing KRAS-specific TCRs from CB25, with triplicates for each treatment. Statistical significance was assessed by a two-tailed t-test (*** = P < 0.0001). Tumor Cell Index was used as a measure of cytotoxicity and showed the superiority of NK cells expressing the KRAS-specific TCR UT-NK15 in killing KRAS+HLA-Cw08:02+BxPC-3 cells relative to untransduced control NK cells.

[0131] Detailed Description

[0132] Following long-standing patent law practice, the words "a" and "an" are used in conjunction with the word "comprising" in this specification, including in the claims, to mean "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 may be implemented with any other method or composition described herein, and that different embodiments may be combined.

[0133] Throughout this specification, unless the context requires otherwise, the words "comprise, comprises and comprising" will be understood to include the stated steps or elements or groups of steps or elements, but not to exclude any other steps or elements or groups of steps or elements. By "consisting of" is meant including and limited to what immediately follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. By "consisting essentially of" is meant including any element immediately following the phrase, and limited to other elements that do not interfere with or affect the activity or action of the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but no other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements.

[0134] References throughout this specification to "one embodiment," "an embodiment," "specific embodiments," "related embodiments," "certain embodiments," "additional embodiments," or "further embodiments," or combinations thereof, indicate that a particular feature, structure, or characteristic associated with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any appropriate manner in one or more embodiments.

[0135] As used herein, the terms "or" and "and / or" are used to describe combinations or multiple components that are mutually exclusive. For example, "x, y, and / or z" can mean "only x," "only y," "only z," "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment.

[0136] Throughout this application, the term "about" is used according to its plain and ordinary meaning in the arts of cellular and molecular biology to indicate that a value includes the standard error of error for the device or method employed to determine the value.

[0137] The term "CD3 receptor complex" or "CD3 co-receptor complex" as used herein refers to a protein complex that functions as a T cell co-receptor in nature and is composed of a CD3 ζ chain, a CD3 γ chain, a CD3 δ chain, and two CD3 ε chains (although in alternative embodiments only one CD3 ε chain is used).

[0138] As used herein, the term "engineered" refers to entities generated by the human hand, including cells, nucleic acids, polypeptides, vectors, and the like. In at least some cases, engineered entities are synthetic and contain elements that do not exist in nature or are not configured in the manner used in the present disclosure. In specific embodiments, vectors are engineered by recombinant nucleic acid technology and cells are engineered by transfection or transduction of engineered vectors. Cells can be engineered to express heterologous proteins that are not naturally expressed by the cell because the heterologous proteins are recombinant or synthetic, or because the cell does not naturally express these proteins.

[0139] The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that, where appropriate, do not produce adverse, allergic or other adverse reactions when administered to animals (such as humans). The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is known to those skilled in the art in view of this disclosure. In addition, for animal (such as human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety and purity standards required by the U.S. FDA Office of Biological Standards.

[0140] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (such as water, alcohol / aqueous solutions, physiological saline solutions, vehicles for injectable drugs, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (such as propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters, such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (such as antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption delay agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquids and nutritional supplements and the like, and combinations thereof, which are known to those of ordinary skill in the art. The pH and exact concentration of each component in the pharmaceutical composition are adjusted according to well-known parameters.

[0141] The term "subject" as used herein generally refers to an individual suffering from or suspected of having cancer. The subject can be any biological or animal subject, which is the object of the method or material, including mammals, such as humans, experimental animals (such as primates, rats, mice, rabbits), livestock (such as cattle, sheep, goats, pigs, turkeys and chickens), domestic pets (such as dogs, cats and rodents), horses and transgenic non-human animals. The subject can be a patient, for example, suffering from or suspected of having a disease (which can be called a medical condition), such as a benign or malignant tumor, or cancer. The subject may be receiving or has been treated. The subject may be asymptomatic. The subject may be a healthy individual, but it is hoped that cancer prevention will be provided. The term "individual" can be used interchangeably in some cases. The "subject" or "individual" used in this article may or may not live in a medical facility, and may be treated as an outpatient of a medical facility. The individual may receive one or more medical compositions via the Internet. The individual may include people or non-human animals of any age, thus including adults and minors (i.e., children) and infants, and including individuals in the womb. The term does not imply a requirement for medical treatment; therefore, individuals may voluntarily or involuntarily become part of an experiment, whether clinical or in support of basic scientific research.

[0142] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or pathological condition, and may include a slight reduction in one or more measurable markers of the disease or condition being treated (such as cancer). Treatment may involve optionally reducing or ameliorating one or more symptoms of a disease or condition, or slowing the progression of a disease or condition. "Treatment" does not necessarily mean completely eradicating or curing a disease or condition, or its associated symptoms. Treatment may mean alleviating at least one symptom of a disease or condition.

[0143] As used herein, "TCR / CD3 complex" refers to a protein complex naturally present on the surface of T cells, comprising the T cell receptor (TCR) α and β chains and / or the T cell receptor γ and δ chains, in addition to the CD3ζ, CD3γ, CD3δ and CD3ε chains.

[0144] I. Implementation of the Disclosure

[0145] Natural killer (NK) cells are an emerging cellular immunotherapy for patients with hematological malignancies and solid tumors. The present disclosure relates in particular to NK cells that have been modified to have enhanced function as immunotherapy compared to unmodified NK cells. These modifications give NK cells greater flexibility when used in combination with other therapeutic agents, and at least in certain embodiments, have T cell-like activity by utilizing CD3 / TCR receptor complexes. In specific embodiments, NK cells are modified to express (i) a single CD3 chain (CD3ζ, CD3ε, CD3δ or CD3γ) or part or all of a human CD3 receptor complex (including any combination of CD3δ, ε (one or two copies of ε), γ and ζ); or (ii) a single CD3 chain or a human CD3 receptor complex (including any combination of CD3δ, ε (one or two molecules), γ and ζ) as a full-length protein or a partial protein heterologously connected to one or more intracellular signaling domains; and (iii) the CD3 complex may or may not include a T cell receptor (αβ or γδ). The present disclosure relates to the use of NK cells expressing CD3 in the diagnosis and treatment of diseases, including the use of these cells in combination with monospecific, bispecific or multispecific antibodies, wherein one epitope of the bispecific and / or multispecific antibody binds to CD3 on NK cells expressing CD3. NK cells expressing CD3 can be pre-compounded with bi / multispecific antibodies ex vivo to redirect their specificity for target antigens and / or combined in vivo. In diagnostic embodiments, the labeled NK cells can be loaded with any kind of bispecific or multispecific antibodies, including at least antibodies comprising anti-CD3 antibodies, and the migration of the loaded, labeled NK cells to the target antigen site bound by another antibody on the bispecific or multispecific antibody can be monitored.

[0146] In certain embodiments of the present disclosure, TCR recognizes antigens and / or epitopes presented by a major histocompatibility complex (multi-histocompatibility complex, MHC). In certain embodiments, the antigen and / or epitope is a peptide, a lipid and / or a 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.

[0147] In certain embodiments, the TCR target antigen can be a primary and / or secondary antigen that provides target antigen specificity to the transduced effector cells. In certain embodiments, the TCR primarily acts as a stabilizer of the CD3 co-receptor complex, while the antibody provides the primary target antigen specificity to the transduced effector cells.

[0148] II. Compositions of the Disclosure

[0149] The present disclosure relates to compositions comprising at least a modified NK cell expressing at least a portion of a TCR / CD3 complex. In some cases, the composition further comprises a monospecific, bispecific or multispecific antibody, included in the same formulation, although in alternative embodiments, the NK cell and the antibody are used as physically separate compositions.

[0150] A. NK cell TCR / CD3 modification

[0151] In a specific embodiment, the composition provided herein includes artificially modified to express part or all of the TCR receptor complex and part or all of the CD3 co-receptor complex NK cells. In a specific embodiment, NK cells are modified to include all components of the CD3 complex, including CD3ζ, CD3ε, CD3γ and CD3δ. Although in a particular case, full-length CD3ζ, CD3ε, CD3γ and CD3δ, including their extracellular domains, transmembrane domains and intracellular domains, are used, in alternative embodiments, only one or more of CD3ζ, CD3ε, CD3γ and CD3δ are used, each part 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 may also be modified to express TCR receptor complexes, although any component of the TCR receptor complex is not used in alternative embodiments.

[0152] In certain embodiments, an amino acid sequence (e.g., a polypeptide) may include an amino acid represented by a single letter "X" or a three letter code "Xaa". In certain embodiments, the amino acid represented by "X" or "Xaa" is any naturally occurring amino acid, such as, but not limited to, arginine (Arg, R), histidine (His, H), lysine (Lys, K), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), glycine (Gly, G), proline (Pro, P), cysteine ​​(Cys, C), alanine (Ala, A), valine (Val, V), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), or tryptophan (Trp, W).

[0153] In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is arginine (Arg, R). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is histidine (His, H). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is lysine (Lys, K). In certain 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 certain 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 certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO:25 or SEQ ID NO:88 is serine (Ser, S). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is threonine (Thr, T). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is asparagine (Asn, N). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is glutamine (Gln, Q). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is glycine (Gly, G). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is proline (Pro, P). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is cysteine ​​(Cys, C). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is alanine (Ala, A). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is valine (Val, V). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is isoleucine (Ile, I).In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is leucine (Leu, L). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is methionine (Met, M). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is phenylalanine (Phe, F). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is tyrosine (Tyr, Y). In certain embodiments, the amino acid represented by "X" or "Xaa" in SEQ ID NO: 25 or SEQ ID NO: 88 is tryptophan (Trp, W).

[0154] In certain embodiments, the specific sequence of any component of the CD3 receptor is used, including wild-type or mutants of these components, as long as the CD3 receptor containing the mutant can signal transduction through the CD3 complex, thereby activating and killing the target. In some cases, the following examples of the sequences of CD3ε, CD3δ, CD3γ and CD3ζ are used for the modification of NK cells.

[0155] CD3ε(UniProtKB-P07766(CD3E_HUMAN))

[0156] Signal peptide

[0157] MQSGTHWRVLGLCLLSVGVW(SEQ ID NO:1)

[0158] Extracellular domain

[0159] sp|P07766|23-126

[0160] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD(SEQ ID NO:2)

[0161] Transmembrane domain

[0162] sp|P07766|127-152

[0163] VMSVATIVIVDICITGGLLLLVYYWS(SEQ ID NO:3)

[0164] Intracellular domain

[0165] sp|P07766|153-207

[0166] KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI(SEQ ID NO:4)

[0167] Example of the Homo sapiens CD3e molecule (CD3E), mRNA in the NCBI reference sequence: Accession number NM_000733.4

[0168] (SEQ ID NO:5)

[0169] The following are complete examples of the nucleic acid and amino acid sequences of CD3ε, respectively (the signal peptide sequence is underlined):

[0170] ATGCAGAGCGGCACCCACTGGAGAGTGCTGGGCCTGTGCCTGCTGAGCGTGGGCGTGTGGGGCCAGGACGGCAACGAGGAGATGGGCGGCATCACCCAGACCCCCTACAAGGTGAGCATCAGCGGCACCACCGTGATCCTGACCTGCCCCCAGTACCCCGGCAGCGAGATCCTGTGGCAGCACAACGACAAGAACATCGGCGGCGACGAGGACGACAAGAACATCGGCAGCGACGAGGACCACCTGAGCCTGAAGGAGTTCAGCGAGCTGGAGCAGAGCGGCTACTACGTGTGCTACCCCAGAGGCAGCAAGCCCGAGGACGCCAACTTCTACCTGTACCTGAGAGCCAGAGTGTGCGAGAACTGCATGGAGATGGACGTGATGAGCGTGGCCACCATCGTGATCGTGGACATCTGCATCACCGGCGGCCTGCTGCTGCTGGTGTACTACTGGAGCAAGAACAGAAAGGCCAAGGCCAAGCCCGTGACCAGAGGCGCCGGCGCCGGCGGCAGACAGAGAGGCCAGAACAAGGAGAGACCCCCCCCCGTGCCCAACCCCGACTACGAGCCCATCAGAAAGGGCCAGAGAGACCTGTACAGCGGCCTGAACCAGAGAAGAATC(SEQ ID NO:37)

[0171] MQSGTHWRVLGLCLLSVGVWGQ DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI(SEQ ID NO:38)

[0172] CD3δ(UniProtKB-P04234(CD3D_HUMAN))

[0173] Signal peptide

[0174] MEHSTFLSGLVLATLLSQVS(SEQ ID NO:6)

[0175] Extracellular domain

[0176] sp|P04234|22-105

[0177] FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA(SEQ ID NO:7)

[0178] Transmembrane domain

[0179] sp|P04234|106-126

[0180] GIIVTDVIATLLLALGVFCFA(SEQ ID NO:8)

[0181] Intracellular domain

[0182] sp|P04234|127-171

[0183] GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK(SEQ ID NO:9)

[0184] Homo sapiens CD3d molecule, delta (CD3-TCR complex), mRNA (cDNA clone MGC:88324IMAGE:30412345), complete cds BC070321.1

[0185] ATGGAACATAGCACGTTTCTCTCTGGCCTGGTACTGGCTACCCTTCTCCGCAAGTGAGCCCCTTCAAGATACCTATAGAGGAACTTGAGGACAGAGTGTTTGTGAATTGCAATACCAGCATCACATGGG TAGAGGGAACGTGGGAACACTGCTCTCAGACATTACAAGACTGGACCTGGGAAAACGCATCCTGGACCCACGAGGAATATATAGGTGTAATGGGACAGATATATACAAGGACAAAGAATCTACCGTGCA AGTTCATTATCGAATGTGCCAGAGCTGTGTGGAGCTGGATCCAGCCACCGTGGCTGGCATCATTGTCACTGATGTCATTGCCACTCTGCTCCTTGCTTTGGGAGTCTTCTGCTTTGCTGGACATGAGACT GGAAGGCTGTCTGGGGCTGCCGACACACAAGCTCTGTTGAGGAATGACCAGGTCTATCAGCCCCTCCGAGATCGAGATGATGCTCAGTACAGCCACCTTGGAGGAAACTGGGCTCGGAACAAGTGA(SEQ IDNO:10)

[0186] The following are complete examples of the nucleic acid and amino acid sequences of CD3δ, respectively (the signal peptide sequence is underlined):

[0187] ATGGAGCACAGCACCTTCCTGAGCGGCCTGGTGCTGGCCACCCTGCTGAGCCAGGTGAGCCCCTTCAAGATCCCCATCGAGGAGCTGGAGGACAGAGTGTTCGTGAACTGCAACACCAGCATCACCTGGGTGGAGGGCACCGTGGGCACCCTGCTGAGCGACATCACCAGACTGGACCTGGGCAAGAGAATCCTGGACCCCAGAGGCATCTACAGATGCAACGGCACCGACATCTACAAGGACAAGGAGAGCACCGTGCAGGTGCACTACAGAATGTGCCAGAGCTGCGTGGAGCTGGACCCCGCCACCGTGGCCGGCATCATCGTGACCGACGTGATCGCCACCCTGCTGCTGGCCCTGGGCGTGTTCTGCTTCGCCGGCCACGAGACCGGCAGACTGAGCGGCGCCGCCGACACCCAGGCCCTGCTGAGAAACGACCAGGTGTACCAGCCCCTGAGAGACAGAGACGACGCCCAGTACAGCCACCTGGGCGGCAACTGGGCCAGAAACAAG(SEQ ID NO:35)

[0188] MEHSTFLSGLVLATLLSQVSP FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK(SEQ ID NO:36)

[0189] CD3γ (T cell surface glycoprotein CD3γ chain gene CD3G P09693)

[0190] Signal peptide

[0191] MEQGKGLAVL ILAIILLQGTLA(SEQ ID NO:11)

[0192] Extracellular domain

[0193] sp|P09693|23-116

[0194] QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS(SEQ ID NO:12)

[0195] Transmembrane domain

[0196] sp|P09693|117-137

[0197] GFLFAEIVSIFVLAVGVYFIA(SEQ ID NO:13)

[0198] Intracellular domain

[0199] sp|P09693|138-182

[0200] GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN(SEQ ID NO:14)

[0201] Homo sapiens CD3g molecule (CD3G), mRNA; NM_000073.3:81-629 Homo sapiens CD3g molecule (CD3G), mRNA

[0202] ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGAGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAATTGA(SEQ ID NO:15)

[0203] The following are complete examples of the nucleic acid and amino acid sequences of CD3γ respectively (the signal peptide sequences are underlined):

[0204] ATGGAACAGGGGAAGGGCCTGGCTGTCCTCATCCTGGCTATCATTCTTCTTCAAGGTACTTTGGCCCAGTCAATCAAAGGAAACCACTTGGTTAAGGTGTATGACTATCAAGAAGATGGTTCGGTACTTCTGACTTGTGATGCAGAAGCCAAAAATATCACATGGTTTAAAGATGGGAAGATGATCGGCTTCCTAACTGAAGATAAAAAAAAATGGAATCTGGGAAGTAATGCCAAGGACCCTCGTGGGATGTATCAGTGTAAAGGATCACAGAACAAGTCAAAACCACTCCAAGTGTATTACAGAATGTGTCAGAACTGCATTGAACTAAATGCAGCCACCATATCTGGCTTTCTCTTTGCTGAAATCGTCAGCATTTTCGTCCTTGCTGTTGGGGTCTACTTCATTGCTGGACAGGATGGAGTTCGCCAGTCGAGAGCTTCAGACAAGCAGACTCTGTTGCCCAATGACCAGCTCTACCAGCCCCTCAAGGATCGAGAAGATGACCAGTACAGCCACCTTCAAGGAAACCAGTTGAGGAGGAAT(SEQ ID NO:33)

[0205] MEQGKGLAVLILAIILLQGTLA QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN(SEQ ID NO:34)

[0206] CD3ζ

[0207] Signal peptide

[0208] sp|P20963|SP

[0209] MKWKALFTAAILQAQLPITEA(SEQ ID NO:16)

[0210] Extracellular domain

[0211] sp|P20963|22 - 30ECD

[0212] QSFGLLDPK(SEQ ID NO:17)

[0213] Transmembrane domain

[0214] sp|P20963|31-51tmd

[0215] LCYLLDGILFIYGVILTALFL(SEQ ID NO:18)

[0216] Intracellular domain

[0217] sp|P20963|52-164ICD

[0218] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:19)

[0219] The following are complete examples of the respective nucleic acid and amino acid sequences of CD3ζ (the signal peptide sequence is underlined):

[0220] ATGAAGTGGAAGGCCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCA CAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGAC CCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO:31)

[0221] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:32)

[0222] Homo sapiens CD247 molecule (CD247; also known as CD3ζ), transcript variant 1, mRNA NCBI Reference Sequence: NM_198053.3

[0223] NM_198053.3:65-559 Homo sapiens CD247 molecule (CD247), transcript variant 1, mRNA

[0224] ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO:20)

[0225] In certain embodiments, NK cells are modified to express one or more of TCR α chains, TCR β chains, TCR γ chains and TCR δ chains, and any combination thereof can be used. In certain cases, NK cells are modified to express T cell receptor (TCR) α β chains or TCR γ δ chains. In some cases, NK cells are modified to express only part or all of the constant region of one or more of TCR α chains, TCR β chains, TCR γ chains and TCR δ chains. NK cells can be modified to express only part or all of the constant region of T cell receptor (TCR) α β chains or TCR γ δ chains. In the case of using a partial constant region, 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 continuous amino acids in any 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 contiguous amino acids of the constant region.

[0226] In certain cases, any sequence contained herein is used to modify NK cells, although in other cases sequences related to these sequences in identity are used. For example, related sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to any sequence contained herein can be used in the present disclosure.

[0227] Specific constructs can be used to express various TCR / CD3 proteins in NK cells, and in various configurations. In a specific case, NK cells can be transduced or transfected with one or more vectors to express any various proteins contained herein, including at least any one or more components of TCR / CD3 complexes. In a specific case, one or more vectors themselves may or may not be polycistronic, by ultimately being able to produce more than an independent polypeptide. In the case of using one or more polycistronic vectors, they can use one or more internal ribosome entry sites (IRES) and / or one or more 2A self-cleavage peptide sites. In the case of using one or more 2A sequences, the following sequence can be used, wherein GSG is an optional linker:

[0228] T2A(GSG)EGRGSLLTCGDVEENPGP(SEQ ID NO:21)

[0229] P2A(GSG)ATNFSLLKQAGDVEENPGP(SEQ ID NO:22)

[0230] E2A(GSG)QCTNYALLKLAGDVESNPGP(SEQ ID NO:23)

[0231] F2A(GSG)VKQTLNFDLLKLAGDVESNPGP(SEQ ID NO:24)

[0232] In the case of expressing multiple protein components from a polycistronic vector, the order in the 5' to 3' direction on the polynucleotide vector can be any order, although in other cases, they are present in a specific order on the vector. The polycistronic vector can express multiple components of the CD3 receptor complex without other heterologous proteins, or the polycistronic vector can express multiple components of the CD3 receptor complex and one or more other heterologous proteins. The polycistronic vector can express multiple components of the TCR receptor complex without other heterologous proteins, or the polycistronic vector can express multiple components of the TCR receptor complex and one or more other heterologous proteins. The polycistronic vector may or may not express one or more components of the TCR receptor complex and one or more components of the CD3 complex. In a specific embodiment, the polycistronic vector includes one or more components of the CD3 receptor complex and one or more heterologous proteins, such as cytokines and engineered antigen receptors, such as CAR.

[0233] exist Figure 2A An example of a polycistronic vector is shown in , where the full length of CD3ε, CD3δ, CD3γ and CD3ζ are present and separated by the same or different 2A self-cleaving peptide sites. Figure 2B The plasmid map further indicates that the polycistronic vector can include a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain of each of CD3ε, CD3δ, CD3γ, and CD3ζ.

[0234] Figure 3 A table is provided showing examples of various TCR expression constructs for engineering TCR expression NK cells. In a specific embodiment of the present disclosure, CD3 receptor components and TCR receptor components are expressed by different vectors in NK cells. In any case, the vector can express TCRs for specific antigens such as cancer antigens or viral antigens. TCR may or may not include at least part of CD3ζ, including the intracellular domain of CD3ζ, and NK cells also express CD3ζ as a molecule separated from TCR and as a part of the CD3 receptor complex. Similarly, CAR may or may not include at least part of CD3ζ, including the intracellular domain of CD3ζ, and NK cells also express CD3ζ as a molecule separated from TCR and as a part of the CD3 receptor complex.

[0235] In a specific embodiment, the TCR of the modified NK cell is not necessarily used as a therapeutic aspect of the cell, but as a structural support or scaffold to promote the function of the CD3 receptor complex or enhance its function. That is, the TCR can be any TCR, and its ability to target a specific desired antigen may not be used. In this case and as an example, a TCR targeting a viral antigen can be used for NK cells that will be used for cancers that are not necessarily associated with the specific virus. In other cases, the TCR is selected based on its ability to target specific cancer antigens. Examples of antigens that TCR can target are provided elsewhere in this article.

[0236] exist Figure 3 , note the following examples of constructs:

[0237]

[0238] In TCRpp65ZicdGDEFL, the corresponding component sequences are as follows, but these specific sequences or other sequences may be used in this and / or other constructs:

[0239] TCRb - Extracellular domain:

[0240] MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD(SEQ ID NO:40)ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQID NO:41)

[0241] CD3ζ intracellular domain (Z-ICD):

[0242] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENP

[0243] GP (SEQ ID NO:42) (where the P2A sequence is located at the C-terminus)

[0244] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO:43) (where the lowercase sequence is the P2A sequence)

[0245] TCRa - extracellular domain:

[0246] MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO:44)

[0247] ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO:45)

[0248] CD3γδε(CD3GDE):

[0249] MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP (SEQ ID NO:46) (where the E2A sequence is at the C-terminus)

[0250]

[0251] IL-15:

[0252] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:48)

[0253] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

[0254] TCR2: refers to TCRpp65 linked to the full-length CD3ζ chain, full-length CD3γ chain, full-length CD3δ chain, and full-length CD3ε chain; it lacks IL-15. The representative sequence is as follows:

[0255]

[0256]

[0257] TCR3: refers to TCRpp65 connected to the intracellular CD3z domain and IL-15. It can also be called TCRpp65Zicd15. Its representative sequence is as follows:

[0258] MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:52)

[0259] In TCRpp65Zicd15, the corresponding component sequences are as follows, but these specific sequences or other sequences can be used herein and / or in other constructs:

[0260] TCRb - extracellular domain:

[0261] MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO:40)

[0262] ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO:41)

[0263] CD3ζ intracellular domain (Z-ICD):

[0264] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENP

[0265] GP (SEQ ID NO:42) (where the P2A sequence is located at the C-terminus)

[0266] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCgccaccaacttctccctgctgaagcaggccggcgacgtggaggagaaccccggcccc (SEQ ID NO:43) (where the lowercase sequence is the P2A sequence)

[0267] TCRa - extracellular domain:

[0268] MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO:44)

[0269] ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO:45)

[0270] CD3ζ intracellular domain (Z-ICD) (in certain embodiments, two or more Z-ICD sequences can be used):

[0271] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP(SEQ IDNO:53)

[0272] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:54)

[0273] IL-15:

[0274] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:48)

[0275] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

[0276] TCR4: refers to TCRpp65, which can also be called TCRpp65βα, and the representative sequence is as follows:

[0277] MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:55)

[0278] For TCR pp65 βα, the corresponding component sequences are as follows, but these specific sequences or other sequences can be used herein and / or in other constructs:

[0279] TCRb - extracellular domain:

[0280] MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO:40)

[0281] ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO:41)

[0282] CD3ζ intracellular domain (Z-ICD):

[0283] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGP(SEQ ID NO:42)

[0284] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:54)

[0285] TCRa - extracellular domain:

[0286] MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS(SEQ ID NO:44)

[0287] ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGC(SEQ ID NO:45)

[0288] CD3ζ intracellular domain (Z-ICD):

[0289] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRPGPQCTNYALLKLAGDVESNPGP(SEQ IDNO:53)

[0290] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:54)

[0291] IL-15:

[0292] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:48)

[0293] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

[0294] Additional representative sequences of TCRpp65βα are as follows:

[0295]

[0296] Z1: refers to full-length CD3ζ, full-length CD3γ, full-length CD3δ, and full-length CD3ε linked to IL15 (see Figure 2A and 2B ), it may also be called CD3ZFLGDEFL15, and a representative sequence may be as follows:

[0297] (SEQ ID NO:58)

[0298]

[0299] Z2: refers to full-length CD3ζ, full-length CD3γ, full-length CD3δ and full-length CD3ε linked to membrane-bound IL21 (membrane-bound IL21 with CD8 transmembrane domain), which can also be called CD3ZGDEFLSP821CD28, and the representative sequence is as follows:

[0300]

[0301] For CD3ZGDEFLSP821CD28, the corresponding component sequences are as follows, but these specific sequences or other sequences may be used herein and / or in other constructs:

[0302] CD3:

[0303] MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:61)

[0304]

[0305] SP CD8:

[0306] MRICLTSDRLAPAAGLAAPRRQAV(SEQ ID NO:63)

[0307] atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg(SEQ ID NO:64)

[0308] IL-21:

[0309] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ IDNO:65)

[0310] CATAAATCTTCCTCTCAAGGTCAGGACCGCCATATGATTCGAATGCGGCAGCTGATTGACATAGTCGATCAACTGAAGAACTATGTGAATGATCTTGTGCCCGAGTTTTTGCCAGCCCCTGAAGACGTAGAAACTAATTGTGAGTGGAGTGCCTTTTCCTGCTTTCAAAAGGCACAGCTGAAATCCGCCAACACGGGCAATAACGAACGGATAATTAACGTATCCATTAAGAAGCTGAAGCGGAAGCCGCCCTCAACCAATGCGGGACGGCGGCAAAAGCATCGCTTGACCTGTCCGTCATGCGACAGCTACGAGAAAAAGCCCCCGAAGGAGTTCTTGGAACGCTTCAAGAGTCTCCTTCAGAAAATGATTCACCAGCACCTGTCCTCACGGACGCACGGAAGCGAGGACAGT(SEQ ID NO:66)

[0311] CD8 hinge:

[0312] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:67)

[0313] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 68)

[0314] CD28 transmembrane domain:

[0315] FWVLVVVGGVLACYSLLVTVAFIIFWV*(SEQ ID NO:69)

[0316] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO:70)

[0317] Z3: refers to full-length CD3ζ, full-length CD3γ, full-length CD3δ and full-length CD3ε linked to membrane-bound IL21 (membrane-bound IL21 with CD28 transmembrane domain), which can also be called CD3ZGDEFL8SP21CD8, and its representative sequence is as follows:

[0318]

[0319] For CD3ZGDEFL8SP21CD8, the corresponding component sequences are as follows, but these specific sequences or other sequences can be used herein and / or in other constructs:

[0320] CD3:

[0321] MLEMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:61)

[0322]

[0323] SP CD8:

[0324] MRICLTSDRLAPAAGLAAPRRQAV (SEQ ID NO:63)

[0325] atgcgcatttgcctgaccagcgatcgcctggcgccggcggcgggcctggcggcgccgcgccgccaggcggtg (SEQ ID NO:64)

[0326] IL-21:

[0327] HKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ IDNO:65)

[0328] cataaatcttcctctcaaggtcaggaccgccatatgattcgaatgcggcagctgattgacatagtcgatcaactgaagaactatgtgaatgatcttgtgcccgagtttttgccagcccctgaagacgtagaaactaattgtgagtggagtgccttttcctgctttcaaaaggcacagctgaaatccgccaacacgggcaataacgaacggataattaacgtatccattaagaagctgaagcggaagccgccctcaaccaatgcgggacggcggcaaaagcatcgcttgacctgtccgtcatgcgacagctacgagaaaaagcccccgaaggagttcttggaacgcttcaagagtctccttcagaaaatgattcaccagcacctgtcctcacggacgcacggaagcgaggacagt (SEQ ID NO:66)

[0329] CD8 hinge:

[0330] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:67)

[0331] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ IDNO:68)

[0332] CD8 transmembrane domain:

[0333] IYIWAPLAGTCGVLLLSLVIT*(SEQ ID NO:72)

[0334] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC(SEQID NO:73)

[0335] In certain embodiments, the present invention provides a CD3 construct comprising a fusion of an intracellular co-stimulatory domain derived from CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM or any combination thereof. In certain embodiments, the intracellular co-stimulatory domain is fused with CD3δ, CD3ε, CD3γ and / or CD3ζ. In certain embodiments, this CD3 fusion construct comprises CD3ζ fused with a DAP10 intracellular co-stimulatory domain. In certain embodiments, this CD3 fusion construct comprises CD3ζ fused with a CD28 intracellular co-stimulatory domain. In certain embodiments, this CD3 fusion construct comprises CD3ζ fused with a DAP10 intracellular co-stimulatory domain and a CD28 intracellular co-stimulatory domain. In certain embodiments, the CD3 zeta fused to 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: 106. In certain embodiments, the CD3 zeta fused to 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: 107. In certain embodiments, the CD3ζ fused to the DAP10 intracellular co-stimulatory domain and the CD28 intracellular co-stimulatory domain is represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:108. In certain embodiments, the CD3ζ fused to the DAP10 intracellular co-stimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:109.In certain embodiments, the CD3ζ fused to the CD28 intracellular co-stimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:110. In certain embodiments, the CD3ζ fused to the DAP10 intracellular co-stimulatory domain and the CD28 intracellular co-stimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 111. In certain embodiments, the CD3ζ fused to the intracellular domain may not include the C-terminal 2A domain. In certain embodiments, the CD3ζ fused to the intracellular domain may not include the N-terminal signal peptide domain.

[0336] ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGCTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:106)

[0337] ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCAAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQ ID NO:107)

[0338] ATGAAGTGGAAGGCGCTTTTCACCGCGGCCATCCTGCAGGCACAGTTGCCGATTACAGAGGCACAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC(SEQID NO:108)

[0339] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP(SEQ ID NO:109)

[0340] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP(SEQ ID NO:110)

[0341] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGP(SEQ ID NO:111)

[0342] 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 co-stimulatory domain and the CD28 intracellular co-stimulatory 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 co-stimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 115. In certain embodiments, the CD28 intracellular co-stimulatory domain is represented by an amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 116. In certain embodiments, the DAP10 intracellular co-stimulatory domain and the CD28 intracellular co-stimulatory domain are represented by 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: 117.

[0343] CTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 112)

[0344] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCCGACTTCGCAGCCTATCGCTCA (SEQ ID NO: 113)

[0345] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCACTTTGCGCACGCCCACGCCGCAGCCCCGCCCAAGAAGATGGCAAAGTCTACATCAACATGCCAGGCAGGGGC (SEQ ID NO: 114)

[0346] LCARPRRSPAQEDGKVYINMPGRG(SEQ ID NO:115)

[0347] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:116)

[0348] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSLCARPRRSPAQEDGKVYINMPGRG(SEQID NO:117)

[0349] UTNK15-DAP10: refers to full-length CD3ζ fused to an intracellular co-stimulatory domain derived from DAP10, full-length CD3γ, full-length CD3δ and full-length CD3ε connected to IL15, which can be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:118. In certain embodiments, the UTNK15-DAP10 amino acid sequence can 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.

[0350]

[0351] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLLCARPRRSPAQEDGKVYINMPGRGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ IDNO:119)

[0352] UTNK15-28: refers to full-length CD3ζ fused to an intracellular co-stimulatory domain derived from CD28, full-length CD3γ, full-length CD3δ and full-length CD3ε connected to IL15, which can be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:120. In certain embodiments, the UTNK15-28 amino acid sequence can 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.

[0353]

[0354]

[0355] UTNK15-28-DAP10: refers to a full-length CD3ζ fused with an intracellular co-stimulatory domain derived from DAP10 and an intracellular co-stimulatory domain derived from CD28, full-length CD3γ, full-length CD3δ and full-length CD3ε linked to IL15, which can be represented by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:122. In certain embodiments, the UTNK15-28-DAP10 amino acid sequence can 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.

[0356]

[0357]

[0358] like Figure 3 As shown and described above, the term "linked" refers to being present on the same polynucleotide vector and does not necessarily mean that the two polypeptides are expressed as one polypeptide. For example, the cytokines produced from the vectors of the present disclosure can ultimately be produced as molecules separate from any one or more TCR / CD3 receptor complex components. The term "fused" or "fusion protein" refers to two polypeptides containing a peptide bond connecting the two molecules, i.e., the two polypeptides are covalently bound by an amide bond and are not separated by a splitting element (e.g., a 2A element).

[0359] A specific example of a TCR that can be used in a cell is the NY-ESO TCR, and specific examples of sequences include at least the following sequence:

[0360] TCRα:

[0361] XQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDS KSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:25)

[0362] TCRβ:

[0363] GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGF YPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ IDNO:26)

[0364] 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.

[0365] aaacaggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaactgcagtttcact gatagcgctatttacaac ctccagtggtttaggcaggaccctgggaaaggtctcacatctctgttgctt attcagtcaagtcagagagag caaacaagtggaagacttaatgcctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgactcagccacctacctc tgtgctgtgaggcccctttatggaggaag ctacatacctacattt ggaagaggaaccagccttatattgttcatccgtat(SEQ ID NO:85)

[0366] 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.

[0367] atccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgattttgattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagcaacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagctttgaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc(SEQ ID NO:86)

[0368] In certain embodiments, the TCR may comprise a TCR α-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.

[0369] atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagcaaacaggaggtgacacagattcctgcagctctgagtgtcccagaaggagaaaacttggttctcaactgcagtttcact gatagcgct atttacaac ctccagtggtttaggcaggaccctgggaaaggtctcacatctctgttgctt attcagtcaagtcaga gagag caaacaagtggaagacttaatgcctcgctggataaatcatcaggacgtagtactttatacattgcagcttctcagcctggtgactcagccacctacctc tgtgctgtgaggcccctttatggaggaagctacatacctacatttggaagaggaaccagccttattgttcatccgtatatccagaaccctgaccctgccgtgtaccagctgagagactctaaatccagtgacaagtctgtctgcctattcaccgatttt gattctcaaacaaatgtgtcacaaagtaaggattctgatgtgtatatcacagacaaaactgtgctagacatgaggtctatggacttcaagagcaacagtgctgtggcctggagc aacaaatctgactttgcatgtgcaaacgccttcaacaacagcattattccagaagacaccttcttccccagcccagaaagttcctgtgatgtcaagctggtcgagaaaagcttt gaaacagatacgaacctaaactttcaaaacctgtcagtgattgggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatgacgctgcggctgtggtccagc(SEQ IDNO:87)

[0370] 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.

[0371] XQEVTQIPAALSVPEGENLVLNCSFT DSAIYN LQWFRQDPGKGLTSLLL IQSSQRE QTSGRLNASLDKSSGRSTLYIAASQPGDSATYL CAVRPLYGGSYIPTF GRGTSLIVHPY (SEQ ID NO:88)

[0372] 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.

[0373] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQID NO:89)

[0374] 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.

[0375] DSAIYN (SEQ ID NO:90)

[0376] 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.

[0377] IQSSQRE (SEQ ID NO:91)

[0378] 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.

[0379] CAVRPLYGGSYIPTF (SEQ ID NO:92)

[0380] In certain embodiments, the TCR may comprise a TCR β chain variable region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:93.

[0381] ggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactgcagtgtgcccaggat atgaaccatgaatac atgtcctggtatcgacaagacccaggcatggggctgaggctgattcattac tcagtt ggtgctggtatcactgaccaaggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctcccagacatctgtgtacttc tgtgccagcagttacgtcgggaacaccgggga gctgtttttt ggagaaggctctaggctgaccgtactggag(SEQ ID NO:93)

[0382] In certain embodiments, the TCR may comprise a TCR β-chain constant region encoded by a nucleotide sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:94.

[0383] Gacctgaaaaacgtgttcccacccaaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtatgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggc(SEQ ID NO:94)

[0384] 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.

[0385] Atgagcatcggcctcctgtgctgtgcagccttgtctctcctgtgggcaggtccagtgaatgctggtgtcactcagaccccaaaattccaggtcctgaagacaggacagagcatgacactgcagtgtgcccaggat atgaaccat gaatac atgtcctggtatcgacaagacccaggcatggggctgaggctgattcattac tcagttggtgctggtatc actgaccaaggagaagtccccaatggctacaatgtctccagatcaaccacagaggatttcccgctcaggctgctgtcggctgctccctcccagacatctgtgtacttc tgtgccagcagttacgtcgggaacaccggggagctgttttttggagaaggctctaggctgaccgtactggaggacctgaaaaacgtgttcccacccAaggtcgctgtgtttgagccatcagaagcagagatctcccacacccaaaaggccacactggtatgcctggccacaggcttctaccccgaccacgtggagctgagctggtgggtgaatgggaaggaggtgcacagtggggtcagcacagacccgcagcccctcaaggagcagcccgccctcaatgactccagatactgcctgagcagccgcctgagggtctcggccaccttctggcagaacccccgcaaccacttccgctgtcaagtccagttctacgggctctcggagaatgacgagtggacccaggatagggccaaacccgtcacccagatcgtcagcgccgaggcctggggtagagcagactgtggcttcacctccgagtcttaccagcaaggggtcctgtctgccaccatcctctatgagatcttgctagggaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatggccatggtcaagagaaaggattccagaggc(SEQ ID NO:95)

[0386] In certain embodiments, the TCR may comprise a TCR β-chain variable region amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:96.

[0387] GVTQTPKFQVLKTGQSMTLQCAQD MNHEY MSWYRQDPGMGLRLIHY SVGAGI TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF CASSYVGNTGELFF GEGSRLTVLE(SEQ ID NO:96)

[0388] In certain embodiments, the TCR may comprise a TCR β chain constant region amino acid sequence that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:97.

[0389] DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:97)

[0390] In certain embodiments, the TCR may comprise a beta chain CDR1 amino acid sequence that is at least or exactly 80% or 100% identical to SEQ ID NO:98.

[0391] MNHEY (SEQ ID NO:98)

[0392] In certain embodiments, the TCR may comprise a beta chain CDR2 amino acid sequence that is at least or exactly 80% or 100% identical to SEQ ID NO:99.

[0393] SVGAGI (SEQ ID NO:99)

[0394] In certain embodiments, the TCR may comprise a beta chain CDR3 amino acid sequence that is at least or exactly 80% or 100% identical to SEQ ID NO:100.

[0395] CASSYVGNTGELFF (SEQ ID NO: 100)

[0396] In certain embodiments, the TCR chain (e.g., TCR alpha chain, beta chain, delta chain and / or gamma chain) may include a signal peptide. In certain embodiments, the signal peptide is encoded by a nucleic acid that is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 101 or SEQ ID NO: 102. In certain embodiments, the signal peptide is at least or exactly 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:103 or SEQ ID NO:104.

[0397] atggagaccctcttgggcctgcttatcctttggctgcagctgcaatgggtgagcagc(SEQ ID NO:101)

[0398] atgagcatcggcctcctgtgctgtgcagccttgtctctcctgtgggcaggtccagtgaatgct(SEQID NO:102)

[0399] METLLGLLILWLQLQWVSS(SEQ ID NO:103)

[0400] MSIGLCCAALSLLWAGPVNA(SEQ ID NO:104)

[0401] In certain embodiments, the TCR recognizes a peptide corresponding to amino acid residues 157-165 of the human cancer testis antigen NY-ESO-1 in the context of HLA-A*02 class I alleles. In certain embodiments, the TCR may target an epitope characterized by an amino acid sequence according to SEQ ID NO:105.

[0402] SLLMWITQC (SEQ ID NO: 105)

[0403] A specific example of a TCR that can be used in a cell is TCRpp65α, and specific examples of sequences include at least the following sequence (the underline indicates a signal peptide sequence):

[0404] ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGACAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCCAGTGATGTAGGCATCTACTTCTGTGCTGGACCCATGAAAACCTCCTACGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ IDNO:27)

[0405] MDSWTFCCVSLCILVAKHTDAGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGPMKTSYDKVIFGPGTSLSVIPNIQNPDPAVYQLRDSKS SDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:28)

[0406] A specific example of a TCR that can be used in a cell is TCRpp65β, and specific examples of sequences include at least the following sequence (the underline indicates a signal peptide sequence):

[0407] ATGGACTCCTGGACCTTCTGCTGTGTGTCCCTTTGCATCCTGGTAGCAAAGCACACAGATGCTGGAGTTATCCAGTCACCCCGGCACGAGGTGACAGAGATGGGACAAGAAGTGACTCTGAGATGTAAACCAATTTCAGGACACGACTACCTTTTCTGGTACAGACAGACCATGATGCGGGGACTGGAGTTGCTCATTTACTTTAACAACAACGTTCCGATAGATGATTCAGGGATGCCCGAGGATCGATTCTCAGCTAAGATGCCTAATGCATCATTCTCCACTCTGAAGATCCAGCCCTCAGAACCCAGGGACTCAGCTGTGTACTTCTGTGCCAGCAGTTCGGCAAACTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO:29)

[0408] MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSSANYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGF FPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:30)

[0409] TCRpp65ZFLGDEFL15

[0410] In certain embodiments, a construct in which TCRpp65 is linked to full-length CD3ζ, full-length CD3γ, full-length CD3δ, full-length CD3ε, and is also linked to IL-15 (and may be referred to as TCRpp65ZFLGDEFL15) may be utilized. A representative sequence of such a construct is as follows:

[0411]

[0412] In TCRpp65ZFLGDEFL15, the corresponding component sequences are as follows, but these specific sequences or other sequences may be used in this and / or other constructs:

[0413] TCRb - Extracellular domain:

[0414] MLEGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSPVTGGIYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEA EISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADATNFSLLKQAGDVEENPGP(SEQ ID NO:75) (and includes the P2A sequence at its C-terminus)

[0415] ATGCTCGAGGGAGTGACCCAGACCCCCAAGTTCCAGGTGCTGAAGACCGGACAGAGCATGACCCTGCAGTGCGCCCAGGACATGAACCACGAGTACATGAGCTGGTACCGGCAGGACCCCGGAATGGGACTGCGGCTGATCCACTACAGCGTGGGAGCCGGAATCACCGACCAGGGAGAGGTGCCCAACGGATACAACGTGAGCCGGAGCACCACCGAGGACTTCCCCCTGCGGCTGCTGAGCGCCGCCCCCAGCCAGACCAGCGTGTACTTCTGCGCCAGCAGCCCCGTGACCGGAGGAATCTACGGATACACCTTCGGAAGCGGAACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGATTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGAGTGAGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGACTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGACGGGCCGAC(SEQ ID NO:76)

[0416] TCRa - extracellular domain:

[0417] MILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCARNTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDAYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSEGRGSLLTCGDVEENPGP(SEQ ID NO:77) (and includes a T2A sequence at its C-terminus)

[0418] ATGATCCTGAACGTGGAGCAGAGCCCCCAGAGCCTGCACGTGCAGGAGGGAGACAGCACCAACTTCACCTGCAGCTTCCCCAGCAGCAACTTCTACGCCCTGCACTGGTACCGGTGGGAGACCGCCAAGAGCCCCGAGGCCCTGTTCGTGATGACCCTGAACGGAGACGAGAAGAAGAAGGGACGGATCAGCGCCACCCTGAACACCAAGGAGGGATACAGCTACCTGTACATCAAGGGAAGCCAGCCCGAGGACAGCGCCACCTACCTGTGCGCCCGGAACACCGGAAACCAGTTCTACTTCGGAACCGGAACCAGCCTGACCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGCCTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC(SEQID NO:78)

[0419] TCR5: called TCRCgdZFLGDEFL15, is the constant region of TCRγ and δ, connected to full-length CD3ζ, full-length CD3γ, full-length CD3δ and full-length CD3ε; and IL-15. The representative sequence is as follows:

[0420] TCR constant gamma-delta (TCRCgd)

[0421]

[0422] CD3:

[0423]

[0424] IL-15:

[0425] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:48)

[0426] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGC CGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAG CTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGA GCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

[0427] TCR6: also known as TCRCabZFLGDEFL15, is the constant region of TCRα and β, connected to full-length CD3ζ, full-length CD3γ, full-length CD3δ and full-length CD3ε; and IL-15. The representative sequence is as follows:

[0428] TCR constant alpha-beta (TCRCab)

[0429] METLLGLLILWLQLQWVSSIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSGATNFSLLKQAGDVEENPGPMSIGLLCCAALSLLWAGPVNADLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:83)

[0430]

[0431] CD3:

[0432] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQCTNYALLKLAGDVESNPGPMEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRNVKQTLNFDLLKLAGDVESNPGPMEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKEGRGSLLTCGDVEENPGPMQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRIGPQCTNYALLKLAGDVESNPGP(SEQ ID NO:79)

[0433]

[0434] IL-15:

[0435] MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS*(SEQ ID NO:48)

[0436] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAGGTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

[0437] In some embodiments, the TCR construct comprises a NY-ESO specific TCR and a CD8α / β co-receptor molecule. In some embodiments, such a construct may comprise a TCRα chain variable region signal peptide, a TCRα chain variable region, a TCRα chain constant region, a 2A element (e.g., a P2A element), a TCRβ chain variable region signal peptide, a TCRβ chain variable region, a TCRβ chain constant region, a 2A element (e.g., an E2A element), a CD8-β polypeptide, a 2A element (e.g., a T2A element), and a CD8-α polypeptide. In some embodiments, the TCR construct comprising the nucleotide coding sequence of a NY-ESO-specific TCR and a CD8α / β co-receptor molecule 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, the TCR construct comprising the amino acid sequence of a NY-ESO-specific TCR and a CD8α / β co-receptor molecule 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.

[0438] In some embodiments, the CD8α co-receptor molecule is transcriptionally linked to any TCR molecule disclosed herein. In some embodiments, the nucleotide coding sequence of the CD8α co-receptor molecule 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 nucleotide coding sequence of the CD8β co-receptor molecule 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 amino acid sequence of the CD8α co-receptor molecule 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 amino acid sequence of the CD8β co-receptor molecule 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.

[0439]

[0440]

[0441] ATGAGGCCACGACTTTGGCTGCTGCTCGCTGCACAGTTGACTGTACTGCATGGCAATAGTGTGTTGCAGCAGACACCTGCATACATCAAGGTTCAGACAAATAAGATGGTTATGCTGAGTTGCGAGGCAAAAATTAGTTTGAGCAATATGCGGATCTACTGGTTGCGACAGAGACAGGCTCCCAGTAGTGATAGTCACCACGAATTCCTGGCTCTTTGGGATTCCGCAAAAGGAACGATTCATGGGGAAGAAGTAGAGCAGGAGAAGATTGCGGTTTTCCGCGATGCATCTCGCTTTATCCTTAATCTTACATCCGTTAAGCCTGAGGACAGTGGGATCTATTTTTGTATGATTGTAGGGTCCCCCGAATTGACATTTGGGAAGGGTACGCAGCTCTCCGTAGTTGACTTTCTGCCCACAACGGCACAACCCACTAAGAAGTCCACCCTGAAGAAGCGCGTCTGTCGCTTGCCCAGACCTGAAACCCAAAAGGGTCCACTCTGTTCCCCTATAACCCTGGGGTTGTTGGTGGCGGGCGTCTTGGTCCTGCTTGTTAGCTTGGGCGTAGCCATTCATCTGTGTTGCCGAAGACGCAGAGCCCGACTTAGATTTATGAAGCAATTCTATAAGTGA(SEQ ID NO:126)

[0442] ATGGCCTTGCCCGTCACTGCGCTTTTGCTCCCGCTCGCTCTTCTCCTGCATGCAGCCCGACCATCTCAATTTAGAGTTTCTCCACTCGACAGGACGTGGAACCTCGGCGAAACCGTCGAACTTAAATGTCAAGTACTTCTCTCAAATCCGACTTCTGGTTGCTCATGGCTCTTTCAGCCGAGAGGAGCAGCTGCCAGCCCCACCTTCCTGCTGTATCTCTCCCAGAACAAGCCGAAGGCCGCCGAAGGGCTCGATACTCAACGATTTAGCGGGAAGCGACTCGGGGACACGTTCGTTCTTACTCTCAGCGATTTTAGAAGAGAGAACGAGGGATATTATTTTTGTTCCGCACTCTCTAACAGCATCATGTACTTCAGTCATTTTGTACCAGTCTTTCTCCCTGCAAAACCAACGACTACTCCAGCACCAAGACCGCCCACTCCCGCACCTACTATTGCAAGCCAACCTTTGAGTCTCCGACCAGAGGCATGCAGACCTGCTGCTGGAGGTGCAGTACATACGCGAGGGTTGGATTTTGCCTGCGATATCTATATCTGGGCCCCCTTGGCCGGCACGTGCGGGGTGCTCCTGCTGAGTCTCGTAATTACTCTTTATTGTAATCATAGAAACCGCAGAAGGGTGTGTAAGTGTCCCCGGCCTGTCGTGAAAAGCGGGGATAAGCCCAGTTTGTCTGCTCGGTACGTC(SEQ ID NO:127)

[0443] MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQFYK(SEQ ID NO:128)

[0444] MALPPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALS NSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV(SEQ ID NO:129)

[0445] In certain embodiments, TCR constructs include PRAME specific TCR chains. In certain embodiments, TCR constructs including PRAME specific TCR chains include TCR α and TCR β chains found in PRAME specific TCR clone 46, clone 54 and / or clone DSK3. In certain embodiments, TCR constructs including PRAME specific TCR chains include TCR α and TCR β chains targeting PRAME epitope SLLQHLIGL (SEQ ID NO: 131) and / or QLLALLPSL (SEQ ID NO: 132).

[0446] In certain 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 SEQ ID NO: 133 (e.g., TCR clone 46 TCRα) and / or SEQ ID NO: 134 (e.g., TCR clone 46 TCRβ). In certain 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 SEQ ID NO: 135 (e.g., TCR clone 46 TCRα) and / or SEQ ID NO: 136 (e.g., TCR clone 46 TCRβ).

[0447] ATGCTTCTGGAACACCTGCTGATTATCCTGTGGATGCAACTCACGTGGGTCTCCGGGCAACAACTGAATCAAAGCCCCCAATCCATGTTTATACAGGAGGGAGAGGACGTAAGTATGAATTGCACATCTTCATCTATCTTTAACACCTGGCTGTGGTACAAACAAGACCCCGGAGAAGGTCCTGTACTTCTCATCGCACTTTACAAAGCAGGTGAGCTTACCAGTAACGGGAGACTCACCGCACAGTTCGGTATTACAAGAAAGGATTCCTTTCTCAACATCTCCGCTTCTATCCCTTCAGACGTCGGAATTTATTTTTGTGCTGGTATCCCTCGAGACAATTACGGTCAAAACTTTGTATTTGGGCCTGGGACTCGGCTGTCAGTTTTGCCGTATATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:133)

[0448] ATGGGCATTAGGCTGCTGTGCAGAGTAGCATTTTGCTTTCTGGCAGTAGGATTGGTCGATGTAAAGGTTACACAGTCCTCACGGTACTTGGTAAAGCGCACTGGTGAAAAGGTCTTTCTGGAATGTGTACAAGATATGGATCACGAAAATATGTTTTGGTACAGGCAAGATCCCGGCCTTGGACTTAGACTGATATATTTCTCCTACGATGTTAAAATGAAGGAGAAGGGCGATATTCCAGAAGGATATTCCGTGAGCCGCGAAAAGAAGGAGCGATTCAGTTTGATACTCGAAAGTGCCTCCACAAACCAAACCTCTATGTACCTTTGCGCGTCAACGCCGTGGCTGGCCGGTGGCAATGAACAATTCTTCGGGCCGGGTACGCGCCTCACTGTCCTGGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:134)

[0449] MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGIPRDNYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:135)

[0450] MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASTPWLAGGNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:136)

[0451] In certain 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 SEQ ID NO: 137 (e.g., TCR clone 54 TCRα) and / or SEQ ID NO: 138 (e.g., TCR clone 54 TCRβ). In certain 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 SEQ ID NO: 139 (e.g., TCR clone 54 TCRα) and / or SEQ ID NO: 140 (e.g., TCR clone 54 TCRβ).

[0452] ATGCTGCTGCTGCTGGTGCCCGTGCTGGAAGTGATCTTCACCCTGGGCGGCACCAGAGCCCAGAGCGTGACACAGCTGGGCAGCCACGTGTCCGTGTCTGAGAGGGCCCTGGTGCTGCTGAGATGCAACTACTCTTCTAGCGTGCCCCCCTACCTGTTTTGGTACGTGCAGTACCCCAACCAGGGGCTGCAGCTGCTCCTGAAGTACACCAGCGCCGCCACACTGGTGAAGGGCATCAACGGCTTCGAGGCCGAGTTCAAGAAGTCCGAGACAAGCTTCCACCTGACCAAGCCCAGCGCCCACATGTCTGACGCCGCCGAGTACTTCTGTGCCGTGAGCGGCCAGACCGGCGCCAACAACCTGTTCTTCGGCACCGGCACCCGGCTGACAGTGATCCCTTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ IDNO:137)

[0453] ATGGGCTTCCGGCTGCTGTGCTGCGTGGCCTTTTGTCTGCTGGGAGCCGGACCTGTGGATAGCGGCGTGACCCAGACCCCCAAGCACCTGATCACCGCCACCGGCCAGAGAGTGACCCTGCGCTGCAGCCCTAGAAGCGGCGACCTGAGCGTGTACTGGTATCAGCAGAGCCTCGACCAGGGCCTGCAGTTCCTGATCCAGTACTACAACGGCGAGGAACGGGCCAAGGGCAACATCCTGGAACGGTTCAGCGCCCAGCAGTTCCCCGATCTGCACAGCGAGCTGAACCTGAGCAGCCTGGAACTGGGCGACAGCGCCCTGTACTTCTGCGCCAGCGCCAGATGGGATAGAGGCGGCGAGCAGTACTTCGGCCCTGGCACCAGACTGACCGTGACCGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:138)

[0454] MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSERALVLLRCNYSSSVPPYLFWYVQYPNQGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHMSDAAEYFCAVSGQTGANNLFFGTGTRLTVIPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:139)

[0455] MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASARWDRGGEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:140)

[0456] 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 SEQ ID NO: 141 (e.g., TCR clone DSK3 TCRα) and / or SEQ ID NO: 142 (e.g., TCR clone DSK3 TCRβ). 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 SEQ ID NO: 143 (e.g., TCR clone DSK3 TCRα) and / or SEQ ID NO: 144 (e.g., TCR clone DSK3 TCRβ).

[0457] ATGAAGAGCCTGAGGGTACTGCTGGTGATATTGTGGCTTCAGCTTAGTTGGGTCTGGTCACAACAAAAGGAAGTTGAGCAAAACTCAGGACCACTGAGTGTACCCGAGGGCGCTATAGCATCACTGAACTGTACCTACTCAGATCGGGGAAGCCAATCCTTTTTCTGGTACAGACAGTATTCCGGGAAGAGTCCTGAGTTGATCATGTTTATATACTCCAATGGCGATAAGGAGGATGGACGCTTCACCGCTCAGCTTAATAAAGCGTCACAGTATGTATCCCTCCTGATTCGGGACTCACAACCATCTGACTCTGCAACATACCTTTGTGCCGTAAAGGACAACGCCGGGAACATGCTCACTTTTGGAGGAGGTACCCGGCTTATGGTAAAACCACATATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ IDNO:141)

[0458] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVKDNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:142)

[0459] ATGGGATTCCGGCTTCTTTGTTGTGTGGCATTTTGTCTGTTGGGTGCGGGTCCAGTCGATAGTGGTGTAACTCAGACACCAAAACACCTTATCACGGCAACTGGGCAACGAGTGACGCTCCGCTGTAGCCCGAGGTCCGGTGATTTGAGTGTGTACTGGTACCAGCAATCTTTGGACCAGGGCTTGCAGTTCCTCATACAGTATTACAATGGTGAAGAAAGAGCGAAGGGTAATATCCTGGAAAGATTCTCCGCACAACAGTTTCCTGATCTCCACAGCGAACTGAACCTGAGTTCTCTCGAGCTCGGGGATAGTGCTTTGTACTTCTGCGCGTCATCCGACGGTGGCGGAGTCTATGAACAATATTTCGGCCCAGGGACTAGGCTTACGGTGACGGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTTGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAATGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:143)

[0460] MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSDGGGVYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQ KATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:144)

[0461] In certain embodiments, the TCR construct comprising the 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 NO: 145-152. In certain embodiments, the TCR construct comprising the PRAME specific TCR chain comprises TCR α and TCR β chains and / or modified forms thereof found in PRAME specific TCR clones T116-49 and / or T402-93. In certain embodiments, the TCR construct comprising the PRAME specific TCR chain comprises TCR α and TCR β chains targeting the PRAME epitope LYVDSLFFL (SEQ ID NO: 167). In certain embodiments, 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 / 063966A1, which is incorporated herein by reference for purposes described herein. In certain embodiments, the TCR construct comprising the PRAME specific TCR chain comprises an amino acid sequence that is at least or just 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.

[0462] ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTGTGGCAGCTGACCTGGGTCCGATCTCAGCAGCCTGTTCAGTCTCCTCAGGCCGTGATCCTGAGAGAAGGCGAGGACGCCGTGATCAACTGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCGAGGCCCCTGTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGCTTCAACGAGAAGAAGCAGCAGTCCAGCCTGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGCACCTACTTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGGCACCCTGCTGACCGTGAATCCCAAT(SEQ ID NO:145)

[0463] ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGAGCCAAGTGAACGGCCAGCAAGTGATGCAGATCCCTCAGTACCAGCACGTGCAAGAAGGCGAGGACTTCACCACCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTACAAGCAGCGGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGGTCAAGTCCGGCGAAGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAACAGCAGCCTGCACATCACCGCCACACAGACCACCGATGTGGGCACCTACTTTTGTGCTGGCGCCCTGCCTAGAGCCGGCAGCTATCAACTGACATTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAAC(SEQ ID NO:146)

[0464] ATGGAGACACTGCTGAAGGTGCTGTCTGGCACACTGCTGTGGCAGCTGACCTGGGTCCGATCTCAGCAGCCTGTTCAGTCTCCTCAGGCCGTGATCCTGAGAGAAGGCGAGGACGCCGTGATCAACTGCAGCAGCTCTAAGGCCCTGTACAGCGTGCACTGGTACAGACAGAAGCACGGCGAGGCCCCTGTGTTCCTGATGATCCTGCTGAAAGGCGGCGAGCAGAAGGGCCACGAGAAGATCAGCGCCAGCTTCAACGAGAAGAAGCAGCAGTCCAGCCTGTACCTGACAGCCAGCCAGCTGAGCTACAGCGGCACCTACTTTTGCGGCACAGCCAATAGCGGCGGCAGCAACTACAAGCTGACCTTCGGCAAGGGCACCCTGCTGACCGTGAATCCCAATATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:147)

[0465] ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGAGCCAAGTGAACGGCCAGCAAGTGATGCAGATCCCTCAGTACCAGCACGTGCAAGAAGGCGAGGACTTCACCACCTACTGCAACAGCAGCACCACACTGAGCAACATCCAGTGGTACAAGCAGCGGCCTGGCGGACACCCTGTGTTTCTGATCCAGCTGGTCAAGTCCGGCGAAGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAACAGCAGCCTGCACATCACCGCCACACAGACCACCGATGTGGGCACCTACTTTTGTGCTGGCGCCCTGCCTAGAGCCGGCAGCTATCAACTGACATTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAACATCCAGAATCCGGAGCCCGCCGTATACCAGCTGAAGGACCCTAGAAGCCAGGACAGCACCCTGTGCCTGTTCACCGACTTCGACAGCCAGATCAACGTGCCCAAGACCATGGAAAGCGGCACCTTCATCACCGACAAGACAGTGCTGGACATGAAGGCCATGGACAGCAAGTCCAACGGCGCAATCGCCTGGTCCAACCAGACCAGCTTCACATGCCAGGACATCTTCAAAGAGACAAACGCCACATACCCCAGCAGCGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCCTTCGAGACAGACATGAACCTGAACTTCCAGAATCTGTCCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:148)

[0466] ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGGACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGTGTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCAGCCAGACCAGCGTGTACTTCTGCGCCATCAGCGACTACGAGGGCACCGAGGCCTTTTTTGGCCAAGGCACAAGACTGACCGTGGTG(SEQ ID NO:149)

[0467] ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAATGTACCGTGGAAGGCACCAGCAATCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGACTGCAGCTGCTGTTTTACAGCGTCGGCATCGGCCAGATCAGCAGCGAGGTTCCACAGAATCTGAGCGCCAGCAGACCCCAGGACAGACAGTTTATCCTGAGCAGCAAGAAGCTGCTGCTGAGCGACAGCGGCTTCTACCTGTGTGCTTGGAGCCTCGGAGCCGGCTACACCGACACACAGTATTTTGGCCCTGGCACCAGACTGACCGTGCTG(SEQ ID NO:150)

[0468] ATGGGCACCAGACTGTTCTTCTACGTGGCCCTGTGTCTGCTGTGGACAGGCCATGTGGATGCCGGAATCACACAGAGCCCCAGACACAAAGTGACCGAGACAGGCACCCCTGTGACACTGAGATGTCACCAGACCGAGAACCATCGGTACATGTATTGGTACAGACAGGACCCCGGCCACGGCCTGAGACTGATCCACTATAGCTACGGCGTGAAGGACACCGACAAGGGCGAAGTGTCTGACGGCTACAGCGTGTCCAGAAGCAAGACCGAGGACTTCCTGCTGACCCTGGAAAGCGCCACAAGCAGCCAGACCAGCGTGTACTTCTGCGCCATCAGCGACTACGAGGGCACCGAGGCCTTTTTTGGCCAAGGCACAAGACTGACCGTGGTGGAAGATCTCCGGAACGTGACCCCCCCTAAAGTGACCCTGTTCGAACCCAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGAGTGTCCACCGACCCTCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAAGGCTCCCCCAAGCCCGTGACCCAGAATATCTCTGCCGAGGCCTGGGGCAGAGCCGACTGTGGAATTACCAGCGCCAGCTACCACCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAAGAAGAACAGC(SEQ IDNO:151)ATGCTGTGTTCTCTGCTGGCTCTGCTGCTGGGCACCTTTTTTGGCGTCAGAAGCCAGACCATCCACCAGTGGCCTGCTACACTGGTGCAGCCTGTTGGAAGCCCTCTGAGCCTGGAATGTACCGTGGAAGGCACCAGCAATCCCAACCTGTACTGGTACAGACAGGCCGCTGGAAGAGGACTGCAGCTGCTGTTTTACAGCGTCGGCATCGGCCAGATCAGCAGCGAGGTTCCACAGAATCTGAGCGCCAGCAGACCCCAGGACAGACAGTTTATCCTGAGCAGCAAGAAGCTGCTGCTGAGCGACAGCGGCTTCTACCTGTGTGCTTGGAGCCTCGGAGCCGGCTACACCGACACACAGTATTTTGGCCCTGGCACCAGACTGACCGTGCTGGAAGATCTCCGGAACGTGACCCCCCCTAAAGTGACCCTGTTCGAACCCAGCAAGGCCGAGATCGCCAACAAGCAGAAAGCCACCCTCGTGTGCCTGGCCAGAGGCTTCTTCCCCGACCATGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGAGTGTCCACCGACCCTCAGGCCTACAAAGAGAGCAACTACAGCTACTGCCTGAGCAGCAGACTGCGGGTGTCCGCCACCTTCTGGCACAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTTCACGGCCTGAGCGAAGAGGACAAGTGGCCCGAAGGCTCCCCCAAGCCCGTGACCCAGAATATCTCTGCCGAGGCCTGGGGCAGAGCCGACTGTGGAATTACCAGCGCCAGCTACCACCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGGCCTGGTGCTGATGGCCATGGTCAAGAAGAAGAACAGC(SEQ ID NO:152)

[0469] METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKI SASFNEKKQQSSLYLTASQLSYSGTYFCGTANSGGSNYKLTFGKGTLLTVNPN(SEQ IDNO:153)

[0470] MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGALPRAGSYQLTFGKGTKLSVIPN(SEQ ID NO:154)

[0471] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ IDNO:155)

[0472] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFPSPESCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQID NO:156)

[0473] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQID NO:157)

[0474] METLLKVLSGTLLWQLTWVRSQQPVQSPQAVILREGEDAVINCSSSKALYSVHWYRQKHGEAPVFLMILLKGGEQKGHEKISASFNEKKQQSSLYLTASQLSYSGTYFCGTANSGGSNYKLTFGKGTLLTVNPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:158)

[0475] MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGALPRAGSYQLTFGKGTKLSVIPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:159)

[0476] MGTRLFFYVALCLLWTGHVDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISDYEGTEAFFGQGTRLTVV(SEQ ID NO:160)

[0477] MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLGAGYTDTQYFGPGTRLTVL(SEQ ID NO:161)

[0478] EDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:162)

[0479] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:163)

[0480] EDLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:164)

[0481] MGTRLFFYVALCLLWTGHVDAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISDYEGTEAFFGQGTRLTVVEDLRNVTPPKVTLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:165)

[0482] MLCSLLALLLGTFFGVRSQTIHQWPATLVQPVGSPLSLECTVEGTSNPNLYWYRQAAGRGLQLLFYSVGIGQISSEVPQNLSASRPQDRQFILSSKKLLLSDSGFYLCAWSLGAGYTDTQYFGPGTRLTVLEDLRNVTPPKVTLFEPSKAEIAN KQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:166)

[0483] In certain embodiments, the TCR construct comprises a gp100-specific TCR chain. In certain embodiments, the TCR construct comprising a gp100-specific TCR chain comprises TCR α and TCR β chains and / or modified forms thereof found in gp100-specific TCR clone Sp (0.01) A. In certain embodiments, the TCR construct comprising a gp100-specific TCR chain comprises TCR α and TCR β chains targeting gp100 epitope KTWGQYWQV (SEQ ID NO: 168). In certain embodiments, gp100-specific TCR sequences, TCR variable domain sequences, CDR sequences and / or TCR constant domain sequences are described in patent publication number US 8,216,565 B2, which is incorporated herein by reference for purposes described herein.

[0484] In certain 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 certain 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.

[0485] ATGAAATCCTTGAGTGTTTCCCTAGTGGTCCTGTGGCTCCAGTTAAACTGGGTGAACAGCCAGCAGAAGGTGCAGCAGAGCCCAGAATCCCTCATTGTCCCAGAGGGAGCCATGACCTCTCTCAACTGCACTTTCAGCGACAGTGCTTCTCAGTATTTTGCATGGTACAGACAGCATTCTGGGAAAGCCCCCAAGGCACTGATGTCCATCTTCTCCAATGGTGAAAAAGAAGAAGGCAGATTCACAATTCACCTCAATAAAGCCAGTCTGCATTTCTCGCTACACATCAGAGACTCCCAGCCCAGTGACTCTGCTCTCTACCTCTGTGCAGCCAATAACTATGCCCAGGGATTAACCTTCGGTCTTGGCACCAGAGTATCTGTGTTTCCCTACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO:169)

[0486] ATGGGCTCCAGACTCTTCTTTGTGGTTTTGATTCTCCTGTGTGCAAAACACATGGAGGCTGCAGTCACCCAAAGTCCAAGAAGCAAGGTGGCAGTAACAGGAGGAAAGGTGACATTGAGCTGTCACCAGACTAATAACCATGACTATATGTACTGGTATCGGCAGGACACGGGGCATGGGCTGAGGCTGATCCATTACTCATATGTCGCTGACAGCACGGAGAAAGGAGATATCCCTGATGGGTACAAGGCCTCCAGACCAAGCCAAGAGAATTTCTCTCTCATTCTGGAGTTGGCTTCCCTTTCTCAGACAGCTGTATATTTCTGTGCCAGCAGCCCTGGGGGGGGGGGGGAACAGTACTTCGGTCCCGGCACCAGGCTCACGGTTTTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACGAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGAAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCATGA(SEQ ID NO:170)

[0487] MKSLSVSLVVLWLQLNWVNSQQKVQQSPESLIVPEGAMTSLNCTFSDSASQYFAWYRQHSGKAPKALMSIFSNGEKEEGRFTIHLNKASLHFSLHIRDSQPSDSALYLCAANNYAQGLTFGLGTRVSVFPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:171)

[0488] MGSRLFFVVLILLCAKHMEAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSPGGGGEQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKRKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS(SEQ ID NO:172)

[0489] QQKVQQSPESLIVPEGAMTSLNCTFSDSASQYFAWYRQHSGKAPKALMSIFSNGEKEEGRFTIHLNKASLHFSLHIRDSQPSDSALYLCAANNYAQGLTFGLGTRVSVFPY(SEQ ID NO:173)

[0490] EAAVTQSPRSKVAVTGGKVTLSCHQTNNHDYMYWYRQDTGHGLRLIHYSYVADSTEKGDIPDGYKASRPSQENFSLILELASLSQTAVYFCASSPGGGGEQYFGPGTRLTVL(SEQ ID NO:174)

[0491] In some embodiments, the TCR construct includes MART-1 specific TCR chains. In some embodiments, the TCR construct including the MART-1 specific TCR chains includes TCR α and TCR β chains and / or modified forms thereof found in MART-1 specific TCR clones F4 and / or F5. In some embodiments, the TCR construct including the MART-1 specific TCR chains includes TCR α and TCR β chains targeting MART-1 epitope AAGIGILTV (SEQ ID NO: 175). In some embodiments, MART-1 specific TCR sequences, TCR variable domain sequences, CDR sequences and / or TCR constant domain sequences are described in patent publication number US 9,128,080 B2, which is incorporated herein by reference for purposes described herein.

[0492] 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.

[0493] ATGTTGCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGACCCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAAGGACAGCTTCCTGAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTACTTCTGTGCTGGTGGGACCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAGGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC(SEQ ID NO:176)

[0494] ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGACAGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTCACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGAACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCTGAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAGAAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTCCTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTTCTGTGCCATCAGTGAGGTAGGGGTTGGGCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO:177)

[0495] ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACTTCGGAGGAGGAAAGCTTATCTTCGGACAGGGAACGGAGTTATCTGTGAAACCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ IDNO:178)

[0496] ATGAGAATCAGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAGGTCCAGTGATTGCTGGGATCACCCAGGCACCAACATCTCAGATCCTGGCAGCAGGACGGCGCATGACACTGAGATGTACCCAGGATATGAGACATAATGCCATGTACTGGTATAGACAAGATCTAGGACTGGGGCTAAGGCTCATCCATTATTCAAATACTGCAGGTACCACTGGCAAAGGAGAAGTCCCTGATGGTTATAGTGTCTCCAGAGCAAACACAGATGATTTCCCCCTCACGTTGGCGTCTGCTGTACCCTCTCAGACATCTGTGTACTTCTGTGCCAGCAGCCTAAGTTTCGGCACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCATGTGGCTTTACCTCGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC(SEQ ID NO:179)

[0497] GQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQDPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGGTGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:180)

[0498] DAGITQSPRHKVTETGTPVTLRCHQTENHRYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLTLESATSSQTSVYFCAISEVGVGQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ IDNO:181)

[0499] QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNFGGGKLIFGQGTELSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:182)

[0500] IAGITQAPTSQILAAGRRMTLRCTQDMRHNAMYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDGYSVSRANTDDFPLTLASAVPSQTSVYFCASSLSFGTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRACGFTSSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ IDNO:183)

[0501] In certain embodiments, TCR constructs include tyrosinase-specific TCR chains. In certain embodiments, the TCR constructs including tyrosinase-specific TCR chains are included in TCR α and TCR β chains and / or their modified forms found in tyrosinase-specific TCR clone TIL 1383I. In certain embodiments, the TCR constructs including tyrosinase-specific TCR chains include TCR α and TCR β chains targeting tyrosinase epitopes, which are represented by the amino acid 368-376 of tyrosinase (reactions of class I MHC (HLA-A2) restricted epitopes (368-376) for tyrosinase). In certain embodiments, tyrosinase-specific TCR sequences, TCR variable domain sequences, CDR sequences, and / or TCR constant domain sequences are described in publications Roszkowski et al. Cancer Res.65 (4): 1570-6 (2005), and the document is incorporated herein by reference for purposes described herein.

[0502] In certain embodiments, the TCR construct comprises a MAGE-A3 specific TCR chain. In certain embodiments, the TCR construct comprising the MAGE-A3 specific TCR chain comprises the TCR α and TCR β chains of amino acid 271-279, such as epitope FLWGPRALV (SEQ ID NO: 184) targeting MAGE-A3. In certain embodiments, the TCR construct comprising the MAGE-A3 specific TCR chain comprises the TCR α and TCR β chains of amino acid 112-120, such as epitope KVAELVHFL (SEQ ID NO: 185) targeting MAGE-A3. In certain embodiments, the MAGE-A3 specific TCR sequence, TCR variable domain sequence, CDR sequence, and / or TCR constant domain sequence are disclosed in International Patent Application Publication No. WO 2012 / 054825A1, which is incorporated herein by reference for purposes described herein. In certain embodiments, the anti-MAGE-A3 112-120 TCR comprises an A118T substitution relative to wild-type (wherein position 118 in the α chain is a threonine). In certain embodiments, the anti-MAGE-A3 112-120 TCR comprises an A118V substitution relative to wild-type (wherein position 118 in the α chain is a valine).

[0503] In certain 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 certain 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.

[0504] ATGGGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTTCTGGTATGTGCAACATCTCAATGAATCCCCTCGGCTACTCCTGAAGAGCTTCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTTTCGACACAAATGCTTACAAAGTCATCTTT(SEQID NO:186)

[0505] ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCCTAGGAACAGGCCTTGTGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAGAATGGGAGAGAATGTTTTGCTGGAATGTGGACAGGACATGAGCCATGAAACAATGTACTGGTATCGACAAGACCCTGGTCTGGGGCTACAGCTGATTTATATCTCATACGATGTTGATAGTAACAGCGAAGGAGACATCCCTAAAGGATACAGGGTCTCACGGAAGAAGCGGGAGCATTTCTCCCTGATTCTGGATTCTGCTAAAACAAACCAGACATCTGTGTACTTCTGTGCTAGCAGTTCAACAAACACAGAAGTCTTCTTT(SEQ IDNO:187)

[0506] ATGGGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGGAGGAGCAATGGCGATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTCTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTATTTACTCAAATCCTTTCCTTTTCTGGTATGTGCAACATCTCAATGAATCCCCTCGGCTACTCCTGAAGAGCTTCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTTTCGACACAAATGCTTACAAAGTCATCTTTGGAAAAGGGACACATCTTCATGTTCTCCCTAACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACACCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTTCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO:188)

[0507] ATGAGAGTTAGGCTCATCTCTGCTGTGGTGCTGTGTTCCCTAGGAACAGGCCTTGTGGACATGAAAGTAACCCAGATGCCAAGATACCTGATCAAAAGAATGGGAGAGAATGTTTTGCTGGAATGTGGACAGGACATGAGCCATGAAACAATGTACTGGTATCGACAAGACCCTGGTCTGGGGCTACAGCTGATTTATATCTCATACGATGTTGATAGTAACAGCGAAGGAGACATCCCTAAAGGATACAGGGTCTCACGGAAGAAGCGGGAGCATTTCTCCCTGATTCTGGATTCTGCTAAAACAAACCAGACATCTGTGTACTTCTGTGCTAGCAGTTCAACAAACACAGAAGTCTTCTTTGGTAAAGGAACCAGACTCACAGTTGTAGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGCAACCACTTCCGCTGCCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGGATTACCTCAGCATCCTATCAACAAGGGGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAACTCGTGA(SEQ ID NO:189)

[0508] ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCCTGGAGCTCAGATGCAGTTATTCATACAGTGCAGCACCTTACCTCTTCTGGTACGTGCAGTATCCTGGCCAGAGCCTCCAGTTTCTCCTCAAATACATCACAGGAGACACCGTTGTTAAAGGCACCAAGGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCGGATACAGGAAACTACAAATACGTCTT(SEQ ID NO:190)

[0509] ATGGGCATCCAGACCCTCTGTTGTGTGATCTTTTATGTTCTGATAGCAAATCACACAGATGCTGGAGTTACCCAGACACCCAGACATGAGGTGGCAGAGAAAGGACAAACAATAATCCTGAAGTGTGAGCCAGTTTCAGGCCACAATGACCTTTTCTGGTACAGACAGACCAAGATACAGGGACTAGAGTTGCTGAGCTACTTCCGCAGCAAGTCTCTTATGGAAGATGGTGGGGCTTTCAAGGATCGATTCAAAGCTGAGATGCTAAATTCATCCTTCTCCACTCTGAAGATTCAACCTACAGAACCCAGGGACTCAGCTGTGTATCTGTGTGCCAGCAGTTTTGGGACAGCTAGTGCAGAAACGCTGTATTTT(SEQ ID NO:191)

[0510] ATGGTCCTAGTGACCATTCTGCTGCTCAGCGCGTTCTTCTCACTGAGAGGAAACAGTGCCCAGTCCGTGGACCAGCCTGATGCTCATGTCACGCTCTCTGAAGGAGCCTCCCTGGAGCTCAGATGCAGTTATTCATACAGTGCAGCACCTTACCTCTTCTGGTACGTGCAGTATCCTGGCCAGAGCCTCCAGTTTCTCCTCAAATACATCACAGGAGACACCGTTGTTAAAGGCACCAAGGGCTTTGAGGCCGAGTTTAGGAAGAGTAACTCCTCTTTCAACCTGAAGAAATCCCCAGCCCATTGGAGCGACTCAGCCAAGTACTTCTGTGCACTGGAGGGCCCGGATACAGGAAACTACAAATACGTCTTTGGAGCAGGTACCAGACTGAAGGTTATAGCACACATCCAGAACCCAGAACCTGCTGTGTACCAGTTAAAAGATCCTCGGTCTCAGGACAGCACCCTCTGCCTGTTCACCGACTTTGACTCCCAAATCAATGTGCCGAAAACCATGGAATCTGGAACGTTCATCACTGACAAAACTGTGCTGGACATGAAAGCTATGGATTCCAAGAGCAATGGGGCCATTGCCTGGAGCAACCAGACAAGCTTCACCTGCCAAGATATCTTCAAAGAGACCAACGCCACCTACCCCAGTTCAGACGTTCCCTGTGATGCCACGTTGACTGAGAAAAGCTTTGAAACAGATATGAACCTAAACTTCCAAAACCTGTCAGTTATGGGACTCCGAATCCTCCTGCTGAAAGTAGCCGGATTTAACCTGCTCATGACGCTGAGGCTGTGGTCCAGTTGA(SEQ ID NO:192)

[0511] ATGGGCATCCAGACCCTCTGTTGTGTGATCTTTTATGTTCTGATAGCAAATCACACAGATGCTGGAGTTACCCAGACACCCAGACATGAGGTGGCAGAGAAAGGACAAACAATAATCCTGAAGTGTGAGCCAGTTTCAGGCCACAATGACCTTTTCTGGTACAGACAGACCAAGATACAGGGACTAGAGTTGCTGAGCTACTTCCGCAGCAAGTCTCTTATGGAAGATGGTGGGGCTTTCAAGGATCGATTCAAAGCTGAGATGCTAAATTCATCCTTCTCCACTCTGAAGATTCAACCTACAGAACCCAGGGACTCAGCTGTGTATCTGTGTGCCAGCAGTTTTGGGACAGCTAGTGCAGAAACGCTGTATTTTGGCTCAGGAACCAGACTGACTGTTCTCGAGGATCTGAGAAATGTGACTCCACCCAAGGTCTCCTTGTTTGAGCCATCAAAAGCAGAGATTGCAAACAAACAAAAGGCTACCCTCGTGTGCTTGGCCAGGGGCTTCTTCCCCTGACACGTGGAGCTGAGCTGGTGGGTGAATGGCAAGGAGGTCCACAGTGGGGTCAGCACGGACCCTCAGGCCTACAAGGAGAGCAATTATAGCTACTGCCTGAGCAGCCGCCTGAGGGTCTCTGCTACCTTCTGGCACAATCCTCGAAACCACTTCCGCTGTCAAGTGCAGTTCCATGGGCTTTCAGAGGAGGACAAGTGGCCAGAGGGCTCACCCAAACCTGTCACACAGAACATCAGTGCAGAGGCCTGGGGCCGAGCAGACTGTGGAATCACTTCAGCATCCTATCATCAGGGGGTTCTGTCTGCAACCATCCTCTATGAGATCCTACTGGGGAAGGCCACCCTATATGCTGTGCTGGTCAGTGGCCTGGTGCTGATGGCCATGGTCAAGAAAAAAAATTCCTGA(SEQ ID NO:193)

[0512] MGPVTCSVLVLLLMLRRSNGDGDSVTQTEGLVTLTEGLPVMLNCTYQTIYSNPFLFWYVQHLNESPRLLLKSFTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCAFDTNAYKVIF(SEQ ID NO:194)

[0513] MRVRLISAVVLCSLGTGLVDMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSTNTEVF(SEQ ID NO:195)

[0514] MGPVTCSVLVLLLMLRRSNGDGDSVTQTEGLVTLTEGLPVMLNCTYQTIYSNPFLFWYVQHLNESPRLLLKSFTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCAFDTNAYKVIFGKGTHLHVLPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNTTYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSSL(SEQ ID NO:196)

[0515] MRVRLISAVVLCSLGTGLVDMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSTNTEVFFGKGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVM(SEQ ID NO:197)

[0516] MVLVTILLLSAFFSLRGNSAQSVDQPDAHVTLSEGASLELRCSYSYSAAPYLFWYVQYPGQSLQFLLKYITGDTVVKGTKGFEAEFRKSNSSFNLKKSPAHWSDSAKYFCALEGPDTGNYKYV(SEQ ID NO:198)

[0517] MGIQTLCCVIFYVLIANHTDAGVTQTPRHEVAEKGQTIILKCEPVSGHNDLFWYRQTKIQGLELLSYFRSKSLMEDGGAFKDRFKAEMLNSSFSTLKIQPTEPRDSAVYLCASSFGTASAETLY(SEQ ID NO:199)

[0518] MVLVTILLLSAFFSLRGNSAQSVDQPDAHVTLSEGASLELRCSYSYSAAPYLFWYVQYPGQSLQFLLKYITGDTVVKGTKGFEAEFRKSNSSFNLKKSPAHWSDSAKYFCALEGPDTGNYKYVFGAGTRLKVIAHIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:200)

[0519] MGIQTLCCVIFYVLIANHTDAGVTQTPRHEVAEKGQTIILKCEPVSGHNDLFWYRQTKIQGLELLSYFRSKSLMEDGGAFKDRFKAEMLNSSFSTLKIQPTEPRDSAVYLCASSFGTASAETLYFGSGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS(SEQ ID NO:201)

[0520] In certain embodiments, the TCR construct comprises a MAGE-A4 specific TCR chain. In certain embodiments, the TCR construct comprising the MAGE-A4 specific TCR chain comprises a TCR α and TCR β chain targeting the epitope GVYDGREHTV (SEQ ID NO: 202). In certain embodiments, the TCR construct comprising the MAGE-A4 specific TCR chain comprises a TCR α and TCR β chain targeting the epitope FMNKFIYEI (SEQ ID NO: 203). In certain embodiments, the MAGE-A4 specific TCR sequence, TCR variable domain sequence, CDR sequence and / or TCR constant domain sequence are described in International Patent Application Publication Nos. WO 2017 / 174824A1 and WO 2021 / 229212 A1, each of which is incorporated herein by reference for the purposes described herein. In certain embodiments, the anti-MAGE-A4 TCR α chain variable domain may have an M4V or M4L amino acid substitution. In certain embodiments, the anti-MAGE-A4 TCR beta chain variable domain may have an N10E amino acid substitution.

[0521] In certain 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 certain 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.

[0522] ATGAAGAAGCACCTGACCACCTTTCTCGTGATCCTGTGGCTGTACTTCTACCGGGGCAACGGCAAGAACCAGGTGGAACAGAGCCCCCAGAGCCTGATCATCCTGGAAGGCAAGAACTGCACCCTGCAGTGCAACTACACCGTGTCCCCCTTCAGCAACCTGCGGTGGTACAAGCAGGACACCGGCAGAGGCCCTGTGTCCCTGACCATCCTGACCTTCAGCGAGAACACCAAGAGCAACGGCCGGTACACCGCCACCCTGGACGCCGATACAAAGCAGAGCAGCCTGCACATCACCGCCAGCCAGCTGAGCGATAGCGCCAGCTACATCTGCGTGGTGTCCGGCGGCACAGACAGCTGGGGCAAGCTGCAGTTTGGCGCCGGAACACAGGTGGTCGTGACCCCCGACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGACCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTCAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGCGGCAGCCGGGCCAAGAGA(SEQ ID NO:204)

[0523] ATGGCCAGCCTGCTGTTCTTCTGCGGCGCCTTCTACCTGCTGGGCACCGGCTCTATGGATGCCGACGTGACCCAGACCCCCCGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCTCCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGACCCTGGCCTGGGCCTGCGGCTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCCAGACAGGCTCAGGCCAAGTTCAGCCTGTCCCTGGAAAGCGCCATCCCCAACCAGACCGCCCTGTACTTTTGTGCCACAAGCGGCCAGGGCGCCTACGAGGAGCAGTTCTTTGGCCCTGGCACCCGGCTGACAGTGCTGGAAGATCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAAATCAGCCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTGTCCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTTTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCTTGGGGGCGCGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGAAAGGCCACACTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:205)

[0524] MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPDIQNPDPA VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSGSRAKR(SEQ ID NO:206)

[0525] MKKHLTTFLVILWLYFYRGNGKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQFGAGTQVVVTPD(SEQ IDNO:207)

[0526] MASLLFFCGAFYLLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQ KATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPANLDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILGGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:208)

[0527] MASLLFFCGAFYLLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEQFFGPGTRLTVLE(SEQ ID NO:209)

[0528] MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIMTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF(SEQ ID NO:210)

[0529] MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTIVTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF(SEQ ID NO:211)

[0530] MKNQVEQSPQSLIILEGKNCTLQCNYTVSPFSNLRWYKQDTGRGPVSLTILTFSENTKSNGRYTATLDADTKQSSLHITASQLSDSASYICVVSGGTDSWGKLQF(SEQ ID NO:212)

[0531] MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYNEQFF(SEQ ID NO:213)

[0532] MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATSGQGAYEEQFF(SEQ ID NO:214)

[0533] In some embodiments, the TCR construct includes a Wilms tumor antigen (WT1) WT1 specific TCR chain. In some embodiments, the TCR construct comprising a WT1 specific TCR chain includes TCR α and TCR β chains for epitope VLDFAPPGA (SEQ ID NO: 215). In some embodiments, the TCR construct comprising a WT1 specific TCR chain includes TCR α and TCR β chains for epitope RMFPNAPYL (SEQ ID NO: 216). In some embodiments, WT1 specific TCR sequences, TCR variable domain sequences, CDR sequences and / or TCR constant domain sequences are described in International Patent Application Publication Nos. WO 2020 / 185796 A1 and WO 2021 / 034976 A1, and each document is incorporated herein by reference for the purposes described herein. In some embodiments, the leader sequence and / or signal peptide can be removed from the TCR amino acid sequence, and the sequence identity percentage can be calculated based on the TCR amino acid sequence without a leader sequence and / or signal peptide.

[0534] In some embodiments, the 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, the 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.

[0535] ATGGAGACACTGCTGGGACTACTGATTCTGTGGCTGCAACTGCAATGGGTGAGCAGCAAACAGGAGGTTACCCAGATTCCTGCTGCTCTGTCTGTTCCTGAAGGCGAGAATCTGGTGCTGAACTGCAGCTTCACAGATAGCGCCATCTACAACCTGCAGTGGTTCAGACAGGATCCTGGAAAAGGCCTGACAAGCCTGCTGCTGATTCAGAGCTCTCAGAGAGAGCAGACATCTGGAAGACTGAATGCTAGCCTGGACAAGTCTAGCGGCAGAAGCACCCTGTATATTGCCGCCTCTCAACCTGGAGATTCTGCCACATACCTGTGTGCTGTGAAGGAGACATCTGGCTCTAGACTGACCTTTGGCGAGGGAACACAACTGACCGTGAATCCTGAC(SEQ ID NO:217)ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAACAACTACTACCTGTTCTGGTACAAGCAGCCCCCCTCTAGGCAGATGATCCTGGTGATCAGACAGGAGGCCTATAAACAGCAGAATGCCACAGAGAACCGGTTCAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAGATCTCTGATTCTCAGCTGGGCGATACAGCCATGTACTTTTGCGCCTTCATCTACCCCAGCTACACAAGCGGCACATACAAGTACATCTTCGGCACCGGCACAAGACTGAAGGTTCTGGCCAAC(SEQ ID NO:218)

[0536] ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCAGAACAGCCCCTCTCTGTCTGTGCAGGAAGGCAGAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTGTGGTACAAGAAGTACCCCGCCGAGGGCCCTACCTTTCTGATCAGCATCTCTAGCATCAAGGACAAGAACGAAGATGGCAGATTCACCGTGTTCCTGAACAAGAGCGCCAAGCACCTGAGCCTGCACATTGTGCCTTCTCAACCTGGAGATTCTGCCGTGTACTTTTGTGCTGCCTCTGGAACAGGCGGAAGCTATATCCCCACATTTGGAAGAGGAACAAGCCTGATCGTGCACCCTTAC(SEQ ID NO:219)

[0537] ATGGCCATGTTACTAGGAGCGAGCGTGCTGATTCTGTGGTTACAGCCTGATTGGGTGAACTCTCAGCAGAAGAACGATGATCAGCAGGTGAAGCAGAACAGCCCCTCTCTGTCTGTGCAGGAAGGCAGAATCAGCATCCTGAATTGCGATTACACCAACAGCATGTTCGACTACTTCCTGTGGTACAAGAAGTACCCCGCCGAGGGCCCTACCTTTCTGATCAGCATCTCTAGCATCAAGGACAAGAACGAAGATGGCAGATTCACCGTGTTCCTGAACAAGAGCGCCAAGCACCTGAGCCTGCACATTGTGCCTTCTCAACCTGGAGATTCTGCCGTGTACTTTTGTGCTGCCTCTGGCATTGGCGACTACAAACTGAGCTTTGGAGCCGGCACAACAGTGACCGTTAGAGCCAAT(SEQ ID NO:220)

[0538] ATGGTGAAGATCCGGCAGTTCCTCCTGGCTATTCTGTGGCTGCAACTGTCTTGTGTGTCTGCTGCCAAGAATGAAGTGGAGCAGTCTCCCCAGAACCTTACAGCCCAGGAAGGCGAGTTTATCACCATCAACTGCAGCTATTCTGTGGGCATTAGCGCCCTGCATTGGCTGCAGCAACACCCTGGAGGAGGAATTGTGTCTCTGTTTATGCTGTCTTCTGGCAAGAAGAAGCACGGCCGGCTGATTGCCACCATCAACATCCAGGAGAAGCACTCTTCTCTGCACATTACAGCCTCTCATCCCAGGGATTCTGCCGTGTACATCTGTGCCGTGAGAACCAGCTACGATAAGGTGATTTTCGGACCAGGCACCTCTCTGAGCGTGATCCCCAAT(SEQ ID NO:221)

[0539] ATGAAGAGCCTGAGAGTCCTGCTGGTGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGAAGTGGAGCAGAATAGCGGCCCTCTGTCTGTTCCTGAAGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGAGGATGGCAGGTTTACAGCCCAGCTGAACAAGGCCAGCCAGTATGTTTCTCTGCTGATCAGAGATAGCCAGCCTAGCGATTCTGCCACCTACCTGTGTGCCGTGAACTTACTTGGAGCTACAGGATACTCTACACTGACCTTCGGCAAAGGCACCATGCTGCTGGTGAGCCCTGAT(SEQ ID NO:222)

[0540] ATGTGGGGCGTTTTCCTTCTGTATGTGAGCATGAAGATGGGCGGCACAACAGGCCAGAACATCGATCAGCCTACCGAGATGACAGCCACAGAAGGAGCTATTGTTCAGATCAACTGCACCTACCAGACAAGCGGCTTCAACGGCCTGTTCTGGTACCAGCAGCATGCTGGAGAAGCTCCTACATTTCTGAGCTACAATGTGCTGGATGGCCTGGAGGAGAAAGGCAGGTTTAGCAGCTTCCTGAGCAGGTCTAAGGGCTATTCTTATCTGCTGCTGAAGGAGCTGCAGATGAAGGATTCCGCCAGCTACCTGTGTGCCGTTAGGGGCATCAATGATTACAAGCTGAGCTTTGGAGCCGGAACAACAGTGACCGTGAGAGCCAAC(SEQ ID NO:223)

[0541] ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCCGTGATTACCGGCTTTCAGAAGCTGGTGTTTGGCACAGGCACCAGACTGCTGGTTTCTCCCAAT(SEQ ID NO:224)

[0542] ATGAGACTGGTGGCACGCGTAACTGTGTTTCTGACCTTTGGCACCATCATCGATGCCAAGACAACCCAGCCTACAAGCATGGACTGTGCCGAGGGAAGAGCTGCTAATCTGCCATGTAATCACAGCACAATCAGCGGCAACGAGTACGTGTACTGGTACCGGCAGATCCACTCTCAAGGACCTCAGTACATCATTCATGGCCTGAAGAACAACGAGACCAACGAGATGGCCAGCCTGATCATCACCGAGGACAGGAAGTCTTCTACCCTGATTCTGCCTCATGCTACACTGAGAGATACCGCCGTGTACTACTGCATTGCCGGAGTGGGAAGAGGCCAGAATTTCGTGTTTGGACCTGGAACAAGACTGAGCGTTCTGCCCTAT(SEQ ID NO:225)

[0543] ATGGAGAAGAACCCCTTGGCAGCACCTCTGCTTATTCTGTGGTTCCACCTGGATTGTGTGAGCAGCATCCTGAATGTGGAGCAGTCTCCTCAGAGCCTGCATGTGCAAGAAGGCGATAGCACCAATTTCACCTGCAGCTTTCCAAGCAGCAACTTCTACGCCCTGCACTGGTACAGATGGGAAACCGCCAAATCTCCTGAAGCCCTGTTTGTGATGACCCTGAATGGCGACGAGAAGAAGAAGGGCAGAATTAGCGCCACCCTGAATACCAAGGAGGGCTACAGCTACCTGTACATCAAGGGCTCTCAACCTGAGGATTCTGCCACCTACCTTTGCGCCTTTCACCCCAATTTCGGCAACGAGAAACTGACCTTTGGAACCGGAACAAGGCTGACCATCATCCCCAAC(SEQ ID NO:226)

[0544] ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCTGTTCAGCCTAGAGGAGATGGCTCTAGCAATACCGGCAAGCTGATCTTTGGCCAGGGAACAACACTGCAGGTGAAGCCTGAT(SEQ ID NO:227)

[0545] ATCCAGAATCCCGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:228)CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACATCTCTTCTCTGTTGGGTGGTTCTGGGCTTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTATAAGGTGACCAAGAGGGGACAGGATGTGGCTCTGAGATGTGACCCTATTAGCGGACATGTGAGCCTGTACTGGTACAGACAAGCTCTGGGACAAGGACCCGAGTTTCTGACCTACTTCAACTATGAGGCCCAGCAGGACAAATCTGGACTGCCCAACGACAGATTCAGCGCCGAAAGACCAGAAGGCTCTATTAGCACACTGACCATCCAGAGAACAGAGCAGAGGGATTCTGCCATGTACAGATGCGCCAGCAGCTTAACAGGCTCTTACGAGCAGTACTTTGGACCTGGCACAAGACTGACAGTGACAGAG(SEQ ID NO:229)

[0546] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCACACCTGAAGCCTCTAGCCCATATGAGCAGTACTTTGGCCCTGGCACCAGATTAACAGTGACAGAG(SEQ IDNO:230)

[0547] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGACCTGGACTGCTTCATTGGATGGCTCTGTGTTTGCTGGGAACAGGACATGGAGATGCTATGGTGATCCAGAACCCCAGGTATCAGGTGACCCAGTTTGGCAAACCAGTGACACTGAGCTGTTCTCAGACCCTGAACCACAACGTGATGTACTGGTACCAGCAGAAGTCTTCTCAGGCCCCTAAGCTGCTGTTCCACTACTACGACAAGGACTTCAACAACGAGGCCGATACCCCTGACAATTTCCAGAGCAGGAGGCCCAATACCAGCTTCTGTTTCCTGGACATTAGAAGCCCTGGACTGGGAGATGCTGCCATGTACCTGTGTGCCACCAGCAATTTACAGGGAAGACAACCTCAGCACTTTGGCGATGGCACAAGGCTGTCTATCCTGGAG(SEQ IDNO:231)

[0548] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGTGGCTGCTGGCGTGATTCAATCTCCTAGACACCTGATCAAGGAGAAGAGAGAAACAGCCACCCTGAAGTGCTACCCCATCCCCAGACACGATACAGTGTACTGGTATCAGCAAGGACCTGGACAAGATCCCCAGTTCCTGATCAGCTTCTACGAGAAGATGCAGAGCGACAAAGGCAGCATCCCAGACAGATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCAGCCTGGAACTGGGCGATTCTGCTCTGTACTTCTGTGCCTCTTCTCTGAGACTGGGAAGAGAAACCCAGTACTTTGGACCCGGCACAAGACTGCTGGTTCTTGAG(SEQ ID NO:232)

[0549] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACAAGACTTCTCTGCTGGGTGGTGCTTGGATTTCTGGGCACAGATCATACAGGAGCTGGAGTTAGCCAGTCTCCTAGGTACAAAGTGGCCAAGAGAGGACAGGATGTGGCTCTGAGATGTGACCCTATTAGCGGACATGTGAGCCTGTTTTGGTACCAGCAAGCTCTGGGACAAGGACCCGAGTTTCTGACCTACTTCCAGAATGAAGCCCAGCTGGATAAATCTGGACTGCCTAGCGACCGGTTCTTCGCCGAAAGACCTGAAGGATCTGTTAGCACCCTGAAGATTCAGAGAACACAGCAGGAGGACTCTGCCGTGTACCTGTGTGCCTCTTCTTTAGGACAGGCCTATGAGCAGTATTTTGGACCTGGCACCAGACTGACCGTGACAGAG(SEQ IDNO:233)CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCACAAGACTTCTCTGCTGGGTGGCCTTTTGTCTGCTGGTGGAAGAGCTGATTGAAGCTGGAGTTGTGCAGTCTCCTAGGTACAAGATCATCGAGAAGAAGCAGCCCGTGGCCTTCTGGTGTAATCCCATTTCTGGCCACAACACCCTGTACTGGTATCTGCAGAATCTGGGACAGGGCCCTGAACTGCTGATCAGATACGAGAACGAAGAAGCCGTGGACGATTCTCAACTGCCTAAGGACCGCTTTTCTGCCGAGAGGCTGAAAGGAGTGGATTCTACCCTGAAGATCCAACCTGCTGAACTGGGCGATTCTGCTGTGTACCTGTGCGCTTCTAGCCTGACAAGAGGAGCTGAAGCCTTTTTTGGACAGGGCACAAGACTGACAGTGGTGGAG(SEQ IDNO:234)

[0550] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGACCTCAGCTTCTTGGATACGTTGTGCTGTGTCTGCTTGGAGCTGGACCTCTTGAAGCTCAGGTTACCCAGAACCCCAGATACCTGATTACCGTGACAGGCAAAAAGCTGACCGTGACATGTAGCCAGAACATGAACCACGAGTACATGAGCTGGTACCGGCAGGATCCTGGATTAGGCCTGAGACAGATCTACTACAGCATGAACGTGGAGGTGACCGATAAAGGCGACGTGCCTGAGGGATACAAGGTGAGCAGAAAGGAGAAGAGGAATTTCCCCCTGATCCTGGAAAGCCCAAGCCCCAATCAGACAAGCCTGTACTTTTGTGCCAGCAGCTTTTCTGGCGGCACATATGAGCAGTACTTCGGCCCTGGCACAAGACTGACAGTTACAGAG(SEQ IDNO:235)

[0551] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGCTGAGCCCTGATCTCCCTGATTCTGCCTGGAATACCAGACTGCTGTGTCATGTGATGCTGTGTCTGCTTGGAGCCGTTTCTGTGGCTGCTGGCGTGATTCAATCTCCTAGACACCTGATCAAGGAGAAGAGAGAAACAGCCACCCTGAAGTGCTACCCCATCCCCAGACACGATACAGTGTACTGGTATCAGCAAGGACCTGGACAAGATCCCCAGTTCCTGATCAGCTTCTACGAGAAGATGCAGAGCGACAAAGGCAGCATCCCAGACAGATTTAGCGCCCAGCAGTTTAGCGACTATCACTCTGAGCTGAACATGAGCAGCCTGGAACTGGGCGATTCTGCTCTGTACTTCTGTGCCAGCAGCTATAGAGGAGGCAGCACATATGAGCAGTACTTTGGCCCTGGCACAAGACTGACAGTGACAGAG(SEQ ID NO:236)

[0552] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGAGCACCAGACTCCTTTGCTGGATGGCTTTGTGTCTGCTTGGAGCTGAGCTGTCTGAAGCTGAAGTTGCCCAGTCTCCCAGATACAAGATCACCGAGAAATCTCAGGCTGTGGCCTTCTGGTGTGACCCTATTTCTGGACACGCCACCCTGTACTGGTATAGGCAAATTCTGGGACAAGGCCCTGAACTGCTGGTGCAATTTCAGGATGAGAGCGTGGTGGACGATTCTCAACTGCCTAAGGACAGGTTTTCTGCCGAGCGGCTGAAAGGAGTTGATAGCACCCTGAAGATCCAACCTGCTGAACTGGGCGATTCTGCTATGTACCTGTGCGCCTCTTCTCAGAGAGATAGCCCTAACGAGAAGCTGTTCTTTGGCTCTGGAACCCAGCTGTCTGTGCTGGAG(SEQ ID NO:237)

[0553] CTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCACCATGGGCTGTAGACTGTTGTGTTGTGCTGTGCTGTGTCTGTTGGGAGCTGTGCCTATGGAAACAGGCGTTACCCAGACACCTAGACATCTGGTTATGGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCATCTGGGCCATAACGCCATGTACTGGTATAAGCAGAGCGCCAAGAAACCACTGGAACTGATGTTCGTGTACAGCCTGGAGGAGAGGGTGGAGAATAATAGCGTGCCCAGCAGATTTAGCCCTGAGTGCCCAAATTCTTCTCACCTGTTCCTGCACCTGCACACATTACAGCCCGAGGATTCTGCCCTGTACCTGTGTGCTTCTTCTCAAGACCCTTACAAGCTGAGCGGCAATACCATCTACTTCGGCGAAGGCTCTTGGCTGACAGTGGTTGAA(SEQ ID NO:238)GATCTGAACAAGGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCTCCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACTCCGGCGTGTGCACCGATCCCCAGCCTCTGAAAGAACAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACACAGATCGTGTCTGCCGAAGCCTGGGGCAGAGCCGATTGCGGCTTTACCTCCGTGTCCTATCAGCAGGGCGTGCTGAGCGCCACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACTTC(SEQ IDNO:239)

[0554] GACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:240)

[0555] ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACATAGGAAACCATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ IDNO:241)

[0556] ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTCAACTGCAGTTACACAGTCAGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGCTTTCTGCACATCACAGCCCCTAAACCTGAAGACTCAGCCACTTATCTCTGTGCTGTGCAGACCATGGACGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ IDNO:242)

[0557] ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAGAGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGATGCCGCGATGTATTTCTGTGCTTCCAGTCCAGGAACCTACAAATACATCTTTGGAACAGGCACCAGGCTGAAGGTTTTAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQID NO:243)

[0558] ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAGTAATTATTATTTGTTCTGGTACAAACAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGACACTGCGATGTATTTCTGTGCTTTCAACCCTTGGGAGAACTATGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:244)

[0559] ATGAAGAGCCTGAGAGTCCTGCTGGTGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGAAGTGGAGCAGAATAGCGGCCCTCTGTCTGTTCCTGAAGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGAGGATGGCAGGTTTACAGCCCAGCTGAACAAGGCCAGCCAGTATGTTTCTCTGCTGATCAGAGATAGCCAGCCTAGCGATTCTGCCACCTACCTGTGTGCCGTGAACATCGGAAATCACGACATGAGATTTGGAGCCGGCACAAGACTGACCGTGAAGCCCAATATCCAGAACCCTGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ IDNO:245)

[0560] ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCTGTGCAGACCATGGATGGCAACCAGTTCTACTTCGGCACAGGCACATCTCTGACCGTTATCCCCAATATCCAGAACCCTGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ IDNO:246)

[0561] ATGGCTTGTCCTGGATTCTTATGGGCTCTGGTGATCAGCACCTGTCTGGAGTTCTCTATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTGACACTGTCTTGCACCTACGATACAAGCGAGAGCGACTACTACCTGTTCTGGTACAAGCAGCCTCCCTCTAGGCAGATGATCCTGGTGATTAGACAGGAGGCCTACAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGAGATGCTGCCATGTACTTTTGTGCCAGCTCTCCAGGCACCTACAAGTACATTTTTGGCACCGGCACCAGACTGAAGGTGCTGGCCAATATCCAGAATCCCGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQID NO:247)

[0562] ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAGCAACTACTACCTGTTCTGGTACAAGCAGCCCCCTTCTAGGCAGATGATCCTGGTGATCAGACAGGAGGCCTATAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGCGATACAGCCATGTACTTTTGTGCCTTCAACCCCTGGGAGAACTATGGCCAGAATTTCGTGTTCGGCCCTGGCACCAGACTGTCTGTTCTGCCTTATATCCAGAACCCCGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA(SEQ ID NO:248)

[0563] ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGACACTGAAGTTACCCAGACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCACAGGGCTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACCTTCACCTACACGCCCTGCAGCCAGAAGACTCAGCCCTGTATCTCTGCGCCAGCAGCCAAGGGACTAGCGGGGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO:249)

[0564] ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTCTCTTTGGGACCTTCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO:250)

[0565] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTTTCAGACGGGGGGGCTACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO:251)

[0566] ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGACCCCATTCTCTCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG(SEQ ID NO:252)

[0567] ATGGGCTGTAGACTGTTGTGTTGTGCTGTGCTGTGTCTGTTGGGAGCTGTGCCTATCGATACAGAGGTGACCCAGACCCCTAAACATCTGGTTATGGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCACATGGGCCATAGGGCCATGTATTGGTATAAGCAGAAGGCCAAGAAACCTCCTGAGCTGATGTTCGTGTACAGCTACGAGAAGCTGAGCATCAACGAGAGCGTGCCCAGCAGATTTTCTCCTGAGTGCCCTAATTCTAGCCTGCTGAATCTGCACCTGCATGCTCTGCAGCCTGAGGATTCTGCTCTGTACCTGTGTGCTTCTTCTCAGGGCACATCTGGAGCTGATACACAGTACTTCGGACCTGGCACAAGACTGACAGTGCTGGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:253)

[0568] ATGTCTATCGGTCTGCTGTGCTGTGCTGCTCTTTCTCTGCTTTGGGCTGGACCTGTGAATGCTGGAGTTACACAAACCCCCAAGTTCCAAGTGCTGAAGACAGGACAGAGCATGACCCTGCAGTGTGCTCAGGACATGAATCACGAGTACATGAGCTGGTACAGACAGGATCCTGGAATGGGCCTGAGGCTGATCCACTACTCTGTTGGAGCCGGAATTACAGATCAGGGAGAAGTGCCAAATGGCTACAACGTGAGCAGGAGCACAACCGAGGACTTCCCCTTAAGACTGTTGTCTGCTGCTCCATCTCAGACAAGCGTGTACTTTTGCGCCAGCTCCTACTCTCTGTGGGATCTGCAGGAAACCCAGTACTTTGGACCAGGCACAAGACTGTTAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:254)

[0569] ATGGGCACATCTCTTCTCTGCTGGATGGCTCTTTGTCTGCTTGGAGCCGATCATGCCGATACAGGAGTTAGCCAGGATCCTAGACACAAGATCACCAAGAGAGGCCAGAATGTGACCTTCCGGTGCGATCCTATCTCTGAGCACAACAGGCTGTACTGGTACAGACAAACACTGGGACAAGGACCTGAGTTCCTGACCTACTTCCAGAACGAAGCCCAGCTGGAGAAGTCTAGACTTCTGAGCGACAGATTTAGCGCCGAGAGACCTAAAGGCAGCTTTAGCACCCTGGAGATCCAGAGAACAGAACAGGGCGATTCTGCCATGTACCTGTGTGCTAGCAGCTTTTCTGATGGAGGCGCCACCGATACACAGTATTTCGGACCTGGCACAAGACTGACAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:255)

[0570] ATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCAGACCTCACAGCCTGACCGATACACAGTACTTTGGCCCTGGCACAAGACTGACAGTGTTAGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC(SEQ ID NO:256)

[0571] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVKETSGSRLTFGEGTQLTVNP(SEQ ID NO:257)

[0572] MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKI SDSQLGDTAMYFCAFIYPSYTSGTYKYIFGTGTRLKVLAN(SEQID NO:258)

[0573] MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGTGGSYIPTFGRGTSLIVHPY(SEQID NO:259)

[0574] MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGIGDYKLSFGAGTTVTVRAN(SEQID NO:260)

[0575] MVKIRQFLLAILWLQLSCVSAAKNEVEQSPQNLTAQEGEFITINCSYSVGISALHWLQQHPGGGIVSLFMLSSGKKKHGRLIATINIQEKHSSLHITASHPRDSAVYICAVRTSYDKVIFGPGTSLSVIPN(SEQ ID NO:261)

[0576] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNLLGATGYSTLTFGKGTMLLVSP(SEQ IDNO:262)

[0577] MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVRGINDYKLSFGAGTTVTVRAN(SEQ ID NO:263)

[0578] MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVITGFQKLVFGTGTRLLVSPN(SEQ ID NO:264)

[0579] MRLVARVTVFLTFGTIIDAKTTQPTSMDCAEGRAANLPCNHSTISGNEYVYWYRQIHSQGPQYIIHGLKNNETNEMASLIITEDRKSSTLILPHATLRDTAVYYCIAGVGRGQNFVFGPGTRLSVLPY(SEQ ID NO:265)

[0580] MEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCSFPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFHPNFGNEKLTFGTGTRLTIIPN(SEQ IDNO:266)

[0581] MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQPRGDGSSNTGKLIFGQGTTLQVKP(SEQ IDNO:267)

[0582] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQID NO:268)

[0583] MGTSLLCWVVLGFLGTDHTGAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLTGSYEQYFGPGTRLTVTE(SEQ ID NO:269)

[0584] MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSATPEASSPYEQYFGPGTRLTVTE(SEQ ID NO:270)

[0585] MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSNLQGRQPQHFGDGTRLSILE(SEQ ID NO:271)

[0586] MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSLRLGRETQYFGPGTRLLVLE(SEQ ID NO:272)

[0587] MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSLGQAYEQYFGPGTRLTVTE(SEQ ID NO:273)

[0588] MGTRLLCWVAFCLLVEELIEAGVVQSPRYKIIEKKQPVAFWCNPISGHNTLYWYLQNLGQGPELLIRYENEEAVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAVYLCASSLTRGAEAFFGQGTRLTVVE(SEQ ID NO:274)

[0589] MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSRDREQESPLHFGNGTRLTVTE(SEQ ID NO:275)

[0590] MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSFSGGTYEQYFGPGTRLTVTE(SEQ ID NO:276)

[0591] MLSPDLPDSAWNTRLLCHVMCLLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSYRGGSTYEQYFGPGTRLTVTE(SEQ ID NO:277)

[0592] MSTRLLCWMALCLLGAELESEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSQRDSPNEKLFFGSGTQLSVLE(SEQ ID NO:278)

[0593] MGCRLLCCAVLCLLGAVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQDPYKLSGNTIYFGEGSWLTVVE(SEQ ID NO:279)

[0594] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPANLDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(SEQ ID NO:280)

[0595] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPANLDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:281)

[0596] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPN(SEQ ID NO:282)

[0597] MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPN(SEQ ID NO:283)

[0598] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCASSPGTYKYIFGTGTRLKVLAN(SEQ ID NO:284)

[0599] MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPY(SEQ IDNO:285)

[0600] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQID NO:286)

[0601] MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFGPGTRLTVLE(SEQ ID NO:287)

[0602] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFGPGTRLLVLE(SEQ IDNO:288)

[0603] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSSDGGATDTQYFGPGTRLTVLE(SEQ ID NO:289)

[0604] MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEKGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFGPGTRLTVLE(SEQ ID NO:290)

[0605] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPANLDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:291)

[0606] In some embodiments, the TCR construct comprises a human papillomavirus (HPV) specific TCR chain. In some embodiments, the TCR construct comprising an HPV-specific TCR chain comprises TCR α and TCR β chains targeting HPV 18E6 protein and / or HPV 18E7 protein. In some embodiments, the HPV 18E6 epitope is amino acids 121-135 and / or amino acids 77-91 of HPV 18E6 protein. In some embodiments, the TCR construct comprising an HPV-specific TCR chain comprises TCR α and TCR β chains targeting HPV 18E7 protein. In some embodiments, the HPV 18E7 epitope is amino acids 11-19. In some embodiments, HPV-specific TCR sequences, TCR variable domain sequences, CDR sequences, and / or TCR constant domain sequences are described in International Patent Application Publication No. WO 2015 / 009604 A1, which is incorporated herein by reference for purposes described herein.

[0607] In some embodiments, the TCR construct comprises a Kristen rat sarcoma virus (KRAS) specific (KRAS specific) TCR chain. In some embodiments, the TCR construct comprising the KRAS specific TCR chain comprises TCR α and TCR β chains targeting epitope GADGVGKSA (SEQ ID NO: 293). In some embodiments, the TCR construct comprising the KRAS specific TCR chain comprises TCR α and TCR β chains targeting epitope GADGVGKSAL (SEQ ID NO: 292). In some embodiments, KRAS specific TCR sequences, TCR variable domain sequences, CDR sequences and / or TCR constant domain sequences are described in U.S. Patent No. 10,611,816B2, which is incorporated herein by reference for purposes described herein. In some embodiments, the leader sequence and / or signal peptide can be removed from the TCR amino acid sequence, and the sequence identity percentage is calculated based on the TCR amino acid sequence without the leader sequence and / or signal peptide. In some embodiments, the KRAS-specific TCR targets a KRAS antigen associated with a cancerous state, such as, but not limited to, G12C, G12D, and / or G12R.

[0608] In some embodiments, a TCR construct comprising a KRAS-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: 294-298. In some embodiments, a TCR construct comprising a KRAS-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: 299-317. In some embodiments, a TCR construct comprising a KRAS-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: 299-303.

[0609] atgggcaccaggctcttcttctatgtggccctttgtctgctgtgggcaggacacagggatgctggaatcacccagagcccaagatacaagatcacagagacaggaaggcaggtgaccttgatgtgtcaccagacttggagccacagctatatgttctggtatcgacaagacctgggacatgggctgaggctgatcta ttactcagcagctgctgatattacagataaaggagaagtccccgatggctatgttgtctccagatccaagacagagaatttccccctcactctggagtcagctacccgctcccagacatctgtgtatttctgcgccagcagtgaccccggcactgaagctttctttggacaaggcaccagactcacagttgta (SEQ ID NO:294)

[0610] atgggcaccaggctcttcttctatgtggccctttgtctgctgtgggcaggacacagggatgctggaatcacccagagcccaagatacaagatcacagagacaggaaggcaggtgaccttgatgtgtcaccagacttggagccacagctatatgttctggtatcgacaagacctgggacatgggctgaggctgatctattactcagcagctgctgatattacagataaaggagaagtccccgatggctatgttgtctccagatccaagacagagaatttccccctcactctggagtcagctacccgctcccagacatctgtgtatttctgcgccagcagtgaccccggcactgaagctttctttggacaaggcaccagactcacagttgtaGAGGACCTCAAGAATGTGTTTCCGCCCGAAGTCGCGGTTTTTGAACCATCAGAAGCCGAGATCTCTCATACACAAAAGGCGACGCTCGTATGCCTcGCGACGGGATTTTATCCGGACCACGTCGAGCTTTCCTGGTGGGTTAAcGGAAAGGAGGTGCATTCCGGAGTTTGCACGGACCCTCAGCCATTGAAGGAACAGCCCGCACTGAACGACAGTAGGTATTGCCTTTCATCTCGCCTGCGCGTGTCTGCGACATTCTGGCAAAACCCAAGAAATCACTTCAGATGTCAAGTTCAGTTCTACGGTCTCAGCGAGAATGATGAGTGGACACAAGATAGGGCTAAACCCGTGACTCAAATAGTCTCTGCCGAGGCCTGGGGGAGGGCGGATTGCGGCTTCACATCAGAATCATACCAACAAGGAGTATTGAGCGCGACAATTCTTTACGAAATTCTGCTTGGGAAAGCGACTCTGTACGCGGTGCTCGTGTCCGCTTTGGTTCTTATGGCAATGGTTAAACGAAAGGATAGTAGGGGC(SEQ ID NO:295)

[0611] atgaaatccttgagagttttactagtgatcctgtggcttcagttgagctgggtttggagccaacagaaggaggtggagcagaattctggacccctcagtgttccagagggagccattgcctctctcaactgcacttacagtgaccgaggttcccagtccttcttctggtacagacaatattctgggaaaagccctgagttgataatgttcatatactccaatggtgacaaagaagatggaaggtttacagcacagctcaataaagccagccagtatgtttctctgctcatcagagactcccagcccagtgattcagccacctacctctgtgccgcggcgatggatagcagctataaattgatcttcgggagtgggaccagactgctggtcaggcct(SEQ ID NO:296)

[0612] atgaaatccttgagagttttactagtgatcctgtggcttcagttgagctgggtttggagccaacagaaggaggtggagcagaattctggacccctcagtgttccagagggagccattgcctctctcaactgcacttacagtgaccgaggttcccagtccttcttctggtacagacaatattctgggaaaagccctgagttgataatgttcatatactccaatggtgacaaagaagatggaaggtttacagcacagctcaataaagccagccagtatgtttctctgctcatcagagactcccagcccagtgattcagccacctacctctgtgccgcggcgatggatagcagctataaattgatcttcgggagtgggaccagactgctggtcaggcctATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGTCCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCAGAATCCTGCTGCTGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGC(SEQ ID NO:297)

[0613]

[0614] MGTRLFFYVALCLLWAGHRDAGITQSPRYKITETGRQVTLMCHQTWSHSYMFWYRQDLGHGLRLIYYSAAADITDKGEVPDGYVVSRSKTENFPLTLESATRSQTSVYFCASSDPGTEAFFGQGTRLTVV(SEQ ID NO:299)

[0615] MGTRLFFYVALCLLWAGHRDAGITQSPRYKITETGRQVTLMCHQTWSHSYMFWYRQDLGHGLRLIYYSAAADITDKGEVPDGYVVSRSKTENFPLTLESATRSQTSVYFCASSDPGTEAFFGQGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO:300)

[0616] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAAMDSSYKLIFGSGTRLLVRP(SEQ ID NO:301)

[0617] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAAMDSSYKLIFGSGTRLLVRPIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:302)

[0618] MGTRLFFYVALCLLWAGHRDAGITQSPRYKITETGRQVTLMCHQTWSHSYMFWYRQDLGHGLRLIYYSAAADITDKGEVPDGYVVSRSKTENFPLTLESATRSQTSVYFCASSDPGTEAFFGQGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGATNFSLLKQAGDVEENPGPMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAAMDSSYKLIFGSGTRLLVRPIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:303)

[0619] MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSS(SEQ ID NO:304)

[0620] MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVV(SEQ ID NO:305)

[0621] MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSS(SEQ ID NO:306)

[0622] MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGRASNQPQHFGDGTRLSIL(SEQ ID NO:307)

[0623] MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDRDQAGTALIFGKGTTLSVSS(SEQ ID NO:308)

[0624] MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSFGQSSTYGYTFGSGTRLTVV(SEQ ID NO:309)

[0625] MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:310)

[0626] MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGEGRVDGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS(SEQ ID NO:311)

[0627] MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDMDQAGTALIFGKGTTLSVSSDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:312)

[0628] MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSLGRASNQPQHFGDGTRLSILEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS(SEQ ID NO:313)

[0629] MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGDRDQAGTALIFGKGTTLSVSSDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:314)

[0630] MGPGLLCWALLCLLGAGLVDAGVTQSPTHLIKTRGQQVTLRCSPKSGHDTVSWYQQALGQGPQFIFQYYEEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSFGQSSTYGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQA

[0631] YKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS(SEQ ID NO:315)

[0632] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAAMDSSYKLIFGSGTRLLVRPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS(SEQ ID NO:316)

[0633] MGTRLFFYVALCLLWAGHRDAGITQSPRYKITETGRQVTLMCHQTWSHSYMFWYRQDLGHGLRLIYYSAAADITDKGEVPDGYVVSRSKTENFPLTLESATRSQTSVYFCASSDPGTEAFFGQGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS(SEQ ID NO:317)

[0634] B. NK cells

[0635] NK cells modified to express TCR / CD3 receptor complexes can be obtained from any suitable source, including fresh or frozen. In certain embodiments, NK cells are not NK cells obtained from iPSC differentiation. In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukocyte separation products (PBSC), NK cell lines (e.g., NK-92), human embryonic stem cells (hESC), induced pluripotent stem cells (iPSCs), bone marrow or cord blood, which are obtained by methods well known in the art. Specifically, NK cells can be separated from cord blood (CB), peripheral blood (PB), bone marrow, stem cells, NK cell lines or mixtures thereof. In a specific embodiment, NK cells are separated from merged CB. CB can be merged from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more units. NK cells can be autologous or allogeneic relative to the recipient individual. The separated NK cells may be haplotype matched with the subject to be administered cell therapy, or they may not match. NK cells can be detected by specific surface markers, such as CD16 and CD56 in humans. For example, in some cases, the source of NK cells is umbilical cord blood, where NK cells may be mixed with other cells and may be depleted of certain cells expressing CD3. In other methods, umbilical cord CB is used to derive NK cells by isolating CD34+ cells.

[0636] NK cells can be preactivated with one or more inflammatory cytokines, and they can be amplified or not amplified. In some cases, NK cells are preactivated before being modified to express CD3 ± TCR or after being modified to express CD3 ± TCR complexes. In a specific embodiment, the preactivation of NK cells can include culturing separated NK cells in the presence of one or more cytokines. NK cells can be stimulated with IL-2 or other cytokines (e.g., IL-7, IL-12, IL-15, IL-18, IL-21, etc.) in conjunction with common γ chains. In a specific embodiment, preactivation cytokines can be selected from IL-12, IL-15, IL-18 and combinations thereof. One or more additional cytokines can be used in the preactivation step. Preactivation can last for a short period of time, such as 5-72 hours, such as 10-50 hours, especially 10-20 hours, such as 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, particularly about 16 hours. The pre-activated culture may comprise IL-12 at a concentration of 0.1-150 ng / mL, such as 0.5-50 ng / mL, especially 1-20 ng / mL, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ng / mL, especially about 10 ng / mL. The pre-activated culture may comprise IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, such as 40-60 ng / mL, especially 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 ng / mL, especially about 50 ng / mL.

[0637] In some cases, NK cells are amplified before or after modification to express CD3 ± TCR complexes. Preactivated NK cells can be amplified in the presence of artificial antigen presenting cells (aAPC) and / or feeder cells / fragments or NK activation beads. Preactivated NK cells can be washed, for example 2 times, 3 times, 4 times or 5 times, particularly 3 times before amplification. aAPC can be engineered to express CD137 ligands and / or membrane-bound cytokines. Membrane-bound cytokines can be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In a specific embodiment, aAPC is engineered to express CD137 ligands and mIL-21. aAPC can be derived from cancer cells, such as leukemia cells. aAPC may not express endogenous class I, class II HLA or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58). In particular, aAPC may be K562 cells, such as K562 cells engineered to express CD137 ligands and mIL-21. aAPC may be irradiated. In certain embodiments, fragments of APC can be used to amplify NK cells. Engineering can be performed by any known method 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 NK is co-cultured with antigen presenting cells. In certain embodiments, retroviral transduction includes co-transduction of more than one construct. In certain embodiments, retroviral transduction occurs after co-culturing with antigen presenting cells for about 5 days or at about 5 days. In certain embodiments, co-culturing with antigen presenting cells continues after NK cell transduction. The expansion time may be about 2-30 days, such as 3-20 days, especially 12-16 days, such as 12, 13, 14, 15, 16, 17, 18 or 19 days, especially about 14 days. Pre-activated NK cells and aAPCs may be present in a ratio of about 3: 1-1: 3, such as 2: 1, 1: 1, 1: 2, especially about 1: 2. The expansion culture may further include cytokines to promote expansion, such as IL-2. IL-2 may be present at the following concentrations: about 10-500 U / mL, such as 100-300 U / mL, especially about 200 U / mL. IL-2 can be supplemented in the expansion culture, such as every 2-3 days. aAPCs can be added to the culture at least a second time, for example at about 7 days of expansion.

[0638] 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, which are secreted by NK cells or tethered to the NK cell membrane. In these cases, the membrane-bound cytokines may be tethered to the NK cell membrane through specific transmembrane domains, such as the transmembrane domains of CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, and DAP12.

[0639] After preparation, the modified NK cells can be immediately infused (including an effective amount of one or more monospecific, bispecific or multispecific antibodies), or the NK cells can be stored, for example, by cryopreservation. In some cases, when NK cells are derived from cryopreservation, the NK cells are inactivated by an inactivating agent (e.g., a kinase inhibitor, such as dasatinib, nilotinib, rapamycin, etc.) before cryopreservation. In some aspects, the cells can be ex vivo as a large colony within about 1, 2, 3, 4 or 5 days for several days, weeks or months.

[0640] C. Loading of NK cells

[0641] In certain embodiments, NK cells are loaded with antibodies before use. NK cells can be loaded in any particular manner, including in culture or immediately before infusion, for example, to produce a complex of NK cells and antibodies. Conditions are sufficiently suitable for allowing an effective amount of antibodies to bind to the NK cell surface. In the case of using a monospecific antibody, the Fc region of the monospecific antibody binds to NK cells, and the antigen-binding domains of the monospecific antibody freely bind to its target antigen. In certain embodiments using multispecific antibodies, one or more antigen-binding domains of the antibody can bind to the NK cell surface by antigens (such as but not limited to CD3, NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.) on the NK cell surface, and another antigen-binding domain freely binds to its target antigen. In certain embodiments using multispecific antibodies, one or more antigen-binding domains of the antibody can bind to one or more target antigens.

[0642] The culture conditions that NK cells become loads may or may not be a specific type with one or more specific parameters. In a specific embodiment, the load of NK cells is carried out at a specific temperature, such as 37 ° C, although the temperature is 36 ° C or 38 ° C, or lower or higher in an alternative embodiment. The duration of the load step can be any suitable amount of time, such as in the range of one minute to 24 hours or longer. For example, the scope may be 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, 1-24 hours, 1-18 hours, 1-12 hours, 1-6 hours, 6-24 hours, 6-18 hours, 6-12 hours, 12-24 hours, 12-18 hours or 18-24 hours. In certain embodiments, the duration of the loading step may be greater than or equal to 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 any range derivable therein. In specific embodiments, the cell culture medium is a basal medium or a complex medium. In some cases, the culture includes one or more reagents used during the pre-activation and / or expansion steps, while in other cases, the culture does not include these. In specific embodiments, the culture includes one or more cytokines, including, for example, IL-12, IL-15, IL-2 and IL-18. In certain embodiments, the culture includes any type of APC.

[0643] In certain embodiments, the antibodies of the compositions described herein are subjected to an effective amount of NK cells of the present disclosure, thereby producing a "chimeric antigen receptor-like" complex. Specifically, the antigen binding domain of the antibody binds to the NK cell, for example, through an antigen that is a cell surface protein. Multiple antibodies can bind to multiple NK cells, thereby forming multiple cell / antibody complexes. The antibody can be of any type, including monospecific, bispecific or multispecific, and in certain cases, the antibody simultaneously engages with the NK cell and the target antigen through the antigen binding domain of the antibody (for example, it is a conjugate in the art of a fusion protein composed of a single chain variable fragment (scFv) of two different antibodies). In the case where the antibody is monospecific, the antigen binding domain of the antibody binds to the target antigen (such as a cancer antigen), while another part of the antibody (such as the Fc region of the antibody) binds to the NK cell. In the case where the antibody is multispecific, one or more antigen binding domains of the antibody bind to the NK cell (such as through an NK cell surface antigen, which may be naturally occurring or genetically modified, such as CD3), and one or more antigen binding domains of the antibody bind to one or more target antigens. For example, a multispecific antibody can be bispecific, trispecific or tetraspecific. In the case where the antibody is trispecific or tetraspecific, the additional antigen binding domains can bind to other cells (such as stem cells).

[0644] 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, NKG2C, costimulatory molecules such as CS1, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46 or KIR, and redirect NK cells to the target, thereby increasing the response and specificity for different tumors. In certain embodiments, the antibody can bind to a transgenic NK cell surface antigen (such as CD3).

[0645] In certain embodiments, the antibody may bind to any suitable antigen (e.g., an antigen described herein, such as those described as targets of CAR and / or TCR). In specific embodiments, the antibody targets CD19. In specific embodiments, the antibody targets CD20. In specific embodiments, the antibody targets CD123. In specific embodiments, the antibody targets EGFR. In specific embodiments, the antibody targets EGFR2. In specific embodiments, the antibody targets C-met (tyrosine protein kinase MET, also known as mesenchymal epithelial transition factor). In specific embodiments, the antibody targets EGFR and C-met. In specific embodiments, the antibody targets TROP-2 (tumor-associated calcium signal transducer 2).

[0646] In certain embodiments, the NK cells of load can be generated by any suitable means, so that the conditions that the suitable area of ​​the antibody can be combined with the suitable surface area of ​​the NK cells are sufficient. In some cases, any specific culture medium can be used. In certain cases, Plasma-Lyte A and / or human serum albumin are used, and in other cases, they are not used. After the complex is formed in the culture, they may or may not be washed before being applied to the subject (e.g., by infusion). In alternative embodiments, NK cells and antibodies are applied separately, and the complex is formed in vivo.

[0647] D. Pre-activation

[0648] In certain embodiments, NK cells are preactivated before being administered to a recipient individual. The preactivation step may or may not occur before any amplification step. In a specific embodiment, NK cells are preactivated by one or more cytokines, and in a specific embodiment, NK cells are preactivated by one or more of IL-12, IL-15, IL-2, and IL-18 and include two, three, or more preactivations. In the case of using less than all three of IL-12, IL-15, IL-2, and IL-18, it 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 replace IL-15.

[0649] In certain embodiments, the pre-activation cytokines may be IL-12, IL-15 and IL-18. One or more additional cytokines may be used in the pre-activation step. Pre-activation can last for a short period of time, such as 5-72 hours, such as 10-50 hours, especially 10-20 hours, such as 12, 13, 14, 15, 16, 17, 18, 19 or 20 hours, and in some cases particularly about 16 hours. The pre-activated culture may include IL-18 and / or IL-15 at a concentration of 10-100 ng / mL, such as 40-60 ng / mL, especially 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 ng / mL, particularly about 50 ng / mL. In some cases, the pre-activated culture includes IL-12 at a concentration of 0.1-150 ng / mL, including a concentration of 1-20 ng / mL, such as a concentration of 10 ng / mL. In alternative embodiments, NK cells can be stimulated with IL-2 or other cytokines that bind to a common γ chain (e.g., IL-7, IL-21, etc.), which may be in addition to IL-12, IL-15, and IL-18, or in place of one or more thereof. In this case, the pre-activation culture may include IL-12 at a concentration of 0.1-150 ng / mL, such as 0.5-50 ng / mL, especially 1-20 ng / mL, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ng / mL, especially about 10 ng / mL.

[0650] E. Amplification

[0651] In a specific embodiment, before being administered to an individual in need, NK cells are expanded to increase their number. The expanded cells may or may not be derived from pre-activated NK cells, so that there may be a pre-activation step before the expansion step. The NK cell expansion step can be any suitable to expand the NK cell population, but in specific cases, the expansion step uses one or more specific reagents, such as in culture, to enhance its expansion. In some cases, NK cells may not be expanded. Before or during the expansion, IL-2 or IL-15 or IL-18 or a combination of any cytokines can be added to the expansion culture. In a specific embodiment, NK cells can be expanded in vitro in a culture bottle or in one of several different bioreactor configurations of continuous perfusion culture medium / additives.

[0652] In certain cases, NK cells (whether preactivated or not) can be washed (e.g., with PBS or Plasma Lyte or human serum albumin or culture medium or a combination thereof) before and / or after expansion, for example 1, 2, 3, 4 or 5 times. In certain embodiments, the cells are particularly washed 3 times. In certain embodiments, NK cells are expanded in the presence of artificial antigen presenting cells (aAPC). In certain embodiments, NK cells are expanded in the presence of fragments of aAPC. aAPC can be engineered to express CD137 ligands and / or membrane-bound cytokines. The membrane-bound cytokine can be membrane-bound IL-21 (mIL-21) or membrane-bound IL-15 (mIL-15). In certain embodiments, aAPC is engineered to express CD137 ligands and mIL-21. aAPC can be derived from cancer cells, such as leukemia cells. aAPC may not express endogenous class I, class II HLA or CD1d molecules. They may express ICAM-1 (CD54) and LFA-3 (CD58) or CD48. In particular, aAPC may be K562 cells, such as K562 cells engineered to express CD137 ligands and mIL-21. Engineering can be performed by any known method in the art, such as retroviral transduction, but any viral or non-viral vector can be used. aAPC may or may not be irradiated. Amplification can last for a specific time, such as about 2-30 days, such as 3-20 days, especially 12-16 days, such as 12, 13, 14, 15, 16, 17, 18 or 19 days, especially about 14 days. Preactivated NK cells and aAPC may be present in a ratio of about 3: 1-1: 3, such as 2: 1, 1: 1, 1: 2, especially about 1: 2. The amplification culture may further include one or more cytokines to promote amplification, such as IL-2. IL-2 may be present at a concentration of about 10-500U / mL, such as 100-300U / mL, especially about 200U / mL. IL-2 can be supplemented during expansion culture, including at a specific frequency, such as every 2-3 days. aAPC can be added to the culture at least a second time, such as at about 7 days of expansion. Any cytokine used in the pre-activation and / or expansion steps can be a recombinant human cytokine.

[0653] In certain embodiments, after expansion, NK cells can be used immediately in any manner, such as in complex with one or more antibodies, or they can be stored, such as by cryopreservation. In certain aspects, cells can be proliferated ex vivo as a large population within about 1, 2, 3, 4 or 5 days for several days, weeks or months.

[0654] Activated and / or amplified NK cells can secrete type I cytokines, such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony stimulating factor (GM-CSF), which activate innate and adoptive immune cells, as well as other cytokines and chemokines. The measurement of these cytokines can be used to determine the activation state of NK cells. In addition, other methods known in the art can also be used to determine the activation of NK cells to characterize the NK cells of the present disclosure.

[0655] Therefore, with respect to the specific pre-activation and amplification aspects of the present disclosure, in a specific embodiment, NK cells are pre-activated by any combination of IL-12, IL-15 and / or IL-18, and then amplified with aAPC, such as K562 cells expressing mIL-21 and CD137 ligands, to provide an efficient cell product. Therefore, methods for treating various diseases using the present NK cells are provided, such as immunotherapy for cancer patients. In an exemplary method, the separated NK cells can be subjected to a brief pre-activation period, such as about 16 hours, with a combination of cytokines such as interleukin-12 (IL-12), IL-15 and / or IL-18, and then amplified using artificial antigen presenting cells (aAPC) (e.g., K562 feeder cells expressing membrane-bound IL-21 and CD137 ligands) and / or exogenous IL-2. IL-2 or IL-15 or IL-18 or any combination of cytokines can be added at least a second time in the amplification culture.

[0656] F. Cryopreservation

[0657] In certain cases, the NK cells and / or antibodies of the present disclosure are stored in a cryopreservation medium composition comprising at least one cryoprotectant, serum (human serum or animal serum) or a non-serum substitute for serum (non-human serum 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 can be any kind, including at least platelet lysate and / or blood product lysate (e.g., human serum albumin). In embodiments in which a composition of one or more (including two or more) cytokines is employed, the cytokine can be a natural or recombinant or synthetic protein. At least one of the cytokines can be a cytokine approved by the U.S. Food and Drug Administration (FDA). 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 a combination thereof. For embodiments of serum, the serum can be serum of animal origin, such as human serum (including human AB serum) or bovine serum. DMSO and other cryoprotectants can account for 4-10%, 4-6%, 4-8%, 5-10%, 5-8%, 6-10%, 6-8%, 8-10%, etc. of the composition when used.For embodiments in which serum is used, serum may comprise 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%, 5- 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%. The composition may comprise at least or no more than 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% serum.In certain embodiments, the platelet lysate included in the composition can be present in any concentration in the composition, but in certain embodiments, the platelet lysate comprises 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%, 10-55%, 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%, 5 0-90%, 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%. The composition may comprise at least or no more than 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% platelet lysate.

[0658] Said composition may have certain components of specific concentration, including cytokines and / or growth factors. In a specific case, for example, any cytokine, including IL-2, IL-21 and / or IL-15, is present in said composition with a specific concentration. IL-2 can be present in said composition with a concentration of 1-5000, 1-1000, 1-500, 1-100, 100-5000, 100-500, 500-5000, 500-1000 or 1000-5000 U / mL. For example, in a specific case, IL-2 is present in said composition with a concentration of at least or no more than 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 U / mL. In specific 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 can be present in the composition at a concentration of at least or no more than 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. IL-15 can 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 or no more than 10, 50, 100, 500, 1000, 1500 or 2000 ng / mL.

[0659] Compositions included herein comprising at least one cryoprotectant, serum or non-serum substitutes for serum, and at least one cytokine and / or at least one growth factor may further include multiple immune cells and / or stem cells, any type of which is acceptable. In a specific embodiment, the cell is a NK cell, a T cell, a B cell, a NKT cell, which is derived from mature bone marrow or peripheral blood cells, a cell line such as a tumor cell line (e.g., NK92 or other NK cell lines), a hematopoietic stem cell, an induced pluripotent stem cell, a MSC (a cell group also referred to as "mesenchymal stem cells" and "mesenchymal stromal cells" in the literature) or a mixture thereof, which may be derived from bone marrow, peripheral blood, skin, adipose tissue or a combination thereof. In an embodiment in which NK cells are used, the NK cells may be amplified NK cells or may not be. Embodiments of the present disclosure also include pharmaceutical compositions comprising any composition of the present disclosure and a suitable pharmaceutically acceptable carrier.

[0660] In certain embodiments, cells and / or antibodies are treated with one or more inactivating agents (eg, kinase inhibitors, such as dasatinib, nilotinib, rapamycin, etc.) prior to cryopreservation.

[0661] In certain embodiments, the technology described herein includes inactivating NK cells, including treating NK cells with an effective amount of one or more inactivators under conditions that produce inactivated NK cells. In certain embodiments, the inactivator is a kinase inhibitor. In certain embodiments, the inactivator is a rapamycin mechanism target (mTOR) inhibitor. In certain embodiments, the mTOR inhibitor is rapamycin, everolimus and / or tesilolimus. In certain embodiments, the mTOR inhibitor is rapamycin. In certain embodiments, the inactivator is a tyrosine kinase (TK) inhibitor. In certain embodiments, the TK inhibitor is lorlatinib, brigatinib, ceritinib, alectinib, crizotinib, bosutinib, ponatinib, nilotinib, dasatinib, imatinib, zanubrutinib, ecalvabrutinib, ibrutinib, capmatinib, pegidatinib, dacomitinib, osimertinib, erlotinib, gefitinib, lapatinib, afatinib, pemigatinib, erdatinib, nidartinib, gefitinib, 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 and / or nilotinib. In some embodiments, the TK inhibitor is dasatinib.

[0662] In certain embodiments, the treatment with an inactivator is carried out at any time point during the NK cell culture. In certain embodiments, the treatment time is about 24 hours to about 96 hours, about 36 hours to about 84 hours, or about 48 hours to about 72 hours. In certain embodiments, the treatment time is about 24 hours, about 48 hours or about 72 hours. In certain embodiments, the NK cell inactivator is treated with a concentration of about 1 to about 1000nM. In certain embodiments, the NK cell inactivator is treated with a concentration of about 5 to about 500nM. In certain embodiments, the NK cell inactivator is treated with a concentration of about 20 to about 200nM. In certain embodiments, the NK cell inactivator is treated with a concentration of about 30 to about 100nM. In certain 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 relative to activated NK cells.

[0663] In certain embodiments, the technology described herein includes a method for maintaining a viability of a cell colony greater than at least 50% after cryopreservation of a colony, comprising the steps of: exposing the colony to an effective amount of one or more inactivating agents (e.g., tyrosine kinase inhibitors) to inactivate cells before cryopreservation, cryopreserving cells, and thawing the colony, wherein the viability of the colony after thawing exceeds at least 50%. In some cases, after the cells are thawed, the viability of the cell colony exceeds at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% after the cryopreservation of the colony.

[0664] III. Heterologous Proteins and Mutations

[0665] In a specific embodiment, the NK cells are modified not only to express one or more components of the TCR / CD3 complex, but also to express one or more other heterologous proteins. The heterologous proteins can promote the activity of NK cells in any way, including at least their activation, persistence, expansion, homing and / or cytotoxicity.

[0666] A. Monospecific, bispecific or multispecific antibodies

[0667] In certain embodiments, NK cells are modified to express one or more monospecific, bispecific, or multispecific antibodies, although in other cases, NK cells do not express these antibodies, but these antibodies are used in conjunction with NK cells.

[0668] In the case where NK cells are modified to express antibodies, the antibodies may be conjugates that bridge specific immune effector cells and specific target cells to destroy target cells. The present disclosure allows the use of modified NK cells with standard T cell conjugates (BiTEs) because they have been modified to express CD3, which is a T cell antigen that the BiTE conjugate binds in many cases. In such cases, the BiTEs used in the present invention can also target cancer or viral antigens, which can be customized according to the medical condition of the intended recipient individual. For example, BiTEs can be customized to cancer antigens that bind to cancer cell features of individual cancers. The anti-CD3 antibodies of BiTEs can target CD3γ chains, CD3δ chains, CD3ε chains, or CD3ζ chains.

[0669] In some cases, in addition to expressing the CD3 complex (with or without TCR) that allows NK cells to be used as a therapy with BiTEs, NK cells can also be modified to express (or not express, but used in combination with) one or more bispecific NK engagers (BiKEs). BiKEs contain antibodies that bind to surface proteins on NK cells, including surface proteins naturally expressed on NK cells (such as, but not limited to, NKp30, NKp44, NKp46, CD16, CD32, CD64, KIR, etc.), and also contain antibodies that bind to desired target antigens. BiKEs can target NK cells through antibodies to NK surface proteins (such as CD16, CS1, CD32, CD64, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR, NKp30, NKp44, NKp46, or KIR). In such cases, the BiKE used in the present invention can also target cancer or viral antigens, which can be customized according to the medical condition of the intended recipient individual. For example, a BiKE can be customized to bind cancer antigens that are characteristic of cancer cells of an individual's cancer.

[0670] 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 epcoritamab. In certain embodiments, the antibody is flotetuzumab. In certain embodiments, the antibody is APV0436. In certain embodiments, the antibody is TNB383B. In certain embodiments of the use of multispecific antibodies, one or more antigen binding domains of the antibody may bind to one or more target antigens. In certain embodiments, the antibody is ervantamab. In certain embodiments, the antibody is cetuximab. In certain embodiments, the antibody is imatulumab.

[0671] In embodiments where the NK cells express a CD3 complex (with or without a TCR) and one or more BiKEs, the CD3 complex components (with or without a TCR) and one or more BiKEs may be transfected or transduced into the cells using one or more vectors. In some cases, one or more CD3 complex components (with or without a TCR) and the BiKE may or may not be on the same polycistronic vector.

[0672] B. Engineered Receptors

[0673] In certain embodiments, NK cells are engineered to express one or more engineered receptors. In some cases, the engineered receptor is an engineered antigen receptor that targets any type of cancer or viral antigen. The receptor can be customized to target the desired antigen according to the medical condition of the intended recipient individual.

[0674] In certain embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). NK cells can be modified to encode at least one CAR, for example, the CAR can be the first generation, the second generation or the third generation or subsequent generations. CAR may be or may not be bispecific, targeting two or more different antigens. CAR may include one or more costimulatory domains. Each costimulatory domain may include, for example, TNFR superfamily members, CD28, CD137 (4-1BB), CD134 (OX40), DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD27, NKG2D, 2B4M, CD40 or any one or more of its combinations. In a specific embodiment, CAR includes CD3ζ. In certain embodiments, CAR lacks one or more specific costimulatory domains; for example, CAR may lack 4-1BB and / or lack CD28.

[0675] In a specific embodiment, the CAR polypeptide in the cell includes an extracellular spacer domain that connects the antigen binding domain to the transmembrane domain, and this can be referred to as a hinge. The extracellular spacer domain may include, 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, CD8-α hinges, CD28, artificial spacers made of polypeptides such as Gly3, or CH1, CH3 domains of IgG (such as human IgG1 or IgG4). In certain cases, the extracellular spacer domain may include (i) hinge, CH2, and CH3 regions of IgG4, (ii) hinge regions of IgG4, (iii) hinge and CH2 of IgG4, (iv) hinge regions of CD8-α or CD4, (v) hinge, CH2, and CH3 regions of IgG1, (vi) hinge regions of IgG1 or (vii) hinge and CH2 of IgG1, (viii) hinge regions of CD28, or a combination thereof. In specific embodiments, the hinge is from IgG1, and in certain aspects, the CAR polypeptide comprises a specific IgG1 hinge amino acid sequence or is encoded by a specific IgG1 hinge nucleic acid sequence.

[0676] The transmembrane domain in CAR can be derived from natural sources or from synthetic sources. In some aspects, the domain of natural origin is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., including at least one of the following transmembrane regions) α chains, β chains or ζ chains of T cell receptors, CD28, CD3ζ, CD3ε, CD3γ, CD3δ, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D and DAP molecules, such as DAP10 or DAP12. Alternatively, in certain embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly includes hydrophobic residues such as leucine and valine. In some aspects, each end of the synthetic transmembrane domain may have a triplet of phenylalanine, tryptophan and valine.

[0677] In certain embodiments, the engineered receptor utilizes one or more homing receptors (which can home to a target, not necessarily due to signal release, such as in the case of utilizing adhesion molecules) and / or one or more chemokine receptors. Examples of chemokine receptors include CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors. In certain instances, the chemokine receptor is a receptor for CCR2, CCR3, CCR5, CCR8, CCR7, CXCR3, L-selectin (CD62L), CXCR1, CXCR2, or CX3CR1.

[0678] C. Cytokines

[0679] In certain embodiments, the expressed cell NK cells are engineered to express one or more heterologous cytokines and / or are engineered to upregulate the normal expression of one or more heterologous cytokines. These 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.

[0680] One or more cytokines can be co-expressed from a vector, including separate polypeptides as any component of the TCR / CD3 complex. For example, interleukin-15 (IL-15) is tissue-restricted and is only observed at any level in serum or systemically under pathological conditions. IL-15 has a variety of properties suitable for adoptive therapy. IL-15 is a homeostatic cytokine that can induce the development and cell proliferation of natural killer cells, promote the eradication of established tumors by relieving the functional inhibition of tumor-resident cells, and inhibit activation-induced cell death (AICD). In addition to IL-15, other cytokines are also considered. These include, but are not limited to, cytokines, chemokines, and other molecules that contribute to cell activation and proliferation for human applications. NK cells expressing IL-15 can continue to provide supportive cytokine signaling, which is useful for their survival after infusion.

[0681] In some embodiments, the cell expresses one or more exogenously provided engineered receptors, wherein the engineered receptors include chemokine receptors and / or cytokine receptors. In some embodiments, the cytokine receptor is an IL-15 receptor. In some embodiments, the cytokine receptor is a non-natural 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.

[0682] In a specific embodiment, the cell expresses one or more exogenous cytokines provided. As an example, the cytokine is IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, GMCSF, or a combination thereof. The cytokine can be provided to NK cells exogenously because it is expressed from an intracellular expression vector. In another case, by manipulating the regulation of endogenous cytokine expression, for example, genetic recombination is carried out at the promoter site of the cytokine, and the endogenous cytokine in the cell is upregulated. In the case where the cytokine is provided to the cell by an expression construct, the cytokine can be encoded from the same vector of one or more components of the CD3 complex (without or with the TCR complex).

[0683] In some embodiments, a specific sequence of IL-15 is used, such as the following (the signal peptide sequence is underlined):

[0684] ATGCGCATTAGCAAGCCCCACCTGCGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAG CCACTTCCTGACCGAGGCCGGCATCCACGTGTTCATCCTGGGCTGCTTCAGCGCCGGACTGCCCAAGACCGAGGCCAACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGACGTGCACCCCAGCTGCAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAACTGCAG GTGATCAGCCTGGAAAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAACGTGACCGAGAGCGGCTGCAAAGAGTGCGAGGAACTGGAAGAGAAGAACATCAAAGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGC(SEQ ID NO:49)

[0685] MRISKPHLRSISIQCYLCLLLNSHFLTEA GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:48)

[0686] D. Antigen

[0687] The modified NK cells of the present disclosure are used for use with monospecific, bispecific or multispecific antibodies targeting one or more specific antigens. In addition, NK cells can be modified by engineered antigen receptors to target one or more specific antigens. In the case where the NK cells are modified by one or more engineered antigen receptors, the antigen targeted by the monospecific, bispecific or multispecific antibody and the antigen targeted by one or more engineered antigen receptors may be the same antigen or may not be the same antigen. In some cases, the antigen targeted by the monospecific, bispecific or multispecific antibody and the antigen targeted by one or more engineered antigen receptors are different antigens, but are associated with the same type of cancer. In some embodiments, the antigen targeted by the monospecific, bispecific or multispecific antibody and the antigen targeted by one or more engineered antigen receptors are different antigens, but each is associated with a solid tumor.

[0688] Antigens targeted by antibodies and / or engineered antigen receptors include those antigens expressed in the context of diseases, conditions or cell types targeted by adoptive cell therapy. These diseases and conditions include proliferative, neoplastic and malignant diseases and disorders, including cancer and tumors, including hematological cancers, immune system cancers, such as lymphomas, leukemias and / or myeloma, such as B, T and myeloid leukemias, lymphomas and multiple myeloma. In some embodiments, relative to normal or non-targeted cells or tissues, antigens are selectively expressed or overexpressed on disease or condition cells (such as tumors or pathogenic cells). In other embodiments, antigens are expressed on normal cells and / or expressed on engineered cells.

[0689] In some embodiments, the antigen can be presented in the context of an HLA protein. In some embodiments, the antigen can be presented in the context of an HLA-Cw0802 protein. In some embodiments, the target cells expressing the antigen of interest can be transgenic to express the antigen, transgenic to express a protein required for antigen presentation, and / or loaded with the antigen. In some embodiments, target cells are transduced with an HLA-Cw0802 and / or b2m construct comprising a polynucleotide 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: 318-323, or encoding 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: 318-323. In some embodiments, the target cells are loaded with peptides comprising the epitopes GADGVGKSA (SEQ ID NO: 293) and / or GADGVGKSAL (SEQ ID NO: 294).

[0690]

[0691] ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATG(SEQ ID NO:319)

[0692]

[0693] MRVMAPRTLILLLSGALALTETWACSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNQFAYDGKDYIALNEDLRSWTAADKAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKKTLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWGPSSQPTIPIVGIVAGLAVLAVLAVLGAVMAVVMCRRKSSGGKGGSCSQAASSNSAQGSDESLIACKA(SEQ IDNO:321)

[0694] MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:322)

[0695] MRVMAPRTLILLLSGALALTETWACSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNQFAYDGKDYIALNEDLRSWTAADKAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKKTLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWGPSSQPTIPIVGIVAGLAVLAVLAVLGAVMAVVMCRRKSSGGKGGSCSQAASSNSAQGSDESLIACKAATNFSLLKQAGDVEENPGPMSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID NO:323)

[0696] In the present method, any suitable antigen may be targeted. In some cases, the antigen may be associated with certain cancer cells but not with non-cancerous cells. Exemplary antigens include, but are not limited to, antigen molecules from infectious pathogens, self / autoantigens, tumor / cancer-associated antigens, and tumor neoantigens (Linnemann et al., 2015). In certain aspects,Antigens include KRAS, TROP-2, NY-ESO, CD19, EBNA, CD123, HER2, CA-125, TRAIL / DR4, CD20, CD22, CD70, CD38, CD123, CLL1, carcinoembryonic antigen, alpha-fetoprotein, CD56, AKT, Her3, epithelial tumor antigen, CD319 (CS1), ROR1, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, CD5, CD23, CD30, HER V-K, IL-11Rα, kappa chain, lambda chain, CSPG4, CD33, CD47, CLL-1, U5snRNP200, CD200, BAFF-R, BCMA, CD99, p53, mutant p53, Ras, mutant ras, c-Myc, cytoplasmic serine / threonine kinases (e.g., A-Raf, B-Raf and C-Raf, cyclin-dependent kinases), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A1 0, 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, phosphatidylinositol 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)) (in specific embodiments,EGFRvIII), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), VEGFR2, cytoplasmic tyrosine kinases (e.g., src family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducer and activator 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 its regulatory subunits, PMSA, PR-3, MDM2, mesothelin, renal cell carcinoma-5T4, SM22-α, 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, fucose GM1, mesothelin, 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, XAGE 1, 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 sequences of antigens are known in the art, for example, in, In the database: KRAS (accession number NC_000012.12), 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), HER 2 (accession number NG_007503.1), CA-125 (accession number NG_055257.1), WT1 (accession number NG_009272.1), Mage-A3 (accession number NG_013244.1), Mage-A4 (accession number NG_013245.1), Mage-A10 (accession number NC_000023.11), TRAIL / DR4 (accession number NC_000003.12) and / or CEA (accession number NC_000019.10).

[0697] As an example, tumor-associated antigens may be derived from prostate, breast, colorectal, lung, pancreas, kidney, mesothelioma, ovary, liver, brain, bone, stomach, spleen, testis, cervix, anus, gallbladder, thyroid or melanoma cancer. Exemplary tumor-associated antigens or tumor cell-derived antigens include MAGE 1, 3 and MAGE 4 (or other MAGE antigens, such as those disclosed in International Patent Publication No. WO 99 / 40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); KRAS (such as those disclosed in U.S. Patent No. 10,611,816); 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 phosphatase, NKX3.1, and six transmembrane epithelial antigen of the prostate (STEAP).

[0698] Other tumor-associated antigens include Plu-1, HASH-1, HasH-2, Cripto, and Criptin. In addition, tumor antigens can be self-peptide hormones, such as full-length gonadotropin-releasing hormone (GnRH), a short peptide of 10 amino acids in length, which is suitable for the treatment of many cancers.

[0699] Antigens may include epitope regions or epitope peptides derived from genes that are mutated in tumor cells or genes that are transcribed at different levels in tumor cells compared to normal cells, such as telomerase, survivin, mesothelin, mutated ras, mutated KRAS, bcr / abl rearrangement, Her2 / neu, mutant or wild-type p53, cytochrome P450 1B1, and abnormally expressed intronic sequences such as N-acetylglucosamine transferase-V; clonal rearrangements of immunoglobulin genes that produce unique idiotypes in myeloma and B-cell lymphoma; tumor antigens including epitope regions or epitope peptides derived from tumor virus processes, such as human papillomavirus proteins E6 and E7; Epstein Barr virus protein LMP2; non-mutated oncofetal proteins with tumor-selective expression, such as carcinoembryonic antigen and alpha-fetoprotein.

[0700] E. Suicide gene

[0701] In certain embodiments, suicide genes are used in conjunction with NK cell therapy to control its use and allow cell therapy to be terminated at a desired event and / or time. Suicide genes are used to transduce cells so as to cause the death of transduced cells when needed. The cells of the present disclosure have been modified to carry one or more vectors encompassed by the present disclosure, which may contain one or more suicide genes. In certain embodiments, the term "suicide gene" as used herein is defined as a gene that, after administration of a prodrug or other agent, converts a gene product into a compound that kills its host cell. In other embodiments, the suicide gene encodes a gene product that, when needed, is targeted by an agent (such as an antibody) that targets the suicide gene product.

[0702] In some cases, cell therapy may use one or more suicide genes of any type when the individual receiving the cell therapy and / or who has received the cell therapy exhibits or is considered at risk of, including imminent, one or more symptoms of one or more adverse events (e.g., cytokine release syndrome, neurotoxicity, allergic reaction / allergy, and / or on-target / off-target tumor toxicity (as examples)). The use of a suicide gene may be part of a planned regimen or may be used only when there is a recognized need for its use. In some cases, cell therapy is terminated by using an agent that targets a suicide gene or its gene product because therapy is no longer needed.

[0703] The use of suicide genes may be initiated when an individual experiences at least one adverse event, and the adverse event may be identified by any means, including routine monitoring (which may be continuous or discontinuous) from the beginning of the cell therapy. Adverse events may be detected during examination and / or testing. For example, in cases where an individual suffers from cytokine release syndrome (also known as cytokine storm), the individual may have elevated inflammatory cytokines (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 leakage; heart / renal / liver dysfunction; or a combination thereof. In cases where an individual has neurotoxicity, the individual may experience confusion, delirium, dysplasia, and / or seizures. In some cases, an 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.

[0704] Examples of suicide genes include engineered non-secretable (including membrane-bound) tumor necrosis factor (TNF)-α mutant polypeptides (see PCT / US19 / 62009, which is incorporated herein by reference in its entirety), and these can be affected by delivering antibodies that bind to TNF-α mutants. Examples of suicide gene / prodrug combinations that may be used are herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir or FIAU; oxidoreductase and cyclohexamide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidylate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytarabine. Escherichia coli (E. coli) purine nucleoside phosphorylase (a so-called suicide gene that converts the prodrug 6-methylpurine deoxynucleoside to the toxic purine 6-methylpurine) can also be used. As examples, other suicide genes include CD20, CD52, inducible caspase 9, purine nucleoside phosphorylase (PNP), cytochrome P450 enzymes (CYP), carboxypeptidase (CP), carboxylesterase (CE), nitroreductase (NTR), guanine nucleoside transferase (XGRTP), glycosidase, methionine-α,γ-lyase (MET), EGFRv3 and thymidine phosphorylase (TP).

[0705] F. Knockout or knockdown of endogenous genes

[0706] In certain embodiments, the NK cells of the present disclosure may include gene editing of NK cells to remove 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more endogenous genes in NK cells. In some cases, gene editing occurs in NK cells expressing one or more heterologous transgenes (e.g., CD3, TCR, etc.), while in other cases, gene editing occurs in NK cells that do not express heterologous transgenes but will eventually express one or more heterologous transgenes. In a specific embodiment, the gene-edited NK cells are amplified NK cells.

[0707] In certain cases, one or more endogenous genes of NK cells are modified, for example, by reducing some or all of the expression to disrupt the expression. In specific cases, one or more genes are knocked down or knocked out using the methods of the present disclosure. In specific cases, multiple genes are knocked down or knocked out in the same steps as the methods of the present disclosure. The genes in the edited NK cells can be of any type, but in specific embodiments, these genes are genes whose gene products inhibit the activity and / or proliferation of NK cells. In specific cases, the genes in the edited NK cells enable the NK cells to work more effectively in the tumor microenvironment. In a specific case, these genes are 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 knocked down in NK cells. In a specific embodiment, the CISH gene is knocked out or knocked down in NK cells. In a specific embodiment, the CD38 gene is knocked out or knocked down in NK cells.

[0708] In some embodiments, gene editing is performed using one or more DNA binding nucleic acids, such as changes made by RNA-guided endonucleases (RGENs). For example, clustered regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins can be used for changes. In general, the term "CRISPR system" generally includes transcripts and other elements that involve the expression of CRISPR-associated ("Cas") genes or guide their activity, including sequences encoding Cas genes, tracr (transactivation CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (partial direct repeats containing "direct repeats" and tracrRNA treatments in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacer sequences" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from CRISPR loci. Methods utilizing CRISPR systems are well known in the art.

[0709] IV. Administration of Therapeutic Compositions

[0710] NK cells expressing CD3 and monospecific, bispecific or multispecific antibodies are administered to individuals in need thereof, including enabling the anti-CD3 antibody of the bispecific or multispecific antibody to bind to CD3 on NK cells expressing CD3 in such a close manner. In some cases, the two components are administered separately to individuals, and in other cases, the two components are compounded together before administration, such as in an ex vivo manner. In another embodiment, NK cells express antibodies. In some cases, the two components are not pre-compounded before administration, but are administered together by any suitable route of administration, such as by co-infusion to the patient.

[0711] Embodiments of the present disclosure relate to methods for treating or preventing medical diseases or disorders using compositions comprising NK cells and antibodies provided herein. The method includes administering to a subject a therapeutically effective amount of NK cells and antibodies modified through CD3 (± TCR), thereby treating or preventing the subject's disease, including reducing the risk of the disease, alleviating the severity of the disease and / or delaying the occurrence of the disease. In certain embodiments of the present disclosure, cancer or infection is treated by transferring a composition comprising an NK cell colony and corresponding antibodies. In at least some cases, due to its release of proinflammatory cytokines, NK cells can reverse the anti-inflammatory tumor microenvironment, and increase adoptive immune response by promoting differentiation, activation and / or recruitment of auxiliary immune cells to malignant sites. In certain embodiments, providing a step may include culturing NK cells with antibody molecules for a specific duration (e.g., from about 5 minutes to about 24 hours or longer), and storing NK cells and antibody molecules for a period of time (e.g., about 1 day, 2 days, 3 days, 4 days, 5 days or more than 5 days) before infusion / administration.

[0712] The cancer useful for the present treatment method includes any malignant cell type, such as those found in solid tumors or hematological tumors. Exemplary solid tumors can include, but are not limited to, tumors selected from the following organs: pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast. Exemplary hematological tumors include myeloid tumors, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma, etc. Further examples of cancers that can 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), peritoneal cancer, gastric cancer (gastric or stomach cancer) (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (kidney or renal cancer), prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer and melanoma. In certain embodiments, cancer is solid tumor cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is a cancer associated with a KRAS positive tumor.

[0713] Cancers may specifically be of the following histologic types, although they are not limited to these: malignant neoplasms; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in the form of adenomatous polyps; adenocarcinoma in familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-cystic sclerosing carcinoma; adrenal gland cortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine gland carcinoma; sebaceous gland carcinoma; cerumen gland carcinoma; 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; acinar cell carcinoma; adenosquamous carcinoma; squamous metaplastic adenocarcinoma; malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant androgenic tumor; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant Lentigo melanoma; acral lentigo melanoma; nodular melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; mesenchymal sarcoma; malignant mixed tumor; mixed tumor of Müllerian duct; Wilms tumor; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroblastoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphoma Basal sarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontogenic sarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astrocytoblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease;Hodgkin's disease; paragranuloma; small lymphocytic lymphoma; large cell diffuse lymphoma; follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; 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-cleaved cell NHL; giant lesion NHL; mantle cell lymphoma; AIDS-related lymphoma ; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphoid leukemia; plasma cell leukemia; erythroid leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloid leukemia. ;

[0714] The treatment methods provided herein may include administering a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. Therapy can be administered in any appropriate manner known in the art. For example, the first cancer therapy and the second cancer therapy can be administered sequentially (at different times) or simultaneously (at the same time). In certain embodiments, the first cancer therapy and the second cancer therapy are administered in separate compositions. In certain embodiments, the first cancer therapy and the second cancer therapy are in the same composition. Embodiments of the present disclosure relate to compositions and methods including therapeutic compositions. Different therapies can be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions or 4 compositions. Various combinations of agents can be used. Other therapies in addition to the therapies of the present disclosure include surgery, chemotherapy, drug therapy, radiotherapy, hormone therapy, immunotherapy (different from the immunotherapy of the present disclosure) or a combination thereof.

[0715] The therapeutic agent of the present disclosure can be administered by the same route of administration or different routes of administration. In certain embodiments, cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly or intranasally. In certain embodiments, antibiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly or intranasally. Appropriate dosage can be determined according to the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual and the response to treatment and the judgment of the attending physician.

[0716] Treatment may include various "unit doses". A unit dose is defined as a therapeutic composition containing a predetermined amount. The amount to be administered, as well as the specific route and formulation, is within the judgment of professionals in the clinical field. A unit dose need not be administered as a single injection, but may include continuous infusion over a set time. In certain embodiments, a unit dose includes a single administrable dose.

[0717] Depending on the desired therapeutic effect, the amount to be administered (both in terms of the number of treatments and the unit dose) depends on the desired therapeutic effect. An effective dose is understood to refer to the amount necessary to achieve a specific effect. In the implementation of certain embodiments, it is contemplated that doses within the range of 10 mg / kg to 200 mg / kg can affect the protective ability of these agents. Thus, dosages of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any derivable range therein, are contemplated. In addition, these doses may be administered multiple times a day, and / or administered over multiple days, weeks, or months.

[0718] In certain embodiments, the effective dose of the pharmaceutical composition is a dose that can provide a blood concentration of about 1 μM to 150 μM. In another embodiment, the effective dose provides a blood concentration 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 derivable range therein). In other embodiments, the dosage can provide a blood concentration of about, at least about, 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 47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100 μM or any derivable range therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in vivo to a metabolized therapeutic agent, in which case the blood concentration may refer to the amount of the metabolized therapeutic agent. Alternatively, in the case where the therapeutic agent is not metabolized in the subject, the blood concentration discussed herein may refer to the unmetabolized therapeutic agent.

[0719] The exact dosage of the therapeutic composition also depends on the judgment of the clinician and is unique to each individual. Factors affecting dosage include the patient's physiological and clinical state, the route of administration, the intended goal of treatment (relieving symptoms or curing), the efficacy, stability and toxicity of the specific therapeutic substance, or other therapies that the subject may be undergoing.

[0720] Those skilled in the art will understand and know that dosage units of μg / kg or mg / kg body weight can be converted and expressed as comparable concentration units of μg / ml or mM (blood concentration), for example 4 μM to 100 μM. It should also be understood that absorption is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions about absorption and concentration measurements are well known, which will enable those skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions about the dosages, effects and results described herein.

[0721] V. Kit

[0722] Certain aspects of the present disclosure also relate to a kit comprising a composition of the present invention or a composition for implementing the method of the present invention. In a specific embodiment, the kit includes NK cells, which may be fresh or frozen, and may have been pre-activated or amplified. The NK cells may or may not have expressed one or more components of the TCR / CD3 complex. In the case where the NK cells do not yet express one or more components of the TCR / CD3 complex, the kit may include reagents for corresponding transfection or transduction of NK cells, including vectors expressing the components, reagents for amplifying primers of the components, etc. In some cases, the NK cells may or may not have expressed one or more heterologous proteins defined herein, and if not yet expressed, the kit may include vectors expressing heterologous proteins, primers for amplifying heterologous proteins, etc.

[0723] The kit may include components packaged or placed in containers such as tubes, bottles, vials, syringes or other suitable container devices. Individual components may also be provided in concentrated amounts in the kit; in certain embodiments, the components are provided individually at the same concentration as they are in solution with the other components. The concentration of the components may be provided at 1x, 2x, 5x, 10x or 20x or higher. VI. Examples

[0724] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered that can be well implemented in the present invention, and therefore can be considered to constitute preferred modes for its implementation. However, it will be appreciated by those skilled in the art that, in view of this disclosure, many changes may be made to the disclosed specific embodiments, and still the same or similar results may be obtained without departing from the spirit and scope of the present invention.

[0725] Example 1

[0726] Preparation and effective use of NK cells expressing CD3

[0727] This embodiment relates to cancer immunotherapy, as a strategy to redirect the specificity of NK cells against one or more target antigens by "arming" or pre-complexing NK cells with bispecific or multispecific antibodies, for example, before infusion or by co-infusion of the two products separately. NK cells are transduced with one or more CD3 chains, including CD3ζ, CD3γ, CD3δ and CD3ε chains, and can be from any source. Cells can be amplified or non-amplified, they can be pre-activated by one or more inflammatory cytokines (such as IL12 / 15 / 18), and / or they 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 homing / chemokine receptors.

[0728] Figure 1A and 1B Various embodiments of NK cells are illustrated, which are engineered for use with bispecific or multispecific antibodies. Figure 1A As shown, in the first generation of NK cells, these cells are engineered to express CD3, which can be activated by bispecific or multispecific antibodies, including bispecific T cell conjugates (BiTEs) comprising anti-CD3 antibodies, which bind to heterologous CD3 expressed on the surface of NK cells. In another embodiment, NK cells expressing CD3 can be bound by BiTEs comprising anti-CD3 antibodies, and these NK cells also express one or more specific cytokines (e.g., IL-15 and / or IL-21), thereby resulting in enhanced efficacy and effectiveness particularly suitable for treating solid tumors. In another embodiment, NK cells are not only engineered to express CD3 so that they can be activated by BiTEs comprising anti-CD3 antibodies, but are also used for use with bispecific or multispecific antibodies (e.g., bispecific NK cell conjugates or BiKEs) comprising antibodies, which can bind to surface antigens naturally present on NK cells, such as CD16, CS1, CD56, NKG2D, NKG2C, DNAM, 2B4, CD2, NCR or KIR. In this way, NK cells react to both NK conjugates and T cell conjugates. In another embodiment, in addition to expressing CD3 to bind a T cell engager, the NK cells also express an engineered antigen receptor, such as a CAR or an engineered TCR.

[0729] Figure 1BVarious embodiments are illustrated in which NK cells are modified to express both CD3 and TCR. On the right, a T cell TCR is illustrated as having α and β chains with antigen binding sites, wherein the TCR is complexed with CD3ζ to achieve signal transduction. The T cell TCR is co-complexed with two CD3ε chains, one CD3δ chain, and one CD3γ chain. In certain embodiments, the NK cell expresses a TCR in which one or more of the cytoplasmic domains of any of the CD3 molecules is a heterologous intracellular domain, such as an intracellular domain from CD16, NKG2D, DAP10, DAP12, NCR, and DNAM-1. As Figure 1B As shown on the left, NK cells are configured to express a CD3 co-receptor component, which in one example is CD3ε. In this case, standard BiTEs (top left, which contain antibodies against tumor antigens and antibodies against CD3) that are typically used with T cells that naturally express CD3 can be used with NK cells that express CD3. In this specific example, the NK cells express a polypeptide containing the extracellular domain of CD3ε (although the extracellular domains of other CD3 components can also be used), and the extracellular domain of CD3ε is connected to the transmembrane domain and / or cytoplasmic domain of another molecule, such as the transmembrane domain and / or cytoplasmic domain of CD3ζ, CD16, NKG2D, DAP10, DAP12, NCR, or DNAM-1.

[0730] As mentioned above, Figure 1C Schematic depiction of the generation of surface expressed single chimeric CD3 constructs that can be used with anti-CD3 BiTEs. As an example, the CD3ε extracellular domain (ECD) is fused to the CD28, CD16, or NKG2D transmembrane domain (TM) and the CD28, CD16, or NKG2D intracellular domain (ICD) with or without the CD3ζ and / or DAP10 intracellular domains. In one example, these constructs are contained within a Moloney murine virus-derived SFG retroviral vector backbone and can be used for viral production along with packaging plasmids. Figure 1C In cases where a CD3-BiTE is used with such a construct, the antibody will accordingly bind to the extracellular domain of CD3ε.

[0731] Embodiments of the present disclosure utilize part or all of the CD3 receptor complex. Figure 2A and 2BAs shown, NK cells can be transfected or transduced with full-length CD3ζ, CD3γ, CD3δ and CD3ε. In such a case, each full-length CD3ζ, CD3γ, CD3δ and CD3ε includes an extracellular domain, a transmembrane domain and an intracellular domain. When different components of the receptor are expressed from the same vector, they can be configured to produce separate polypeptides, such as using IRES or 2A elements. In any case, any expression construct can be configured to express one or more cytokines, including at least IL-15.

[0732] Figure 4 Shown is the CD3 expression on NK cells at day 4 after CMV TCR complex transduction. The figure provides a FACS graph of CD3 expression on NK cells 4 days after CMV TCR complex transduction. Untransduced (NT) NK cells (CD56+CD3-) are used as negative controls, and T cells (CD3+CD56-) are used as positive controls.

[0733] Figure 5 The TCR expression on NK cells at day 4 after CMV TCR complex transduction of NK is shown. Specifically, a FACS graph showing TCRα / β expression on NK cells 4 days after CMV TCR complex transduction is provided. Untransduced (NT) NK cells (CD56+CD3-TCRa / b-) were used as negative controls, and T cells (CD3+TCRa / b+CD56-) were used as positive controls.

[0734] Figure 6 The TCR / CD3 expression on NK cells at day 6 after CMV TCR complex transduction is shown. Specifically, the FACS graph shows dual CD3 and TCRa / b expression on NK cells 6 days after CMV TCR complex transduction. Untransduced (NT) NK cells (CD56+CD3-TCRa / b-) were used as negative controls, and T cells (CD3+TCRa / b+CD56-) were used as positive controls.

[0735] exist Figure 7 In the figure, the binding of CD3-CD19BiTE on NK cells at different concentrations by CD3 / TCR is shown. Specifically, various cells (non-transduced (NT) NK cells, T cells or three different NK-TCR cells) were incubated with two different concentrations (0.5μg / μl or 4μg / μl) of blinatumomab CD3-CD19 bispecific conjugate (BiTe) at 37°C for 1 hour. Then, at room temperature, biotin-labeled CD19 antigen (from Miltenyi Biotech TMThe cells were then incubated with the CD19 CAR detection reagent (CD19 CAR detection reagent) for 20 minutes, followed by the addition of anti-biotin antibody for 15 minutes. This strategy was used to detect any BiTe that engaged CD3+ cells. Figure 7 The histogram in shows the level of CD19 bound to the CD3-CD19 bispecific engager (BiTe), which correlates with CD3 expression on NK-TCR and T cells.

[0736] Figure 8 The production of NK-TCR cytokines after stimulation with plate-bound CD3 antibodies is shown. Specifically, CD3-OKT3 clone 20 μg / ml was incubated overnight at 4°C in a flat-bottom 96-well plate to form plate-bound antigen. The next day, T cells or NK cells (NT or transduced with TCR) were added to the wells for incubation for 4 hours, and Brefeldin A was added (which prevents cytokine release, traps cytokines in the cytoplasm so that cytokines can be detected by intracellular cytokine staining). They were then collected for surface and intracellular staining to assess cytokine production and degranulation (TNFα and CD107a). Figure 8 The FACS graph in Figure 2 shows a double positive population of TNFα and CD107a in NK cells transduced with TCR. Non-transduced (NT) NK cells (CD56+CD3-) were used as negative controls, and T cells (CD3+CD56-) were used as positive controls.

[0737] Fig. 9 Phosphorylation of CD3ζ in NK TCR / CD3 cells after cross-linking of CD3 is shown. The various cells tested included non-transduced (NT) NK cells; non-transduced (NT) T cells, and three NK cells transduced with different CD3-TCRs (where CD1, CD2, or CD3 represent different donors). Each group of NK cells was transduced with CD3ZFLGDEFL15 (see Figure 2A and 2B NK cells were incubated on ice for 20 minutes at 20 μg / ml concentration of CD3 OKT3 clone (Miltenyi, 130-093-387). Crosslinking was then performed with Fab2 IgG1 antibody at various time points and stained to check for phosphorylation of CD3z. This analysis of CD3ζ is useful because, as an internalization signal from the surface, it can only be crosslinked with CD3 monoclonal antibodies if NK cells express it. NK cells that were not transduced with CD3 will not show any phosphorylation or activation after stimulation.

[0738] NK cells transduced with CD3-TCR also displayed basal levels of tone signaling that increased after CD3 OKT3 stimulation and resembled T cells, whereas non-transduced NK cells showed no CD3ζ phosphorylation either at basal levels or after CD3 OKT3 stimulation.

[0739] Figure 10 shows that the cytotoxic activity of CD3-CD19 BiTE against Raji cells was increased after pre-incubation with NK cells expressing TCR / CD3. NK cells were either transduced with CD3-TCR#1 (CD3ZFLGDEFL15 (see Figure 2A and 2B )) were either transduced with CD3-TCR#2 (Z2, also known as CD3ZGDEFL8SP21CD8, which includes full-length CD3ζ, full-length CD3γ, full-length CD3δ, and full-length CD3ε and is linked to membrane-bound IL21 (with a CD8 transmembrane domain for membrane-bound IL21)). NK cells transduced with the CD3 / TCR construct or untransduced NK cells were loaded with blinatumomab and incubated for 1 hour and then washed with PBS. They were then incubated with CD19+ B cell lymphoma cells at different effector cell:target cell ratios ( Fig. 10A It's a 1:1 ratio. Fig. 10B The effector cells were CD-3-TCR NK cells and the target cells were Raji cells. At two E:T ratios, the CD3-TCR transduced NK cells loaded with blinatumomab showed enhanced anti-tumor activity compared with the non-transduced NK cells loaded with blinatumomab or the CD3 / TCR transduced NK cells not loaded with blinatumomab.

[0740] Example 2

[0741] NY-ESO TCR in NK cells

[0742] This example relates to the generation and use of NY-ESO TCR in NK cells. Fig.11In the , there is an example for producing these cells. The summary overview shows a situation in which NK cells are first transduced with a uTNK15 construct that incorporates the signaling domain from the CD3 complex, NK co-stimulatory molecules, and IL-15, and then undergo a second transduction step to introduce TCR molecules, thereby generating NK cells that co-express CD3 and NK signaling molecules, IL-15, and TCR complexes. In one embodiment, NK cells are derived from umbilical cord blood and expanded in complete medium with irradiated (100Gy) universal antigen presenting cell (uAPC) feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200U / ml). To generate universal T cell-like NK cells (uTNK15 cells) that can secrete IL-15, NK cells are purified and transduced with a retroviral construct containing the CD3 complex with NK co-stimulatory molecules and IL-15 genes 4 days after isolation. Then, 48 hours after the initial transduction, NK cells expressing uTNK15 were transduced with TCRs targeting the selected antigen.

[0743] exist Fig.12 The expression of NY-ESO TCR on NK cells transduced with uTNK15 is shown in Figure . NK cells were derived from umbilical cord blood and expanded with irradiated (100 Gy) universal antigen presenting cell (uAPC) feeder cells (2:1 feeder cell:NK ratio) and recombinant human IL-2 (200 U / ml) in complete medium. To generate universal T cell-like NK cells that can secrete IL-15, 4 days after isolation, NK cells were purified and transduced with a retroviral construct containing CD3 complexed with NK co-stimulatory molecules and IL-15 gene. Then, 48 hours after the initial transduction, NK cells expressing uTNK15 were transduced with TCR targeting the selected antigen. After 48 hours, the expression of CD3 and NY-ESO TCR on various uTNK15 constructs was evaluated using flow cytometry. Non-transduced (NT) NK cells served as 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 using various TCR constructs was Fig.13 NT NK cells were used as negative control.

[0744] Fig.14Expression of NY-ESO TCR on untransduced and transduced T cells is shown. T cells were isolated from umbilical cord blood (same donor as NK cells, as paired positive control) and activated with CD3 / CD28 microbeads at a concentration of 25 μl / 1 million in RPMI complete medium for 48 hours. T cells were then transduced with a retroviral construct containing the NY-ESO TCR. 48 hours after transduction, flow cytometry showed high expression of the NY-ESO TCR on transduced T cells compared to untransduced T cells.

[0745] NK cells transduced with NY-ESO TCR killed target cells loaded with NY-ESO peptide in a dose-dependent manner ( Fig.15 ). 7 days after TCR transduction, chromium 51 CR killing assay was performed to determine the killing ability of TCR-engineered NK and T cells against LCL cells loaded with different concentrations of NY-ESO peptide for 2 hours. uTNK15 cells transduced with NY-ESO TCR showed enhanced killing of peptide-loaded LCL cells compared to untransduced NK cells. T cells transduced with NY-ESO TCR showed enhanced killing of peptide-loaded LCL cells compared to untransduced T cells.

[0746] Fig.16 NY-ESO was shown to be endogenously expressed on myeloma, sarcoma, and melanoma cell lines. Flow cytometry was used to determine NY-ESO expression on U266 (myeloma), Saos-2 (sarcoma), and A375 (melanoma) cell lines. U266, Saos-2, and A375 cell lines showed higher levels of NY-ESO expression than the Raji cell line used as a negative control.

[0747] T cells transduced with NY-ESO TCR killed NY-ESO-expressing tumor targets at higher E:T ratios ( Fig.17 ). 7 days after TCR transduction, chromium 51 CR killing assay was performed to determine the killing ability of NY-ESO TCR engineered T cells against myeloma, osteosarcoma and melanoma cell lines expressing NY-ESO. T cells transduced with NY-ESO TCR showed enhanced killing of NY-ESO positive cell lines compared to untransduced T cells.

[0748] Fig.18 NK cells transduced with NY-ESO TCR showed that they could kill NY-ESO expressing tumor targets even at low E:T ratios. 51CR killing assay was performed to determine the killing ability of NY-ESO TCR-engineered NK cells against myeloma, osteosarcoma, and melanoma cell lines expressing NY-ESO. NK cells transduced with NY-ESO TCR showed enhanced killing of NY-ESO-positive cell lines compared with non-transduced NK cells even at very low effector-target ratios.

[0749] Figure 19 shows that NK cells transduced with NY-ESO have a similar phenotype to NT NK cells. CytoF imaging showed that untransduced NK cells and uTNK15 cells transduced with NY-ESO TCR had similar phenotypes. Fig.19A The u-map with similar clusters is shown. Fig.19B Heatmap showing similar expression of various markers on NT and uTNK15 cells transduced with NY-ESO TCR.

[0750] Fig. 20 A table showing the percentage of CD3+ and CD3+TCR+NK cells in each uTNK15 product is provided. The composition of single positive CD3 NK cells (CD3+) and double positive CD3 / TCR NK cells (CD3+TCR+) was assessed using flow cytometry. Untransduced NK cells contained less than 1% CD3+ and CD3+TCR+NK cells, while the uTNK15 cell products transduced with TCR contained more than 60% CD3+ and more than 25% CD3+TCR+NK cells.

[0751] Figure 21A provides a FACS diagram showing the successful expression of CD3 on NK cells 4 days after transduction of TCR constant α-β (TCRCab; TCR6 construct). Untransduced (NT) NK cells (CD56+CD3-) were used as negative controls. In Figure 21B, NT NK and uTNK15NK cells were incubated with 10 μg / μl of blinatumomab (a kind of CD3-CD19 bispecific conjugate (BiTe)) at 37°C for 1 hour. Then, at room temperature, biotin-labeled CD19 antigen (CD19 CAR detection reagent from Miltenyi) was added for 20 minutes, followed by the addition of anti-biotin antibody for 15 minutes. This strategy is used to detect any BiTe engaged with CD3+ cells. The histogra...

Claims

1. A composition comprising engineered NK cells modified to express one or more transgenic polynucleotides encoding: a) a CD3 protein complex comprising a single chain or a part or all of CD3δ, CD3ε, CD3γ or CD3ζ, or any combination thereof, b) optionally at least one cytokine, and c) at least one engineered T cell receptor (TCR) comprising one or more of a TCR alpha chain, a TCR beta chain, a TCR gamma chain and / or a TCR delta chain, The engineered TCR targets the KRAS antigen.

2. The composition of claim 1, wherein the NK cells are modified to express TCR αβ chain or TCR γδ chain.

3. The composition of claim 1, wherein the NK cells are modified to express CD3ζ, two copies of CD3ε, CD3δ and part or all of CD3γ.

4. The composition of claim 1, wherein the NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ.

5. The composition of claim 1, wherein any one or more of CD3ζ, CD3ε, CD3δ and CD3γ are heterologously linked to one or more intracellular signaling domains.

6. The composition of claim 5, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, and combinations thereof.

7. The composition of claim 6, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

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

115.

9. The composition of claim 7, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

115.

10. The composition of claim 6, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

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

116.

12. The composition of claim 10, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

116.

13. The composition of claim 6, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

14. The composition of claim 13, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

117.

15. The composition of claim 13, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

117.

16. The composition of claim 1, wherein the composition further comprises one or more monospecific, bispecific or multispecific antibodies, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody.

17. The composition of claim 16, wherein the NK cells express antibodies.

18. The composition of claim 16, wherein the NK cells are complexed with antibodies.

19. The composition of claim 16, wherein the antibody is Imgatuzumab, Amivantamab and / or Cetuximab.

20. The composition of claim 1, wherein the NK cells are derived from cord blood (CB), peripheral blood (PB), bone marrow, stem cells or a mixture thereof.

21. The composition of claim 1, wherein the NK cells are pre-activated.

22. The composition of claim 21, wherein the NK cells are pre-activated with one or more cytokines.

23. The composition of claim 22, wherein the cytokine is IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or a combination thereof.

24. The composition of claim 1, wherein NK cells are expanded.

25. The composition of claim 24, wherein the NK cells are expanded in the presence of IL-2.

26. The composition of claim 1, wherein the NK cells are modified to express one or more additional heterologous proteins.

27. The composition of claim 26, wherein the additional heterologous protein is an engineered antigen receptor, cytokine, homing receptor and / or chemokine receptor.

28. The composition of claim 1, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO: 299 and a sequence at least 85% identical to SEQ ID NO:

301.

29. The composition of claim 1, wherein the TCR comprises one or more sequences that are at least 85% identical to SEQ ID NOs: 299-317.

30. The composition of claim 1, wherein the target KRAS antigenic epitope comprises or consists of GADGVGKSA (SEQ ID NO: 293) and / or GADGVGKSAL (SEQ ID NO: 292).

31. The composition of claim 30, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO:304 and a sequence at least 85% identical to SEQ ID NO:

305.

32. The composition of claim 30, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO:306 and a sequence at least 85% identical to SEQ ID NO:

307.

33. The composition of claim 30, wherein the TCR comprises a sequence at least 85% identical to SEQ ID NO:308 and a sequence at least 85% identical to SEQ ID NO:

309.

34. The composition of claim 27, wherein the heterologous protein is a cytokine.

35. The composition of claim 34, wherein the cytokine is selected from the group consisting of IL-15, IL-12, IL-2, IL-18, IL-21, IL-23, IL-7, GMCSF, or a combination thereof.

36. The composition of claim 35, wherein the cytokine is a membrane-bound cytokine.

37. The composition of claim 35, wherein the cytokine is IL-15.

38. The composition of claim 36, wherein the membrane-bound cytokine comprises a transmembrane domain from CD8, CD28, CD27, B7H3, IgG1, IgG4, CD4, DAP10, or DAP12.

39. The composition of claim 27, wherein the NK cells express chimeric antigen receptors and cytokines.

40. The composition of claim 16, wherein the monospecific, bispecific or multispecific antibody comprises an antibody that targets a cancer antigen.

41. The composition of claim 40, wherein the cancer antigen is EGFR, C-met and / or TROP-2 antigen.

42. A composition comprising a complex comprising: a) NK cells modified to express part or all of the CD3 receptor complex and modified to express the T cell receptor (TCR) αβ chain or TCR γδ chain; and b) a monospecific, bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody that binds to CD3 on NK cells, Among them, TCR targets KRAS antigen.

43. The composition of claim 42, wherein the NK cells are modified to express a TCR αβ chain that is at least 85% identical to SEQ ID NO: 299 and SEQ ID NO: 301, and the antibody is imatinib, ervantumab and / or cetuximab.

44. The composition of claim 43, wherein the NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ.

45. A composition as claimed in claim 44, wherein any one or more of CD3ζ, CD3ε, CD3δ and / or CD3γ are heterologously linked to one or more intracellular signaling domains.

46. ​​The composition of claim 45, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

47. The composition of claim 46, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

48. The composition of claim 47, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

115.

49. The composition of claim 47, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

115.

50. The composition of claim 46, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

51. The composition of claim 50, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

116.

52. The composition of claim 50, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

116.

53. The composition of claim 46, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

54. The composition of claim 53, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

117.

55. The composition of claim 53, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

117.

56. The composition of claim 42, wherein the complex is contained in a pharmaceutically acceptable excipient.

57. The composition of claim 56, wherein the complex is deposited in a delivery device.

58. A method of treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of any one of the compositions of claims 1-57.

59. A method for treating cancer in an individual, comprising the step of administering to the individual a therapeutically effective amount of any of the compositions of claims 16, 17, 18, 19, 40, 41 or 42-57, wherein the NK cells and the antibody are administered to the individual simultaneously, and optionally wherein the NK cells and the antibody are administered in the same formulation and / or are pre-complexed prior to administration to the individual.

60. The method of claim 58, wherein the NK cells and the antibody are administered to the individual at different times.

61. The method of claim 59, wherein the NK cells and antibodies are administered by infusion.

62. The method of claim 59, wherein the NK cells are autologous to the individual.

63. The method of claim 59, wherein the NK cells are allogeneic to the individual.

64. A method of redirecting the specificity of NK cells against cancer antigens for use in treating an individual using a monospecific, bispecific or multispecific antibody, wherein the bispecific or multispecific antibody comprises an anti-CD3 antibody, comprising the steps of administering to the individual the antibody and a NK cell expressing part or all of the CD3 receptor complex and expressing part or all of the TCR αβ chain or the TCR γδ chain, Among them, TCR targets KRAS antigen.

65. The method of claim 64, wherein the NK cells are modified to express full-length CD3ζ, CD3ε, CD3δ and / or CD3γ.

66. The method of claim 65, wherein any one or more of CD3ζ, CD3ε, CD3δ and / or CD3γ is heterologously linked to one or more intracellular signaling domains.

67. The method of claim 66, wherein the intracellular signaling domain is selected from the group consisting of: CD16, NKG2D, DAP10, DAP12, 2B4, 4-1BB, CD2, CD28, DNAM, and combinations thereof.

68. The method of claim 67, wherein the intracellular signaling domain comprises a DAP10 intracellular signaling domain.

69. The method of claim 68, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

115.

70. The method of claim 68, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

115.

71. The method of claim 67, wherein the intracellular signaling domain comprises a CD28 intracellular signaling domain.

72. The method of claim 71, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

116.

73. The method of claim 71, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

116.

74. The method of claim 67, wherein the intracellular signaling domain comprises DAP10 and CD28 intracellular signaling domains.

75. The method of claim 74, wherein the intracellular signaling domain comprises an amino acid sequence at least about 85% identical to SEQ ID NO:

117.

76. The method of claim 74, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:

117.

77. The method of claim 64, wherein the TCR chain is a TCR αβ chain and is at least 85% identical to SEQ ID NO: 299 and SEQ ID NO:

301.

78. The method of claim 64, wherein the TCR chain is a TCR αβ chain and is at least 85% identical to one or more of SEQ ID NOs: 299-317.

79. The method of claim 64, wherein the target KRAS antigenic epitope comprises or consists of GADGVGKSA (SEQ ID NO: 293) and / or GADGVGKSAL (SEQ ID NO: 292).

80. The method of claim 78, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID NO:304 and a sequence at least 85% identical to SEQ ID NO:

305.

81. The method of claim 78, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID NO:306 and a sequence at least 85% identical to SEQ ID NO:

307.

82. The method of claim 78, wherein the TCR chain comprises a sequence at least 85% identical to SEQ ID NO:308 and a sequence at least 85% identical to SEQ ID NO:

309.

83. The method of claim 64, further comprising the step of modifying the NK cells to express one or more additional heterologous proteins.

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