IL-12 POLYPEPTIDES, IL-15 POLYPEPTIDES, IL-18 POLYPEPTIDES, CD8 POLYPEPTIDES, COMPOSITIONS THEREOF, AND METHODS OF USE THEREOF - Patent application

Abstract

The present disclosure relates to cells capable of co-expressing one or any combination of T cell receptors ("TCRs"), CD8 polypeptides, interleukin 12 (IL-12) polypeptides, interleukin 15 (IL-15) polypeptides, and / or interleukin 18 (IL-18) polypeptides, and their use in adoptive cell therapy ("ACT"). The present disclosure further provides modified CD8 sequences, IL-12 sequences, IL-15 sequences, IL-18 sequences, vectors thereof, compositions, transformed T cells, and related methods.

Description

[Technical field]

[0001] Related Applications This application is an international application claiming priority to U.S. Provisional Patent Application No. 63 / 336,004, filed April 28, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Reference to an Electronically Submitted Sequence Listing An official copy of the Sequence Listing was submitted via EFS-Web as an ASCII formatted sequence listing with file name "3000011-030977_Sequence-Listing_ST26", created on April 26, 2023, and 563,430 bytes in size, and filed concurrently herewith. The Sequence Listing contained in this ASCII formatted document is a part of this specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to cells capable of co-expressing one or any combination of T cell receptors ("TCRs"), CD8 polypeptides, interleukin 12 (IL-12) polypeptides, interleukin 15 (IL-15) polypeptides, and / or interleukin 18 (IL-18) polypeptides, and their use in adoptive cell therapy ("ACT"). The present disclosure further provides modified CD8 sequences, IL-12 sequences, IL-15 sequences, IL-18 sequences, vectors thereof, compositions, transformed cells, and related methods. [Background technology]

[0004] CD8 and CD4 are transmembrane glycoproteins characteristic of different populations of T lymphocytes, whose antigen responses are restricted by class I and class II MHC molecules, respectively. Both of them play major roles in the differentiation and selection of T cells during thymic development and in the activation of mature T lymphocytes in response to antigen-presenting cells. Both CD8 and CD4 are immunoglobulin superfamily proteins. They determine antigen restriction by binding to MHC molecules at interfaces quite distinct from the regions that present antigenic peptides, but the structural basis of their similar functions seems quite different. Their sequence similarity is low. CD4 is expressed on the cell surface as a monomer, while CD8 is expressed as an αα homodimer (e.g., Figure 55C) or as an αβ heterodimer (e.g., Figure 55A). In humans, the CD8αα homodimer can be functionally replaced by the CD8αβ heterodimer. CD8 interacts with the acidic loop in the α3 domain of class I MHC, thereby increasing the avidity of T cells with their targets. CD8 is also involved in phosphorylation, which binds the cytoplasmic tail of its α chain to the tyrosine kinase p56 lck and activates the CTL.

[0005] Interleukin-12 (IL-12 or IL12) is a heterodimeric cytokine that is important for T cell differentiation and activation of T cells and natural killer cells. The two subunits of IL-12, α (p35) and β (p40), are encoded by separate genes.

[0006] Interleukin-15 (IL-15 or IL15), a pleiotropic cytokine, is a member of the four α-helical bundle cytokine family. (Waldmann TA and Tagaya Y, Ann. Rev. Immunol. 17:19-49, 1999, the contents of which are incorporated herein by reference). Wild-type IL-15, a 14-15 kDa glycoprotein, shares partial structural homology with IL-2. (Ibid.). Wild-type IL-15 can be expressed in two isoforms, one with a 48 amino acid signal peptide and the other with a 21 amino acid signal peptide. (Ibid.). The mature form of wild-type IL-15 consists of 114 amino acids. (Ibid.). Expression of wild-type IL-15 is regulated during transcription, translation, and intracellular trafficking. (Ibid.). Wild-type IL-15 utilizes a unique receptor, IL-15Rα. In lymphocytes, this receptor binds IL-15 with high affinity and binds IL-2Rβ (also called IL-2 / IL-15Rβ) and IL-2Rγ (γ c (Ibid., Okada S at al., Immunol. and Cell Biol. 93:461-471, 2015, the contents of which are incorporated herein by reference).

[0007] Interleukin-18 (IL-18 or IL18) is a cytokine that stimulates several cell types, including CD4 T cells, CD8 T cells, and natural killer cells.

[0008] Adoptive cell therapy (ACT) is a promising approach for the treatment of diseases such as cancer. T cell therapy has been successful in treating various cancers. Li et al. Signal Transduction and Targeted Therapy 4(35):(2019), the contents of which are incorporated herein by reference. However, the cells used in ACT often do not persist in the tumor microenvironment and rapidly lose their ability to kill tumor cells. Thus, there is a need for T cells and natural killer cells that persist long in the tumor microenvironment and / or exhibit a sustained ability to kill tumor cells. It is also desirable to develop methods for producing T cells and natural killer cells with enhanced specific cytotoxicity for immunotherapy. Summary of the Invention

[0009] In embodiments, an IL-12p35 / IL-12p40 fusion polypeptide may be provided. In embodiments, the IL-12p35 / IL-12p40 fusion polypeptide may be soluble and / or secreted by cells transduced to express it. In embodiments, a nucleic acid encoding an IL-12p35 / IL-12p40 fusion polypeptide may be provided. In embodiments, a vector comprising an IL-12p35 / IL-12p40 fusion polypeptide may be provided. In embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide. In embodiments, an IL-12p35 / IL-12p40 fusion polypeptide may comprise a fusion polypeptide of an IL-12α (p35) (IL-12αp35, IL-12α, IL-12p35) polypeptide and an IL-12β (p40) (IL-12βp40, IL-12β, IL-12p40) polypeptide.

[0010] In some embodiments, the IL-12p35 polypeptide may be located C-terminal to the IL-12p40 polypeptide in an IL-12p35 / IL-12p40 fusion polypeptide. (FIG. 67A). In another embodiment, the IL-12p35 polypeptide may be located N-terminal to the IL-12p40 polypeptide in an IL-12p35 / IL-12p40 fusion polypeptide. (FIG. 67B). In some embodiments in FIG. 67A and FIG. 67B, the linker is optional. In some embodiments in FIG. 67A and FIG. 67B, the line may represent a direct bond without an intervening sequence, or may represent an intervening sequence, such as, but not limited to, a linker, a Furin, a sequence encoding a 2A polypeptide, a factor Xa site, an untranslated sequence, a translated sequence, a sequence comprising one or more restriction endonuclease sites, or a combination thereof. The term "IL-12p35 / IL-12p40 fusion polypeptide" is not intended to imply a particular order of the polypeptides in the fusion polypeptide, unless otherwise specified. In some embodiments, the order of the IL-12p35 / IL-12p40 fusion polypeptide can be such that IL-12P40 is N-terminal to IL-12p35, as shown in Figure 67A.

[0011] In some embodiments, the vectors described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0012] In some embodiments, the vectors described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0013] In embodiments, an IL-15 polypeptide may be provided. In embodiments, the IL-15 polypeptide may be soluble and / or secreted by cells transduced to express it. In embodiments, a nucleic acid encoding an IL-15 polypeptide may be provided. In embodiments, a vector comprising an IL-15 polypeptide may be provided. In embodiments, the cells described herein may comprise an IL-15 polypeptide. In embodiments, the IL-15 polypeptide may comprise the entire mature IL-15 polypeptide. In embodiments, the IL-15 may be mutated and / or truncated.

[0014] In some embodiments, the vectors described herein may include an IL-15 polypeptide and a CD8 polypeptide described herein. In some embodiments, the vectors described herein may include an IL-15 polypeptide, a CD8 polypeptide, a cellular receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0015] In some embodiments, the cells described herein may comprise an IL-15 polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-15 polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0016] In embodiments, an IL-18 polypeptide may be provided. In embodiments, the IL-18 polypeptide may be soluble and / or secreted by cells transduced to express it. In embodiments, a nucleic acid encoding an IL-18 polypeptide may be provided. In embodiments, a vector comprising an IL-18 polypeptide may be provided. In embodiments, the cells described herein may comprise an IL-18 polypeptide. In embodiments, the IL-18 polypeptide may comprise the entire mature IL-18 polypeptide. In embodiments, the IL-18 may be mutated and / or truncated.

[0017] In some embodiments, the vectors described herein may include an IL-18 polypeptide and a CD8 polypeptide described herein. In some embodiments, the vectors described herein may include an IL-18 polypeptide, a CD8 polypeptide, a cellular receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0018] In some embodiments, the cells described herein may comprise an IL-18 polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-18 polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0019] In some embodiments, the vectors described herein may include any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. In some embodiments, the vectors described herein may include IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, CD8 polypeptides, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0020] In some embodiments, the cells described herein may include any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. In some embodiments, the cells described herein may include IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, CD8 polypeptides, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0021] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0022] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0023] In certain aspects, the cells described herein can be isolated and / or recombinant cells.

[0024] In embodiments, (i) a polypeptide of SEQ ID NO:309 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:309, (ii) a polypeptide of SEQ ID NO:305 and a polypeptide of SEQ ID NO:307, wherein the C-terminus of SEQ ID NO:305 is linked to the N-terminus of SEQ ID NO:307, or the C-terminus of SEQ ID NO:307 is linked to the N-terminus of SEQ ID NO:305, with or without a linker between the sequences. or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305, and a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, wherein the polypeptide is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. The C-terminus of a polypeptide that is about 100% identical is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, or the C-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307 is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. (iii) a polypeptide of SEQ ID NO:311, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:311; (iv) a polypeptide of SEQ ID NO:315, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98% to SEQ ID NO:315;Nucleic acids encoding polypeptides that are at least about 99% or about 100% identical, or any combination of (v)(i), (ii), (iii) and (iv) can be provided.

[0025] In embodiments, (i) SEQ ID NO:310 or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:310, (ii) SEQ ID NO:306 and SEQ ID NO:308, where the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, or the 3' end of SEQ ID NO:308 is linked to the 5' end of SEQ ID NO:306, with or without a sequence encoding a linker between them, or SEQ ID NO:30 6, and a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, in which the 3' end of the sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306 (iii) SEQ ID NO:312, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:312; (iv) SEQ ID NO:316, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:316; or (v)(i),(ii),Nucleic acids comprising any combination of (iii) and (iv) may be provided.

[0026] In some embodiments, the nucleic acids described herein may further comprise a nucleic acid encoding (a) at least one TCR polypeptide comprising an alpha chain and a beta chain; (b) at least one CD8 polypeptide comprising (i) an alpha chain, (ii) a beta chain, or (iii) an alpha chain and a beta chain; or (c) at least one TCR polypeptide comprising an alpha chain and a beta chain, and at least one CD8 polypeptide comprising (i) an alpha chain, (ii) a beta chain, or (iii) an alpha chain and a beta chain.

[0027] In embodiments, there may be provided a nucleic acid encoding (a) (i) a T cell receptor (TCR) comprising an α chain and a β chain, and a CD8 polypeptide comprising an α chain and a β chain, or (ii) a TCR comprising an α chain and a β chain, and a CD8 polypeptide comprising an α chain and no β chain; (b) at least one interleukin; or (c) both (a) and (b), wherein the TCR α chain and the TCR β chain are set forth in SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36. , 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; the CD8 alpha chain is selected from SEQ ID NO: 7, 258, 259, 262 or a variant thereof; and the CD8 beta chain is also selected from SEQ ID NO: 8, 9, 10, 11, 12, 13. or 14, wherein the at least one interleukin is (i) a polypeptide of SEQ ID NO:309 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:309; (ii) a polypeptide of SEQ ID NO:305 and a polypeptide of SEQ ID NO:307, wherein the C-terminus of SEQ ID NO:305 is linked to the N-terminus of SEQ ID NO:307, or the C-terminus of SEQ ID NO:307 is linked to the N-terminus of SEQ ID NO:305, with no linker between the sequences. and polypeptides that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305, and polypeptides that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, in which case the polypeptides are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305.The C-terminus of a polypeptide that is at least about 99% or about 100% identical is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, or the C-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. (iii) a polypeptide of SEQ ID NO:311 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:311; (iv) a polypeptide of SEQ ID NO:315 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:315; or (v) any combination of (i), (ii), (iii) and (iv).

[0028] In embodiments, a nucleic acid encoding (a) (i) a T cell receptor (TCR) comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and a β chain, or (ii) a TCR comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and no β chain, (b) at least one interleukin, or (c) both (a) and (b), wherein the TCR α chain and the TCR β chain are selected from SEQ ID NOs: 15 and 16, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, and 71 and 303, and the alpha chain of CD8 is SEQ ID NO: 7, 258, 259, 262 or a variant thereof, the beta chain of CD8 is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, and the at least one interleukin is (i) a polypeptide of SEQ ID NO: 309 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 309, (ii) a polypeptide of SEQ ID NO: 305 and a polypeptide of SEQ ID NO: 307, in which case SEQ ID NO: and a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305, and a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307. or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305 is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307;the C-terminus of a polypeptide that is at least about 99% or about 100% identical to SEQ ID NO: 305 is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 305, with or without a linker between the sequences; (iii) a polypeptide of SEQ ID NO: 311, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 311; (iv) a polypeptide of SEQ ID NO: 315, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 315; or (v) any combination of (i), (ii), (iii) and (iv).

[0029] In embodiments, a nucleic acid may be provided that includes: (a) a sequence that is at least about 80% identical to the sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301; (b) a sequence that encodes at least one interleukin; or (c) both (a) and (b).

[0030] In embodiments, a nucleic acid may be provided that includes: (a) a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301; (b) a sequence encoding at least one interleukin; or (c) both (a) and (b).

[0031] In embodiments, the sequence encoding at least one interleukin is selected from the group consisting of: (i) SEQ ID NO:310 or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:310; (ii) SEQ ID NO:306 and SEQ ID NO:308, wherein the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, or the 3' end of SEQ ID NO:308 is linked to the 5' end of SEQ ID NO:306, and a linker is encoded between them. or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, and a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, in which case the sequence is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306. or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306. (iii) SEQ ID NO:312, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:312; (iv) SEQ ID NO:316, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:316;or (v) any combination of (i), (ii), (iii) and (iv).

[0032] In embodiments, a vector may be provided that comprises nucleic acid encoding at least one CD8α, at least one TCR α chain, at least one TCR β chain, at least one interleukin, and optionally at least one CD8 β chain.

[0033] In several embodiments, a vector may be provided comprising nucleic acid encoding N1, N2, N3, N4, N5, L1, L2, L3 and L4, wherein N1 encodes a CD8 β chain, is present or absent, N2 encodes a CD8 β chain, N3 encodes a TCR β chain, N4 encodes a TCR α chain, N5 encodes at least one interleukin, and L1 to L4 each encode at least one linker, each of L1 to L4 being independently the same or different, and each of L1 to L4 being independently present or absent.

[0034] In embodiments, the vector may comprise Formula I or Formula II. 5'-N1-L1-N2-L2-N3-L3-N4-L4-N5 - 3' [I] 5'-N5-L1-N1-L2-N2-L3-N3-L4-N4 - 3' [II]

[0035] In embodiments, N1 may encode SEQ ID NO:8, 9, 10, 11, 12, 13, or 14.

[0036] In embodiments, N2 may encode SEQ ID NO: 7, 258, 259, 262, or a variant thereof.

[0037] In some embodiments, N4 and N3 are selected from the group consisting of SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91, 92 and 93, 93 and 94, 94 and 95, 95 and 96, 97 and 98, 98 and 99, 100 and 101, 102 and 103, 103 and 104, 104 and 105, 105 and 106, 106 and 107, 108 and 109, 109 and 108, 109 and 110, 110 and 111, 112 and 113, 113 and 114, 114 and 115 It can code for 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90 and 91 and 92.

[0038] In embodiments, N5 is (i) a polypeptide of SEQ ID NO:309 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:309, (ii) a polypeptide of SEQ ID NO:305 and a polypeptide of SEQ ID NO:307, wherein the C-terminus of SEQ ID NO:305 is linked to the N-terminus of SEQ ID NO:307, or the C-terminus of SEQ ID NO:307 is linked to the N-terminus of SEQ ID NO:305, with or without a linker between the sequences. or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305, and a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, in which case the polypeptide may be at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. or the C-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307 is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, or the C-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. (iii) a polypeptide of SEQ ID NO:311, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:311; (iv) a polypeptide of SEQ ID NO:315, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%,(v) may encode a polypeptide that is at least about 99% or about 100% identical to (i), (ii), (iii), and (iv), or any combination of (i), (ii), (iii), and (iv).

[0039] In embodiments, the vectors described herein may further comprise (i) a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) located between N1 and L1, between L1 and N2, between N2 and L2, between L2 and N3, between N3 and L3, between L3 and N4, between N4 and L4, between L4 and N5, or any combination thereof, or (ii) a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) located between N5 and L1, between L1 and N1, between N1 and L2, between L2 and N2, between N2 and L3, between L3 and N3, between N3 and L4, between L4 and N4, or any combination thereof. In embodiments, the 2A peptide may be P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96). In some embodiments, the IRES may be selected from the group consisting of an IRES from a picornavirus, an IRES from a flavivirus, an IRES from a pestivirus, an IRES from a retrovirus, an IRES from a lentivirus, an IRES from an insect RNA virus, and an IRES from a cellular mRNA.

[0040] In embodiments, the vectors described herein may further comprise (i) a nucleic acid encoding furin located between N1 and L1, between L1 and N2, between N2 and L2, between L2 and N3, between N3 and L3, between L3 and N4, between N4 and L4, between L4 and N5, or any combination thereof; or (ii) a nucleic acid encoding furin located between N5 and L1, between L1 and N1, between N1 and L2, between L2 and N2, between N2 and L3, between L3 and N3, between N3 and L4, between L4 and N4, or any combination thereof.

[0041] (a) T cells and / or natural killer (NK) cells may be provided that comprise (i) a T cell receptor (TCR) comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and a β chain, or (ii) a TCR comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and no β chain, and (b) at least one interleukin, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91, 92 and 93, 93 and 94, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 100 and 101, 102 and 10 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; the CD8 alpha chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof; and the CD8 beta chain is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14; At least one of the at least one interleukin is (i) a polypeptide of SEQ ID NO:309 or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:309, (ii) a polypeptide of SEQ ID NO:305 and a polypeptide of SEQ ID NO:307, wherein the C-terminus of SEQ ID NO:305 is linked to the N-terminus of SEQ ID NO:307, or the C-terminus of SEQ ID NO:307 is linked to the N-terminus of SEQ ID NO:305, with no interleukin between them. Linkers may be present or absent, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305, and a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, in which case the polypeptide is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305.The C-terminus of a polypeptide that is at least about 99% or about 100% identical is linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, or the C-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307 is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. (iii) a polypeptide of SEQ ID NO:311, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:311; (iv) a polypeptide of SEQ ID NO:315, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:315; or (v) any combination of (i), (ii), (iii) and (iv).

[0042] In embodiments, there may be provided a T cell and / or natural killer (NK) cell comprising: (a) (i) a T cell receptor (TCR) comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain and a beta chain, or (ii) a TCR comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain but not a beta chain, and (b) at least one interleukin, wherein the TCR alpha and TCR beta chains are selected from SEQ ID NOs: 15 and 16, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, and 71 and 303; the CD8 alpha chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof, and the CD8 beta chain, if present, is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. In embodiments, the at least one interleukin may comprise a polypeptide of SEQ ID NO:309, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polypeptide of SEQ ID NO:309.In embodiments, the at least one interleukin is a polypeptide of SEQ ID NO:305 and a polypeptide of SEQ ID NO:307, where the C-terminus of SEQ ID NO:305 is linked to the N-terminus of SEQ ID NO:307, or the C-terminus of SEQ ID NO:307 is linked to the N-terminus of SEQ ID NO:305, with or without a linker between them, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305, and a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:307, where the C-terminus of SEQ ID NO:305 is linked to the N-terminus of SEQ ID NO:307, with or without a linker between them, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:305. The C-terminus of a polypeptide that is about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 307 may be linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 307, or the C-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 307 may be linked to the N-terminus of a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 305, with or without a linker between the sequences. In embodiments, at least one interleukin may comprise a polypeptide of SEQ ID NO:311, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polypeptide of SEQ ID NO:311.In embodiments, the at least one interleukin may comprise a polypeptide of SEQ ID NO:315, or a polypeptide that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the polypeptide of SEQ ID NO:315.

[0043] In embodiments, there may be provided a nucleic acid encoding (a) (i) a T cell receptor (TCR) comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and a β chain, or (ii) a TCR comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and no β chain; (b) at least one interleukin; or (c) both (a) and (b), wherein the TCR α chain and the TCR β chain are selected from the group consisting of SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 72 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 90 and 91, 92 and 93, 93 and 94, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 100 and 101 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; the CD8 alpha chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof; and the CD8 beta chain is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. and at least one interleukin is selected from the group consisting of (i) SEQ ID NO:310 or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:310, (ii) SEQ ID NO:306 and SEQ ID NO:308, wherein the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, or the 3' end of SEQ ID NO:308 is linked to the 5' end of SEQ ID NO:306, with or without a sequence encoding a linker between them, or a sequence A sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, and a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, wherein the 3' end of the sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306 isor the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, and there is no linkage between the sequences. (iii) SEQ ID NO:312, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:312; (iv) SEQ ID NO:316, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:316; or (v) encoded by a nucleic acid comprising a sequence selected from any combination of (i), (ii), (iii), and (iv).

[0044] In some embodiments, a nucleic acid may be provided that includes (a) (i) a T cell receptor (TCR) comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain and a beta chain, or (ii) a TCR comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain and no beta chain, and (b) at least one dominant interleukin, wherein the TCR alpha chain and the TCR beta chain are selected from SEQ ID NOs: 15 and 16, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, and 71 and 303, and the CD8 alpha chain is selected from SEQ ID NOs: and wherein the beta chain of CD8 is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, and the at least one interleukin is (i) SEQ ID NO: 310 or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 310, (ii) SEQ ID NO: 306 and SEQ ID NO: 308, wherein the 3' end of SEQ ID NO: 306 is linked to the 5' end of SEQ ID NO: 308, or The 3' end of SEQ ID NO:308 is linked to the 5' end of SEQ ID NO:306, with or without a sequence encoding a linker between them, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, and a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, in which case the sequence is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306. or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, ...(iii) SEQ ID NO:312, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:312, (iv) SEQ ID NO:316, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:316, or (v) encoded by a nucleic acid comprising a sequence selected from any combination of (i), (ii), (iii) and (iv).

[0045] In embodiments, a nucleic acid may be provided that includes (a) a sequence that is at least about 80% identical to the sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301, and (b) a sequence that encodes at least one interleukin. In embodiments, a nucleic acid may be provided that includes (a) a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301, and (b) a sequence encoding at least one interleukin. In embodiments, the nucleic acid encoding at least one interleukin is (i) SEQ ID NO:310 or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:310, (ii) SEQ ID NO:306 and SEQ ID NO:308, where the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, or the 3' end of SEQ ID NO:308 is linked to the 5' end of SEQ ID NO:306, with or without a linker-encoding sequence between them, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 100% identical to SEQ ID NO:306, (iii) SEQ ID NO:306 and SEQ ID NO:308, where the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, with or without a linker-encoding sequence between them, and sequences that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, in which the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, orThe 3' end of a sequence that is at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 306 may be linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 306, with or without a linker-encoding sequence between them; (iii) SEQ ID NO: 312, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 312; (iv) SEQ ID NO: 316, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 316; or (v) any combination of (i), (ii), (iii) and (iv).

[0046] In embodiments, there may be provided a T cell and / or natural killer (NK) cell comprising: (a) (i) a T cell receptor (TCR) comprising an α chain and a β chain, and a CD8 polypeptide comprising an α chain and a β chain, or (ii) a TCR comprising an α chain and a β chain, and a CD8 polypeptide comprising an α chain and no β chain, and (b) at least one interleukin, wherein the TCR α chain and the TCR β chain are set forth in SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 35, 36 and 37, 38 and 39, 40 and 41, 42 and 43, 44 and 45, 46 and 47, 48 and 49, 50 and 51, 52 and 53, 54 and 55, 56 and 57, 58 and 59, 60 and 61, 62 and 63, 64 and 65, 66 and 67, 68 and 69, 70 and 71, 72 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 82 and 83, 84 and 85, 85 and 86, 87 and 88, 88 and 89, 90 and 91, 92 and 93, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 1 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; the CD8 alpha chain is selected from SEQ ID NO: 7, 258, 259, 262 or a variant thereof; 10, 11, 12, 13 or 14, wherein at least one interleukin is selected from the group consisting of (i) SEQ ID NO: 310 or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO: 310, (ii) SEQ ID NO: 306 and SEQ ID NO: 308, wherein the 3' end of SEQ ID NO: 306 is linked to the 5' end of SEQ ID NO: 308, or the 3' end of SEQ ID NO: 308 is linked to the 5' end of SEQ ID NO: 306, with a linker between them. Coding sequences may or may not be present, or sequences that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, and sequences that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, in which case they are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306.The 3' end of a sequence that is at least about 99% or about 100% identical is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306. (iii) SEQ ID NO:312, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:312; (iv) SEQ ID NO:316, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:316; or (v) encoded by a nucleic acid comprising a sequence selected from any combination of (i), (ii), (iii), and (iv).

[0047] In embodiments, there may be provided a T cell and / or natural killer (NK) cell comprising: (a) (i) a T cell receptor (TCR) comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain and a beta chain, or (ii) a TCR comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain but not a beta chain, and (b) at least one interleukin, wherein the TCR alpha and TCR beta chains are selected from SEQ ID NOs: 15 and 16, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, and 71 and 303; the CD8 alpha chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof, and the CD8 beta chain, if present, is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. In embodiments, at least one interleukin can be encoded by a nucleic acid comprising a sequence selected from SEQ ID NO:310, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:310.In embodiments, the at least one interleukin is SEQ ID NO:306 and SEQ ID NO:308, where the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, or the 3' end of SEQ ID NO:308 is linked to the 5' end of SEQ ID NO:306, with or without a linker-encoding sequence between them, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, and a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, where the 3' end of SEQ ID NO:306 is linked to the 5' end of SEQ ID NO:308, with or without a linker-encoding sequence between them, or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308, or the 3' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:308 is linked to the 5' end of a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identical to SEQ ID NO:306, with or without a sequence encoding a linker between them. In embodiments, at least one interleukin can be encoded by a nucleic acid comprising a sequence selected from SEQ ID NO:312, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:312.In embodiments, at least one interleukin can be encoded by a nucleic acid comprising a sequence selected from SEQ ID NO:316, or a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO:316.

[0048] In some embodiments, a method of preparing T cells and / or natural killer cells for immunotherapy may be provided, comprising isolating T cells and / or natural killer cells from a blood sample of a human subject, activating the isolated T cells and / or natural killer cells, transducing the activated T cells and / or natural killer cells with a nucleic acid described herein or a vector described herein, and expanding the transduced T cells and / or natural killer cells. In some embodiments, the method may further comprise isolating T cells from the transduced T cells and / or natural killer cells, and expanding the isolated CD4+CD8+ transduced T cells. In some embodiments, the blood sample may comprise peripheral blood mononuclear cells (PMBCs). In some embodiments, the activation may comprise contacting the T cells and / or natural killer cells with an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the T cells may be CD4+ T cells. In some embodiments, the T cells may be CD8+ T cells. In embodiments, the T cells can be γδ T cells or αβ T cells. In embodiments, activation, expansion, or both is performed in the presence of a combination of IL-2 and IL-15, optionally with zoledronate.

[0049] In embodiments, a method of increasing the persistence, longevity, functionality, naivety, ability to kill antigen-presenting cells, or a combination thereof of T cells and / or natural killer (NK) cells may be provided, the method comprising isolating T cells and / or natural killer (NK) cells from a blood sample of a human subject, activating the isolated T cells and / or natural killer (NK) cells, transducing the activated T cells and / or natural killer (NK) cells with a nucleic acid described herein, a vector described herein, or a combination thereof to obtain transduced T cells and / or natural killer (NK) cells, and obtaining the transduced T cells and / or natural killer (NK) cells, wherein the persistence, functionality, naivety, longevity, ability to kill antigen-presenting cells, or a combination thereof of the transduced T cells and / or natural killer (NK) cells is increased compared to that of a control cell. In embodiments, the method may further comprise expanding the transduced T cells and / or natural killer (NK) cells. In some embodiments, the control cells may include untransduced T cells and / or natural killer (NK) cells, T cells and / or natural killer (NK) cells transduced with a TCR only, or a combination thereof. In some embodiments, the control cells may include untransduced T cells and / or natural killer (NK) cells, T cells and / or natural killer (NK) cells transduced with a TCR only, T cells and / or natural killer (NK) cells transduced with a TCR and CD8 only, or a combination thereof.In embodiments, the persistence, longevity, functionality, naivety, ability to kill antigen-presenting cells, or combinations thereof of the transduced T cells and / or natural killer (NK) cells and control cells may be determined after one challenge with antigen-presenting cells, two challenges with antigen-presenting cells, three challenges with antigen-presenting cells, four challenges with antigen-presenting cells, five challenges with antigen-presenting cells, six challenges with antigen-presenting cells, seven challenges with antigen-presenting cells, or eight or more challenges with antigen-presenting cells. In embodiments, the persistence, longevity, functionality, naivety, ability to kill antigen-presenting cells, or combinations thereof of the transduced T cells and / or natural killer (NK) cells and control cells may be determined after three challenges with antigen-presenting cells, four challenges with antigen-presenting cells, five challenges with antigen-presenting cells, or six or more challenges with antigen-presenting cells.

[0050] In embodiments, a method of increasing interferon gamma (IFNγ) secretion by T cells and / or natural killer (NK) cells may be provided, the method comprising isolating T cells and / or natural killer (NK) cells from a blood sample of a human subject, activating the isolated T cells and / or natural killer (NK) cells, transducing the activated T cells and / or natural killer (NK) cells with a nucleic acid described herein, a vector described herein, or a combination thereof to obtain transduced T cells and / or natural killer (NK) cells, and obtaining the transduced T cells and / or natural killer (NK) cells, wherein IFNγ secretion of the transduced T cells and / or natural killer (NK) cells is increased compared to that of a control cell. In embodiments, the method may further comprise expanding the transduced T cells and / or natural killer (NK) cells. In some embodiments, the control cells may include untransduced T cells and / or natural killer (NK) cells, T cells and / or natural killer (NK) cells transduced with TCR only, or a combination thereof. In some embodiments, the control cells may include untransduced T cells and / or natural killer (NK) cells, T cells and / or natural killer (NK) cells transduced with TCR only, T cells and / or natural killer (NK) cells transduced with TCR and CD8 only, or a combination thereof. In some embodiments, IFNγ secretion by the transduced T cells and / or natural killer (NK) cells and the control cells may be determined after one challenge with antigen presenting cells, two challenges with antigen presenting cells, three challenges with antigen presenting cells, four challenges with antigen presenting cells, five challenges with antigen presenting cells, six challenges with antigen presenting cells, seven challenges with antigen presenting cells, or eight or more challenges with antigen presenting cells.In some embodiments, IFNγ secretion by transduced T cells and / or natural killer (NK) cells and control cells may be determined after three challenges with antigen-presenting cells, four challenges with antigen-presenting cells, five challenges with antigen-presenting cells, or six or more challenges with antigen-presenting cells.

[0051] In some embodiments, the antigen-presenting cells can present antigens on the cell surface, and the transduced T cells and / or natural killer (NK) cells, as well as the control cells, can have the ability to kill the antigen-presenting cells. In some embodiments, the antigen can include a peptide. In some embodiments, the peptide can be in a complex with an MHC molecule on the cell surface.

[0052] In embodiments, a polypeptide, a fusion polypeptide, or multiple polypeptides encoded by the nucleic acids described herein may be provided.

[0053] In embodiments, a polypeptide, fusion polypeptide, or polypeptides described herein may be isolated, recombinant, or both isolated and recombinant.

[0054] In some embodiments, a polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 305, 307, 309, 311, 313, or 315, and (a) at least one TCR polypeptide comprising an α chain and a β chain, (b) at least one CD8 polypeptide comprising (i) an α chain, (ii) a β chain, or (iii) an α chain and a β chain, or (c) at least one TCR polypeptide comprising an α chain and a β chain, and at least one CD8 polypeptide comprising (i) an α chain, (ii) a β chain, or (iii) an α chain and a β chain, may be provided. In some embodiments, the cell may be an αβ T cell, a γδ T cell, a natural killer T cell, a natural killer (NK) cell, or any combination thereof. In some embodiments, the αβ T cell may be a CD4+ T cell. In some embodiments, the αβ T cells may be CD8+ T cells. In some embodiments, the γδ T cells may be Vγ9Vδ2+ T cells.

[0055] In embodiments, a nucleic acid can be provided that encodes a fusion polypeptide of formula III: N-terminal-P6-PL-P7-C-terminal [III] wherein P6 and P7 are each independently a first polypeptide and a second polypeptide, PL is a linker, and PL comprises SEQ ID NO: 337 or 339, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337 or 339.

[0056] In embodiments, a nucleic acid can be provided that includes formula IV: 5'-N6-NL-N7-3' [IV] wherein N6 and N7 each independently encode a first polypeptide and a second polypeptide; NL encodes a linker; and NL comprises SEQ ID NO: 338 or 340, or a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 338 or 340.

[0057] In embodiments, the nucleic acids described herein may be isolated, recombinant, or both isolated and recombinant.

[0058] In embodiments, the vectors described herein may be isolated, recombinant, or both isolated and recombinant.

[0059] In embodiments, the T cells and / or natural killer (NK) cells described herein may be isolated, recombinant, engineered, or a combination thereof.

[0060] In some embodiments, a vector may be provided that includes the nucleic acid described herein. In some embodiments, the vector described herein may further include a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) disposed between the nucleic acid encoding the CD8 alpha chain and the nucleic acid encoding the CD8 beta chain. In some embodiments, the vector may further include a nucleic acid encoding a 2A peptide or an IRES disposed between the nucleic acid encoding the TCR alpha chain and the nucleic acid encoding the TCR beta chain. In some embodiments, the vector may further include a nucleic acid encoding a 2A peptide or an IRES disposed between the nucleic acid encoding the TCR chain or the CD8 chain and the nucleic acid encoding the interleukin described herein. In some embodiments, the 2A peptide may be P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96). In some embodiments, the IRES may be selected from the group consisting of an IRES from a picornavirus, an IRES from a flavivirus, an IRES from a pestivirus, an IRES from a retrovirus, an IRES from a lentivirus, an IRES from an insect RNA virus, and an IRES from a cellular mRNA. In some embodiments, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence selected from Woodchuck PRE (WPRE) (SEQ ID NO: 264), Woodchuck PRE (WPRE) variant 1 (SEQ ID NO: 256), Woodchuck PRE (WPRE) variant 2 (SEQ ID NO: 257), or Hepatitis B virus (HBV) PRE (HPRE) (SEQ ID NO: 437). In some embodiments, the post-transcriptional regulatory element (PRE) sequence may be Woodchuck PRE (WPRE) variant 1 comprising the nucleic acid sequence of SEQ ID NO: 256. In some embodiments, the post-transcriptional regulatory element (PRE) sequence may be Woodchuck PRE (WPRE) variant 2 comprising the nucleic acid sequence of SEQ ID NO: 257.In some embodiments, the vector may further comprise a promoter selected from cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (MNDU3), ubiquitin C promoter, EF-1 alpha promoter, or mouse stem cell virus (MSCV) promoter. In some embodiments, the promoter may be a mouse stem cell virus (MSCV) promoter. In some embodiments, the vector may be a viral vector or a non-viral vector. In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be selected from adenovirus, poxvirus, alphavirus, arenavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, picornavirus, and any combination thereof. In some embodiments, the viral vector may be pseudotyped with an envelope protein of a virus selected from natural feline endogenous virus (RD114), a version of RD114 (RD114TR: a version of RD114), gibbon ape leukemia virus (GALV), a version of GALV (GALV-TR: a version of GALV), amphotropic murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl plague virus (FPV), Ebola virus (EboV), or baboon retrovirus envelope glycoprotein (BaEV), and lymphocytic choriomeningitis virus (LCMV). In some embodiments, the vector may be a lentiviral vector. In some embodiments, the vector may further comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0061] In embodiments, T cells and / or natural killer cells may be provided that express a polypeptide described herein and / or comprise a vector described herein and / or are produced by a method described herein. In embodiments, the T cells described herein may be αβ T cells, γδ T cells, natural killer T cells, or any combination thereof. In embodiments, the αβ T cells may be CD4+ T cells. In embodiments, the αβ T cells may be CD8+ T cells. In embodiments, the γδ T cells may be Vγ9Vδ2+ T cells.

[0062] In some embodiments, a composition comprising the T cells and / or natural killer cells described herein may be provided.In some embodiments, the composition may be a pharmaceutical composition.In some embodiments, the composition may further comprise an adjuvant, an excipient, a carrier, a diluent, a buffer, a stabilizer, or a combination thereof. In embodiments, the adjuvant can be an anti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon alpha, interferon beta, CpG oligonucleotides and derivatives thereof, poly(I:C) and derivatives thereof, RNA, sildenafil, particle formulations comprising poly(lactide-co-glycolide) (PLG), virosomes, interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 21 (IL-21), interleukin 23 (IL-23), or any combination thereof. In embodiments, the adjuvant may be IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof.

[0063] In some embodiments, a method of treating a patient with cancer may be provided, comprising administering to the patient a composition as described herein, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, stomach cancer, and prostate cancer. In some embodiments, a method of eliciting an immune response in a patient with cancer may be provided, comprising administering to the patient a composition as described herein, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, stomach cancer, and prostate cancer. In embodiments, T cells and / or natural killer cells can kill cancer cells that present peptides in complexes with MHC molecules on the cell surface.

[0064] In some embodiments, the CD8 polypeptides described herein may include a CD8α immunoglobulin (Ig)-like domain, a CD8β region, a CD8α transmembrane domain, and a CD8α cytoplasmic domain. In another embodiment, the CD8β region may be a CD8β stalk region or domain.

[0065] In embodiments, the CD8 polypeptide described herein comprises: (a) an immunoglobulin (Ig)-like domain that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) an immunoglobulin (Ig)-like domain that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:2; (c) a CD8 beta region that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:3; and (d) a cytoplasmic domain that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.

[0066] In embodiments, the CD8 polypeptides described herein have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0067] In embodiments, the CD8 polypeptides described herein have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0068] In embodiments, the CD8 polypeptides described herein can include one or more signal peptides having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NO:6, SEQ ID NO:293 or SEQ ID NO:294 fused to the N-terminus or C-terminus of a CD8 polypeptide described herein.

[0069] In some embodiments, the CD8 polypeptides described herein may include (a) SEQ ID NO:1, which includes 1, 2, 3, 4, or 5 amino acid substitutions; (b) SEQ ID NO:2, which includes 1, 2, 3, 4, or 5 amino acid substitutions; (c) SEQ ID NO:3, which includes 1, 2, 3, 4, or 5 amino acid substitutions; and (d) SEQ ID NO:4, which includes 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the substitutions may be conservative or non-conservative. In some embodiments, the amino acid substitutions may be conservative amino acid substitutions.

[0070] In embodiments, the CD8 polypeptide described herein can be a CD8α polypeptide or a modified CD8α polypeptide.

[0071] In embodiments, the CD8 polypeptide described herein can be a CD8αβ polypeptide or a modified CD8α polypeptide.

[0072] In embodiments, the CD8 beta polypeptide may comprise the amino acid sequence of any one of SEQ ID NOs: 8, 9, 10, 11, 12, 13, or 14.

[0073] In some embodiments, the TCR alpha and beta chains are selected from the group consisting of SEQ ID NOs: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 82 and 83, 84 and 85, 86 and 87, 88 and 89, 89 and 90, 90 and 91, 91 and 92, 92 and 93, 93 and 94, 94 and 95, 95 and 96, 96 and 97, 97 and 98, 98 and 99, 99 and 100, 99 and 101, 99 and 102, 99 and 103, 99 and 104, 99 and 105, 99 and 106, 99 and 107, 99 and 108, 109 and 109, 109 and 109 In one embodiment, the aryl group may be selected from 1 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, or 91 and 92.

[0074] In embodiments, the isolated nucleic acid may comprise a nucleic acid sequence encoding a T cell receptor comprising an alpha and beta chain, and a CD8 polypeptide comprising an alpha and beta chain. In embodiments, the CD8 polypeptide may comprise a CD8 alpha chain and / or a CD8 beta chain, and the CD8 alpha chain and / or the CD8 beta chain may be independently modified or unmodified. The isolated nucleic acid may comprise a nucleic acid at least about 80% identical to the nucleic acid sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301. The isolated nucleic acid can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301.

[0075] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0076] In embodiments, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO:267.

[0077] In embodiments, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO:279.

[0078] In embodiments, the isolated polypeptide can be encoded by a nucleic acid described herein or by a nucleic acid that, due to codon degeneracy, encodes the same polypeptide.

[0079] In embodiments, the isolated polypeptide may comprise an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302. The amino acid sequence may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302. In another embodiment, SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302 comprises 1, 2, 3, 4, 5, 10, 15, or 20 or more amino acid substitutions or deletions. In yet another embodiment, SEQ ID NO: 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 296, 298, 300, or 302 comprises up to 1, 2, 3, 4, 5, 10, 15, or 20 amino acid substitutions or deletions.

[0080] In embodiments, the isolated polypeptide may comprise the amino acid sequence of SEQ ID NO:268.

[0081] In embodiments, the isolated polypeptide may comprise the amino acid sequence of SEQ ID NO:280.

[0082] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0083] In embodiments, the disclosure provides a vector comprising a nucleic acid encoding a polypeptide described herein.

[0084] In embodiments, one or more vectors may contain a nucleic acid encoding an IL-12p35 / IL-12p40 fusion polypeptide.

[0085] In embodiments, one or more vectors may contain a nucleic acid encoding an IL-15 polypeptide.

[0086] In embodiments, one or more vectors may contain a nucleic acid encoding an IL-18 polypeptide.

[0087] In embodiments, one or more vectors can comprise a nucleic acid encoding a CD8 polypeptide. In embodiments, the CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0088] In embodiments, one or more vectors can include a nucleic acid encoding a CD8 α polypeptide. In embodiments, the CD8 polypeptide can include a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0089] In embodiments, one or more vectors can include a nucleic acid encoding a CD8 β polypeptide. In embodiments, the CD8 polypeptide can include a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0090] In some embodiments, the one or more vectors may comprise one or more nucleic acids encoding a T cell receptor (TCR) comprising an alpha and beta chain. In some embodiments, the one or more vectors may comprise one or more nucleic acids encoding a T cell receptor (TCR) comprising a gamma and delta chain. In other embodiments, the one or more vectors may comprise one or more nucleic acids encoding a chimeric antigen receptor (CAR).

[0091] In embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain may independently be modified or unmodified.

[0092] In some embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of a TCR comprising an alpha and beta chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of a TCR comprising a gamma and delta chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of a CAR, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 alpha chain and / or a CD8 beta chain, and the CD8 alpha chain and / or the CD8 beta chain may be independently modified or unmodified.

[0093] In some embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of (i) a TCR comprising an alpha and beta chain, (ii) a CD8 polypeptide, and (iii) an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of (i) a TCR comprising a gamma and delta chain, (ii) a CD8 polypeptide, and (iii) an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding one or any combination of (i) a CAR, (ii) a CD8 polypeptide, and (iii) an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0094] In some embodiments, a vector may be provided that includes one or more nucleic acids encoding (i) a TCR comprising an alpha chain and a beta chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding (i) a TCR comprising a gamma chain and a delta chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding (i) a CAR, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide.

[0095] In some embodiments, a vector may be provided that includes one or more nucleic acids encoding a TCR comprising an α chain and a β chain, and a CD8 polypeptide. In some embodiments, a cell may be provided that includes a TCR comprising a γ chain and a δ chain, and a nucleic acid encoding a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding a CAR and a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0096] In embodiments, two or more vectors may be co-transduced into one or more cells, co-expressed in one or more cells, or any combination thereof, In embodiments, the cells may include αβ T cells, γδ T cells, natural killer (NK) cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0097] In embodiments, the two or more vectors can comprise a nucleic acid encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain can be independently modified or unmodified.

[0098] In embodiments, a vector may comprise a nucleic acid encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain may independently be modified or unmodified.

[0099] In some embodiments, the nucleic acid may be polycistronic and one or more polycistronic nucleic acids may be utilized. Expression of multiple (e.g., 2, 3, 4, 5, or more) polypeptides from a polycistronic nucleic acid may be achieved by any suitable method, such as, for example, i) splicing of pre-mRNA, ii) proteolytic cleavage sites, iii) fusion proteins, iv) inclusion of one or more 2A peptide-encoding nucleic acids (such as, but not limited to, P2A, T2A, E2A, and F2A), v) inclusion of one or more internal ribosome entry sites (IRES). Each of these methods has some advantages and disadvantages for providing multiple transcription units. The most widely used of the five methods are the self-cleaving 2A peptide and the IRES. In some embodiments, the nucleic acid may be monocistronic and one or more monocistronic nucleic acids may be utilized.

[0100] In embodiments, the 2A peptide may be selected from P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

[0101] In some embodiments, the IRES may be selected from the group consisting of an IRES from a picornavirus, an IRES from a flavivirus, an IRES from a pestivirus, an IRES from a retrovirus, an IRES from a lentivirus, an IRES from an insect RNA virus, and an IRES from a cellular mRNA.

[0102] In several embodiments, the vector may contain a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) positioned between the nucleic acid encoding the modified CD8 α polypeptide and the nucleic acid encoding the CD8 β polypeptide.

[0103] In embodiments, the vector may comprise a nucleic acid encoding a 2A peptide positioned between a nucleic acid encoding a TCR alpha chain and a nucleic acid encoding a TCR beta chain.

[0104] In embodiments, a vector may include a nucleic acid encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α and β chain, a TCR comprising a γ and δ chain, and / or a CAR, and the vector may include a nucleic acid encoding either a polypeptide or a fusion polypeptide, or a 2A peptide or an internal ribosome entry site (IRES) disposed between each of the nucleic acids encoding the polypeptides or fusion polypeptides. In embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain may be independently modified or unmodified.

[0105] In some embodiments, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence. In some embodiments, the post-transcriptional regulatory element (PRE) sequence may be selected from a Woodchuck Hepatitis Virus PRE (WPRE) (e.g., but not limited to, a wild-type WPRE, such as SEQ ID NO: 264, or a mutant WPRE, such as, but not limited to, WPREmut1 (SEQ ID NO: 256) or WPREmut2 (SEQ ID NO: 257)), or a Hepatitis B Virus (HBV) PRE (HPRE) (SEQ ID NO: 385), a variant thereof, or any combination thereof.

[0106] In some embodiments, the vector may further comprise one or more promoters, which may be selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer (MNDU3), a ubiquitin C promoter, an EF-1 alpha promoter, a murine stem cell virus (MSCV) promoter, a promoter from CD69, a nuclear factor of activated T-cells (NFAT) promoter, an IL-2 promoter, a minimal IL-2 promoter, or a combination thereof.

[0107] In embodiments, the vector may include one or more Kozak sequences. In embodiments, the Kozak sequence may initiate, increase, or enhance translation, or a combination thereof. In embodiments, the Kozak sequence may be GCCACC. In embodiments, the Kozak sequence may be ACCATGG. In embodiments, the Kozak sequence may be GCCNCCATGG, where N is a purine (A or G) (SEQ ID NO: 384).

[0108] In embodiments, the vector may contain one or more factor Xa sites.

[0109] In some embodiments, the vector may include one or more enhancers. In some embodiments, the enhancer may include Conserved Non-Coding Sequence (CNS) 0, CNS1, CNS2, CNS3, CNS4, or a portion thereof, or any combination thereof.

[0110] In embodiments, the vector may be a viral vector or a non-viral vector.

[0111] In embodiments, the vector may be selected from an adenovirus, a poxvirus, an alphavirus, an arenavirus, a flavivirus, a rhabdovirus, a retrovirus, a lentivirus, a herpesvirus, a paramyxovirus, a picornavirus, or a combination thereof.

[0112] In embodiments, the vector may be pseudotyped with an envelope protein of a virus selected from native feline endogenous virus (RD114), chimeric RD114 (RD114TR), gibbon ape leukemia virus (GALV), chimeric GALV (GALV-TR), amphotropic murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl plague virus (FPV), Ebola virus (EboV), or baboon retrovirus envelope glycoprotein (BaEV), and lymphocytic choriomeningitis virus (LCMV), or a combination thereof.

[0113] In embodiments, the disclosure provides one or more cells transformed with and / or expressing one or more vectors comprising a nucleic acid encoding a polypeptide.

[0114] In embodiments, the cells may include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0115] In embodiments, the T cells may be CD4+ T cells.

[0116] In embodiments, the T cells may be CD8+ T cells.

[0117] In embodiments, the T cells may be CD4+ / CD8+ T cells.

[0118] In embodiments, the T cells may be αβ T cells.

[0119] In embodiments, the T cells may be γδ T cells.

[0120] In some embodiments, the T cells may be αβ T cells and may express a CD8 polypeptide as described herein. In some embodiments, the CD8 polypeptide may include a CD8α chain and / or a CD8β chain, which may be independently modified or unmodified. In some embodiments, the T cells may be αβ T cells and may express a modified CD8 polypeptide as described herein, e.g., a modified CD8α polypeptide, or a modified CD8α polypeptide comprising a CD8β stalk region, e.g., m1CD8α of constructs #11 and #12 (FIG. 4), or CD8α* (FIG. 55B). In some embodiments, the T cells may be αβ T cells and may express one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, and / or a CAR. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0121] In some embodiments, the T cells may be γδ T cells and may express a CD8 polypeptide as described herein. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, which may be independently modified or unmodified. In some embodiments, the T cells may be γδ T cells and may express a modified CD8 polypeptide as described herein, e.g., a modified CD8 α polypeptide, or a modified CD8 α polypeptide comprising a CD8 β stalk region, e.g., m1CD8α as in constructs #11 and #12 (FIG. 4), or CD8α* (FIG. 55B). In some embodiments, the T cells may be γδ T cells and may express one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, and / or a CAR. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0122] In embodiments, a cell may be provided that comprises one or more nucleic acids encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain may independently be modified or unmodified.

[0123] In some embodiments, a cell may be provided that includes one or more nucleic acids encoding one or any combination of a TCR comprising an alpha and beta chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, a cell may be provided that includes one or more nucleic acids encoding one or any combination of a TCR comprising a gamma and delta chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, a cell may be provided that includes one or more nucleic acids encoding one or any combination of a CAR, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 alpha chain and / or a CD8 beta chain, and the CD8 alpha chain and / or the CD8 beta chain may be independently modified or unmodified.

[0124] In some embodiments, a cell may be provided that includes one or more nucleic acids encoding (i) a TCR comprising an alpha and beta chain, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a cell may be provided that includes one or more nucleic acids encoding (i) a TCR comprising a gamma and delta chain, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a cell may be provided that includes one or more nucleic acids encoding (i) a CAR, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0125] In some embodiments, a cell may be provided that comprises one or more nucleic acids encoding (i) a TCR comprising an alpha chain and a beta chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a cell may be provided that comprises one or more nucleic acids encoding (i) a TCR comprising a gamma chain and a delta chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, a cell may be provided that comprises one or more nucleic acids encoding (i) a CAR, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide.

[0126] In some embodiments, a cell may be provided that includes a TCR comprising an alpha and beta chain, and one or more nucleic acids encoding a CD8 polypeptide. In some embodiments, a cell may be provided that includes a TCR comprising a gamma and delta chain, and one or more nucleic acids encoding a CD8 polypeptide. In some embodiments, a cell may be provided that includes a CAR, and one or more nucleic acids encoding a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 alpha chain and / or a CD8 beta chain, and the CD8 alpha chain and / or the CD8 beta chain may be independently modified or unmodified.

[0127] In embodiments, the one or more nucleic acids may be contained in and / or expressed from a vector.

[0128] In embodiments, the cells may include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0129] In some embodiments, a population of cells as described herein may be provided. As a non-limiting example, the present disclosure provides an exogenous CD8 co-receptor comprising a polypeptide as described herein, e.g., an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 99%, or about 100% to SEQ ID NO:5, 7, 258, 259, 8, 9, 10, 11, 12, 13, or 14; an IL-12p35 / IL-12p40 fusion polypeptide, e.g., an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 99%, or about 100% to SEQ ID NO:309; a polypeptide, e.g., an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 99%, or about 100% to SEQ ID NO: 311 or 313; an IL-18 polypeptide, e.g., an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 99%, or about 100% to SEQ ID NO: 315; and / or a T cell receptor. In some embodiments, the population of cells can include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0130] In some embodiments, a method of preparing cells for immunotherapy may include isolating cells from a blood sample of a human subject, activating the isolated cells, transducing the activated cells with one or more vectors, and expanding the transduced cells. In some embodiments, the cells may include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0131] In embodiments, a method of treating a patient with cancer can include administering to the patient a composition comprising a population of expanded cells, where the cells kill cancer cells that present a peptide in a complex with an MHC molecule on the cell, the peptide is selected from SEQ ID NOs: 98-255, and the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, renal cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, gastric cancer, prostate cancer, or a combination thereof. In embodiments, the cells can include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0132] In embodiments, the composition may further comprise an adjuvant.

[0133] In embodiments, the adjuvant may be selected from anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon alpha, interferon beta, CpG oligonucleotides and derivatives thereof, poly(I:C) and derivatives thereof, RNA, sildenafil, particle formulations comprising poly(lactide-co-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, IL-23, or any combination thereof.

[0134] In embodiments, a method of eliciting an immune response in a patient having cancer can include administering to the patient a composition comprising a population of expanded cells, where the cells kill cancer cells that present a peptide in a complex with an MHC molecule on the cell, where the peptide is selected from SEQ ID NOs: 98-255, and the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, colorectal cancer, bladder cancer, renal cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, gastric cancer, prostate cancer, or a combination thereof. In embodiments, the cells can include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof. [Brief description of the drawings]

[0135] [Figure 1] 1 shows a representative CD8α subunit, e.g., SEQ ID NO: 258 (CD8α1). In this embodiment, CD8α1 contains five domains: (1) a signal peptide, (2) an Ig-like domain-1, (3) a stalk region, (4) a transmembrane (TM) domain, and (5) a cytoplasmic tail (Cyto) containing an lck-binding motif.

[0136] [Diagram 2] FIG. 2 shows a sequence alignment between CD8α1 (SEQ ID NO: 258) and m1CD8α (SEQ ID NO: 7).

[0137] [Diagram 3] FIG. 3 shows a sequence alignment between CD8α2 (SEQ ID NO: 259) and m2CD8α (SEQ ID NO: 262), in which the cysteine ​​substitution at position 112 is indicated with an arrow.

[0138] [Figure 4]Figure 4 shows an exemplary vector according to an aspect of the present disclosure. In some embodiments, the vector may also include additional elements, such as those described herein, including but not limited to, one or more promoters or one or more post-transcriptional regulatory elements. In Figure 4, in some embodiments, the lines may represent direct junctions without intervening sequences, or may represent intervening sequences, such as but not limited to, linkers, furin, sequences encoding 2A polypeptides, factor Xa sites, untranslated sequences, translated sequences, sequences including one or more restriction endonuclease sites, or combinations thereof.

[0139] [Diagram 5] FIG. 5A shows the titers of the viral vectors shown in FIG.

[0140] Figure 5B shows the titers of additional viral vectors according to embodiments of the present disclosure: Construct #13; Construct #14; Construct #15; Construct #16; Construct #17; Construct #18; Construct #19; Construct #21; Construct #10n; Construct #11n; and TCR:R11KEA (SEQ ID NO:15 and SEQ ID NO:16) (Construct #8), which binds to PRAME-004 (SLLQHLIGL) (SEQ ID NO:147). Note that constructs #10 and #10n are different batches of the same construct (SEQ ID NO:291 and 292), and constructs #11 and #11n are also different batches of the same construct (SEQ ID NO:285 and 286).

[0141] [Figure 6] FIG. 6 shows the production of T cells.

[0142] [Figure 7] FIG. 7A shows the expression of activation markers in CD3+CD8+ cells before and after activation.

[0143] FIG. 7B shows the expression of activation markers in CD3+CD4+ cells before and after activation.

[0144] [Figure 8]Figure 8A shows the amplification fold of cells transduced with various constructs from donor #1. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control). Note that constructs #9 and #9b are different batches of the same construct (SEQ ID NOs: 287 and 288).

[0145] Figure 8B shows the fold expression of cells transduced with various constructs from donor #2. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR=R11KEA.WPRE wt (TCR with wild type WPRE) (construct #8); NT = non-transduced T cells (negative control).

[0146] [Figure 9A] FIG. 9A shows a flow plot of cells transduced with construct #9.

[0147] [Figure 9B] FIG. 9B shows a flow plot of cells transduced with construct #10 according to one embodiment of the present disclosure.

[0148] [Figure 9C] FIG. 9C shows a flow plot of cells transduced with construct #11.

[0149] [Figure 9D] FIG. 9D shows a flow plot of cells transduced with construct #12.

[0150] [Figure 10]Figure 10 shows the % CD8+CD4+ of cells transduced with various constructs from donor #1 and donor #2. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0151] [Figure 11] Figure 11 shows the % Tet of CD8+CD4+ cells transduced with various constructs. Constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0152] [Figure 12] Figure 12 shows the Tet MFI (CD8+CD4+Tet+) of cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0153] [Figure 13] Figure 13 shows the CD8α MFI (CD8+CD4+Tet+) of cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR=R11KEA.WPREwt (TCR with wild type WPRE); NT=non-transduced T cells (negative control).

[0154] [Figure 14]Figure 14 shows the percentage of CD8+CD4+ (of CD3+) cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0155] [Figure 15] Figure 15 shows the percentage of CD8+Tet+ (of CD3+) cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0156] [Figure 16] Figure 16 shows the Tet MFI (CD8+Tet+) of cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0157] [Figure 17] Figure 17 shows the CD8α MFI (CD8+Tet+) of cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR=R11KEA.WPREwt (TCR with wild type WPRE); NT=non-transduced T cells (negative control).

[0158] [Figure 18]Figure 18 shows the % Tet+ (of CD3+) among cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR = R11KEA.WPREwt (TCR with wild type WPRE); NT = non-transduced T cells (negative control).

[0159] [Figure 19] Figure 19 shows the VCN (upper panel) and CD3+Tet+ / VCN (lower panel) of cells transduced with various constructs. The constructs are as follows: Construct #9b; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; TCR=R11KEA.WPREwt (TCR with wild type WPRE); NT=non-transduced T cells (negative control).

[0160] [Figure 20A] Figures 20A-20C present data showing that constructs (#10, #11, and #12) are equivalent to TCR alone in mediating cytotoxicity against target-positive cell lines expressing antigen at different levels (UACC257 at 1081 copies per cell, A375 at 50 copies per cell). [Figure 20B] Same as above. [Figure 20C] Same as above.

[0161] [Figure 21] Figures 21A-21B show data showing that IFNγ secretion in response to UACC257 was comparable among the constructs, while in A375, expression of #10 was the highest among all constructs. However, when comparing #9 and #11 expressing wild type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 induced a stronger cytokine response measured as IFNγ quantified in the supernatant from Incucyte plates. Construct #9; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; Construct #8 = R11KEA TCR only.

[0162] [Figure 22] Figure 22 shows an exemplary experimental design for evaluating DC maturation and cytokine secretion by PBMC-derived products in response to targets UACC257 and A375. N=2.

[0163] [Figure 23] Figures 23A-23B show data showing that IFNγ secretion in response to A375 is increased in the presence of iDCs. In triple co-cultures with iDCs, IFNγ secretion is higher in construct #10 compared to the other constructs. However, when comparing construct #9 and construct #11 expressing wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with #11 strongly induced cytokine responses measured as IFNγ quantified in culture supernatants of three-dimensional co-cultures using donor D600115, E:T:iDC::1:1 / 10:1 / 4. Construct #9; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; Construct #8 = R11KEA TCR only.

[0164] [Figure 24] Figures 24A-24B show data showing that IFNγ secretion in response to A375 is increased in the presence of iDC. In triple co-cultures with iDC, IFNγ secretion was higher in construct #10 compared to other constructs. IFNγ was quantified in culture supernatants of three-dimensional co-cultures using donor D150081, E:T:iDC::1:1 / 10:1 / 4. Construct #9; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; Construct #8 = R11KEA TCR only.

[0165] [Diagram 25]Figures 25A-25B show data showing that IFNγ secretion in response to UACC257 is increased in the presence of iDCs. In triple co-cultures with iDCs, IFNγ secretion is higher in construct #10 compared to the other constructs. However, when comparing construct #9 and construct #11 expressing wild-type and modified CD8 co-receptor sequences, respectively, T cells transduced with construct #11 strongly induced cytokine responses measured as IFNγ quantified in culture supernatants of three-dimensional co-cultures using donor D600115, E:T:iDC::1:1 / 10:1 / 4. Construct #9; Construct #10; Construct #11; Construct #12; Construct #1; Construct #2; Construct #8 = R11KEA TCR only.

[0166] [Figure 26] FIG. 26 shows T cell production according to one embodiment of the present disclosure.

[0167] [Figure 27] FIG. 27A shows the expression of activation markers before and after activation in CD3+CD8+ cells.

[0168] FIG. 27B shows expression of activation markers before and after activation in CD3+CD4+ cells according to one embodiment of the present disclosure.

[0169] [Figure 28] FIG. 28 shows the amplification fold of cells transduced with the various constructs.

[0170] [Figure 29A] 29A-29B show the % CD8+CD4+ cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 29B] Same as above.

[0171] [Figure 30A] 30A-30B show the % Tet of CD8+CD4+ cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 30B] Same as above.

[0172] [Figure 31A] 31A-31B show the Tet MFI (CD8+CD4+Tet+) of cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 31B] Same as above.

[0173] [Figure 32A] 32A-32B show the percentage of CD8+CD4- (of CD3+) cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 32B] Same as above.

[0174] [Figure 33A] 33A-33B show the % CD8+Tet+ (of CD3+) cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 33B] Same as above.

[0175] [Figure 34A] 34A-34B show the Tet MFI (CD8+Tet+) of cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 34B] Same as above.

[0176] [Figure 35A] 35A-35B show the % Tet+ (of CD3+) cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 35B] Same as above.

[0177] [Figure 36A] 36A-36B show the VCN of cells transduced with various constructs according to one embodiment of the present disclosure. [Figure 36B] Same as above.

[0178] [Figure 37]FIG. 37 shows T cell production according to one embodiment of the present disclosure.

[0179] [Figure 38] FIG. 38 shows the % Tet CD8+CD4+ among cells transduced with the various constructs.

[0180] [Figure 39] FIG. 39 shows the Tet MFI of CD8+CD4+Tet+ among cells transduced with various constructs.

[0181] [Diagram 40] FIG. 40 shows the Tet MFI of CD8+Tet+ cells transduced with various constructs.

[0182] [Diagram 41] FIG. 41 shows the % Tet+ CD3+ cells transduced with various constructs.

[0183] [Diagram 42] FIG. 42 shows the vector copy number (VCN) of cells transduced with the various constructs.

[0184] [Diagram 43] Figure 43 shows the % proportion of T cell subsets in cells transduced with various constructs. FACS analysis was gated on CD3+TCR+.

[0185] [Figure 44A] Figures 44A-44B show the % proportion of T cell subsets in cells transduced with various constructs. FACS analysis was gated on CD4+CD8+ in Figure 44A and CD4-CD8+TCR+ in Figure 44B. [Figure 44B] Same as above.

[0186] [Figure 45A]Figures 45A-45B show data demonstrating that construct #13 and construct #10 are equivalent to TCR alone in mediating cytotoxicity against a UACC257 target positive cell line expressing high levels of antigen (1081 copies per cell). Construct #15 was also effective but caused slower killing compared to construct #13 and construct #10. The effector:target ratio used to generate these results was 4:1. [Figure 45B] Same as above.

[0187] [Diagram 46] Figure 46 shows that IFNγ secretion in response to UACC257 cell line was higher with construct #13 compared to construct #10. IFNγ was quantified in the supernatant from the Incucyte plates. The effector:target ratio used to generate these results was 4:1.

[0188] [Figure 47] Figure 47 shows ICI marker frequencies (2B4, 41BB, LAG3, PD-1, TIGIT, TIM3, CD39+CD69+, and CD39-CD69-).

[0189] [Figure 48-1] Figures 48A-48G show increased expression of IFNγ, IL-2, and TNFα using CD4+CD8+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis was gated on CD3+CD4+CD8+ cells against UACC257, E:T of 4:1. [Figure 48-2] Same as above. [Figure 48-3] Same as above. [Figure 48-4] Same as above.

[0190] [Figure 49-1]Figures 49A-49G show increased expression of IFNγ, IL-2, MIP-1β and TNFα using CD4-CD8+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis was gated on CD3+CD4-CD8+ cells against UACC257, E:T of 4:1. [Figure 49-2] Same as above. [Figure 49-3] Same as above. [Figure 49-4] Same as above.

[0191] [Figure 50-1] Figures 50A-50G show increased expression of IL-2 and TNFα using CD3+TCR+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. FACS analysis was gated on CD3+TCR+ cells against UACC257, E:T of 4:1. [Figure 50-2] Same as above. [Figure 50-3] Same as above. [Figure 50-4] Same as above.

[0192] [Figure 51] 51A-51C show the results of FACS analysis gated on CD4+CD8+ cells for A375, CD4:1 E:T.

[0193] [Figure 52] 52A-52C show the results of FACS analysis gated on CD4-CD8+ cells for A375, CD4:1 E:T.

[0194] [Figure 53] 53A-53C show the results of FACS analysis gated on CD3+TCR+ cells for A375, CD4:1 E:T.

[0195] [Figure 54] FIG. 54 shows T cell production according to one embodiment of the present disclosure.

[0196] [Figure 55] Figures 55A-55C show the interaction between peptide / MHC complexes of T cells and antigen presenting cells (APCs) by binding a complex of TCR and a CD8αβ heterodimer (Figure 55A, e.g., generated by transducing T cells with constructs #2, #3, #4, #10, #13, #14, #15, #16, #17, #18, or #21), a complex of TCR and a homodimeric CD8α (CD8αα*) in which the stalk region has been replaced with a CD8β stalk region (Figure 55B, e.g., generated by transducing T cells with constructs #11, #12, or #19), and a complex of TCR and a CD8α homodimer (Figure 55C, e.g., generated by transducing T cells with constructs #1, #5, #6, #7, or #9).

[0197] [Figure 56] FIG. 56 shows the levels of IL-12 secretion by dendritic cells (DCs) in the presence of CD4+ T cells and immature dendritic cells transduced with constructs #10 or #11, according to one embodiment of the present disclosure.

[0198] [Figure 57] FIG. 57 shows the level of TNF-α secretion by dendritic cells (DCs) in the presence of CD4+ T cells and immature dendritic cells transduced with constructs #10 or #11, according to one embodiment of the present disclosure.

[0199] [Figure 58] FIG. 58 shows the levels of IL-6 secretion by dendritic cells (DCs) in the presence of CD4+ T cells and immature dendritic cells transduced with constructs #10 or #11, according to one embodiment of the present disclosure.

[0200] [Figure 59]FIG. 59 shows a scheme for determining the level of cytokine secretion by dendritic cells (DCs) in the presence of PBMCs and target cells transduced with various constructs according to one embodiment of the present disclosure.

[0201] [Figure 60] FIG. 60 shows the levels of IL-12 secretion by dendritic cells (DCs) in the presence of PBMCs and target cells transduced with various constructs according to one embodiment of the present disclosure.

[0202] [Figure 61] FIG. 61 shows the levels of TNF-α secretion by dendritic cells (DCs) in the presence of PBMCs and target cells transduced with various constructs, according to one embodiment of the present disclosure.

[0203] [Figure 62] FIG. 62 shows the levels of IL-6 secretion by dendritic cells (DCs) in the presence of PBMCs and target cells transduced with various constructs according to one embodiment of the present disclosure.

[0204] [Figure 63A] Figures 63A-63C show IFNγ production from transduced CD4+ selected T cells obtained from donor #1 (Figure 63A), donor #2 (Figure 63B), and donor #3 (Figure 63C) according to one embodiment of the present disclosure. [Figure 63B] Same as above [Figure 63C] Same as above

[0205] [Fig. 63D] Figure 63D shows the EC50 values ​​(ng / ml) in Figures 63A to 63C.

[0206] [Fig. 64A]Figures 64A-64C show IFNγ production from transduced PBMCs obtained from donor #4 (Figure 64A), donor #1 (Figure 64B), and donor #3 (Figure 64C), and their respective EC50 values ​​(mg / ml), according to one embodiment of the present disclosure. [Fig. 64B] Same as above. [Fig. 64C] Same as above.

[0207] [Fig.64D] Figure 64D shows a comparison of EC50 values ​​(ng / ml) between the various donors in Figures 64A-64C.

[0208] [Figure 65A] Figures 65A-65C show IFNγ production from transduced PBMCs (Figure 65A), CD8+ selected T cells (Figure 65B), and CD4+ selected T cells (Figure 65C) from a single donor, and their respective EC50 values ​​(ng / ml), according to one embodiment of the present disclosure. [Figure 65B] Same as above. [Figure 65C] Same as above.

[0209] [Figure 66] 66 shows schematic examples of IL-12, IL-15, and IL-18 secreted from cells transduced to express them, according to embodiments of the present disclosure. In embodiments, the secreted IL-12, IL-15, and IL-18 may each act in cis on the secreting cell and / or in trans on other cells, e.g., other T cells.

[0210] [Figure 67] FIG. 67A shows an exemplary construct comprising IL-12β (or a sequence encoding it) linked to the 5′ end of IL-12α (or a sequence encoding it) via a linker (or a sequence encoding a linker), according to an embodiment of the present disclosure.

[0211] Figure 67B shows an exemplary construct including IL-12α (or a sequence encoding it) linked to the 5' end of IL-12β (or a sequence encoding it) via a linker (or a sequence encoding a linker), according to embodiments of the present disclosure. In embodiments, each linker is independently optional. In embodiments of Figures 67A and 67B, the lines may represent a direct bond with no intervening sequence, or may represent an intervening sequence, such as, but not limited to, a linker, a non-translated sequence, a translated sequence, a sequence including one or more restriction endonuclease sites, or a combination thereof.

[0212] [Figure 68] Figure 68 shows an exemplary vector construct according to embodiments of the present disclosure. In embodiments, the WPRE of the construct shown in Figure 68 can include a Xa site. In embodiments of Figure 68, the line can represent a direct bond with no intervening sequence, or can represent an intervening sequence, such as, but not limited to, a linker, a non-translated sequence, a translated sequence, a sequence including one or more restriction endonuclease sites, or a combination thereof.

[0213] [Figure 69] Figure 69 shows an exemplary vector construct that may be provided in embodiments. In Figure 69, IL-12 FP represents an IL-12p35 / IL-12p40 fusion polypeptide. In some embodiments, the vector may also include additional elements, such as those described herein, including but not limited to, one or more promoters or one or more post-transcriptional regulatory elements. In Figure 69, in some embodiments, the lines may represent direct junctions without intervening sequences, or may represent intervening sequences, including but not limited to, linkers, furin, sequences encoding 2A polypeptides, factor Xa sites, untranslated sequences, translated sequences, sequences including one or more restriction endonuclease sites, or combinations thereof.

[0214] [Figure 70]Figure 70 shows an exemplary vector construct that may be provided in embodiments. In some embodiments, the vector may also include additional elements, such as those described herein, including but not limited to, one or more promoters or one or more post-transcriptional regulatory elements. In Figure 70, in some embodiments, the lines may represent direct junctions without intervening sequences, or may represent intervening sequences, such as but not limited to, linkers, furin, sequences encoding 2A polypeptides, factor Xa sites, untranslated sequences, translated sequences, sequences including one or more restriction endonuclease sites, or combinations thereof.

[0215] [Figure 71] Figure 71 shows an exemplary vector construct that may be provided in embodiments. In some embodiments, the vector may also include additional elements, such as those described herein, including but not limited to, one or more promoters or one or more post-transcriptional regulatory elements. In Figure 71, the lines may represent direct junctions without intervening sequences, or may represent intervening sequences, including but not limited to, linkers, furin, sequences encoding 2A polypeptides, factor Xa sites, untranslated sequences, translated sequences, sequences including one or more restriction endonuclease sites, or combinations thereof.

[0216] [Figure 72] Figures 72-74 show examples of total cell counts (Figure 72), fold expansion (Figure 73), and viability (Figure 74) for non-transduced cells (NT), TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR). Data are grouped (n=4) and presented as the mean, taken at harvest on day 7. [Figure 73] Same as above. [Figure 74] Same as above.

[0217] [Figure 75] Figures 75-77 show examples of total cell counts (Figure 75), fold expansion (Figure 76), and viability (Figure 77) for non-transduced cells (NT), TCR-only transduced cells (TCR), secreted IL-15 only transduced cells (sIL15 only), and secreted IL-15 and TCR transduced cells (sIL15+TCR). Data are grouped (n=3) and presented as the mean, taken at harvest on day 7. [Figure 76] Same as above. [Figure 77] Same as above.

[0218] [Figure 78] Figure 78 shows an example of TCR expression frequency on CD8+ cells by non-transduced cells (NT), TCR only transduced cells (TCR), IL-18 only transduced cells (IL18), IL-18 and TCR transduced cells (IL18+TCR), IL-12 only transduced cells (IL12), and IL-12 and TCR transduced cells (IL12+TCR). Data are grouped (n=4) and presented as the mean, and were obtained using a tetramer+cytokine panel.

[0219] [Figure 79] Figure 79 shows an example of the frequency of expression of IL-12 and TCR on CD8+ cells by non-transduced cells (NT), TCR only transduced cells (TCR), IL-18 only transduced cells (IL18), IL-18 and TCR transduced cells (IL18+TCR), IL-12 only transduced cells (IL12), and IL-12 and TCR transduced cells (IL12+TCR). Data are grouped (n=4) and presented as the mean, obtained using a tetramer+cytokine panel.

[0220] [Figure 80]FIG. 80 shows exemplary concentrations (in pg / mL) of IL-18 secreted by cells transduced with 10 μL, 5 μL, 2.5 μL, or 1.25 μL of vector encoding an IL-18 polypeptide under the control of the MSCV promoter. Data were collected using an L-18 ELISA assay. Human IL-18 ELISA was performed using the Human Total IL-18 Quantikine QuicKit ELISA from R&D Systems, following the manufacturer's protocol, using a Synergy2 microplate reader with plate reading at a wavelength of 450 nm. Data analysis was performed using Prism / GraphPad statistical software. Data were grouped (n=2) and expressed as the mean. PMA / ionomycin stimulation was performed using eBioscience Cell Stimulation cocktail (500X), which consisted of PMA / ionomycin at a final concentration of approximately 1X. The dotted line indicates the detection limit of the IL-18 ELISA assay.

[0221] [Figure 81]Figure 81 shows exemplary concentrations (in pg / mL) of secreted IL-15 in the supernatants of cells from two donors transduced with varying amounts of vector encoding secreted IL-15. The vector amounts used were 20 μL, 10 μL, 5 μL, 2.5 μL, or 1.25 μL per 1e6 cells. Non-transduced cells (NT) were assayed as a control. Data were obtained via ELISA, and assays were performed in triplicate for each transduction condition for each donor. Data were expressed as averages. The concentrations of IL-15 produced by cells transduced with 1.25 μL of vector (for donor D150081) and by non-transduced cells were too low to appear on the graph. Donor D319060 had very low detection values ​​for IL-15 produced by cells transduced with 1.25 μL of vector, as shown in Figure 81. Human IL-15 ELISA was performed using the Human IL-15 Quantikine ELISA Kit from R&D Systems according to the manufacturer's protocol using a Synergy2 microplate reader with plate reading at a wavelength of 450 nm. Data analysis was performed using Prism / GraphPad statistical software.

[0222] [Figure 82]FIG. 82 shows an example of the killing kinetics of UACC257 tumor cells expressing red fluorescent protein (RFP) (UACC257-RFP) by non-transduced cells (NT), TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR). UACC257 cells express high levels of the antigen PRAME (antigen preferentially expressed in melanoma). Cells were challenged with UACC257 cells at about 0 hours, about 70 hours, about 140 hours, about 240 hours, and about 320 hours at an effector:target ratio of 4:1. Fold tumor growth of UACC257-RFP cells alone is shown as a control. Data were grouped (n=4), expressed as the mean, and TCR+ normalized. Data was collected using an IncuCyte.

[0223] [Figure 83] Figure 83 shows the exemplary data presented in Figure 82, but omits the data for cells transduced with IL-18 only (IL18) and cells transduced with IL-12 only (IL12).

[0224] [Figure 84] Figure 84 shows exemplary tumor proliferation index of UACC257-RFP cells cultured with non-transduced cells (NT), TCR only transduced cells (TCR), IL-18 only transduced cells (IL18), IL-18 and TCR transduced cells (IL18+TCR), IL-12 only transduced cells (IL12), and IL-12 and TCR transduced cells (IL12+TCR). Cells were cultured at a 4:1 effector:target ratio. The tumor proliferation index of UACC257-RFP cells alone (tumor only condition) is shown as a control. Data are grouped (n=4), expressed as the mean, and TCR+ normalized. The integral area under the curve (AUC) normalized to the tumor only condition is shown.

[0225] [Figure 85] Figure 85 shows the exemplary data presented in Figure 84, but omits the data for cells transduced with IL-18 only (IL18) and cells transduced with IL-12 only (IL12).

[0226] [Figure 86] FIG. 86 shows an example of the killing kinetics of A375 tumor cells expressing red fluorescent protein (RFP) (A375-RFP) by non-transduced cells (NT), TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR). A375 cells express low levels of the antigen PRAME. Cells were challenged with A375 cells at about 0 hours, about 70 hours, about 140 hours, about 240 hours, and about 320 hours at an effector:target ratio of 8:1. Fold tumor growth of A375-RFP cells alone is shown as a control. Data are grouped (n=4), presented as the mean, and TCR+ normalized. Data was collected using an IncuCyte.

[0227] [Figure 87] Figure 87 shows the exemplary data presented in Figure 86, but omits the data for cells transduced with IL-18 only (IL18) and cells transduced with IL-12 only (IL12).

[0228] [Figure 88]Figure 88 shows exemplary tumor proliferation index of A375-RFP cells cultured with non-transduced cells (NT), TCR only transduced cells (TCR), IL-18 only transduced cells (IL18), IL-18 and TCR transduced cells (IL18+TCR), IL-12 only transduced cells (IL12), and IL-12 and TCR transduced cells (IL12+TCR). Cells were cultured at an effector:target ratio of 8:1. The tumor proliferation index of A375-RFP cells alone (tumor only condition) is shown as a control. Data are grouped (n=4), expressed as the mean, and TCR+ normalized. The integral area under the curve (AUC) normalized to the tumor only condition is shown.

[0229] [Figure 89] Figure 89 shows the exemplary data presented in Figure 88, but omits the data for cells transduced with IL-18 only (IL18) and cells transduced with IL-12 only (IL12).

[0230] [Figure 90] FIG. 90 shows exemplary killing kinetics of MCF7 tumor cells expressing green fluorescent protein (GFP) (MCF7-GFP) by non-transduced cells (NT), as well as cells transduced with IL-18 and TCR (IL18+TCR) and cells transduced with IL-12 and TCR (IL12+TCR). MCF7 cells are negative for the antigen PRAME. Cells were challenged with MCF7 cells at about 0 hours at an effector:target ratio of 4:1. Fold tumor growth of MCF7-RFP cells alone is shown as a control. Data were grouped (n=4), expressed as the mean, and TCR+ normalized. Data were collected using an IncuCyte. Co-cultures were imaged approximately every 24 hours for a total of approximately 120 hours.

[0231] [Figure 91]Figure 91 shows exemplary tumor proliferation index of MCF7-GFP cells cultured with non-transduced cells (NT), as well as with IL-18 and TCR-transduced cells (IL18+TCR), and with IL-12 and TCR-transduced cells (IL12+TCR). Cells were cultured at an effector:target ratio of 4:1. The tumor proliferation index of MCF7-RFP cells alone (tumor-only condition) is shown as a control. Data were grouped (n=4) and TCR+ normalized. Results were expressed as the mean. The area under the integral curve (AUC) normalized to the tumor-only condition is shown.

[0232] [Figure 92] FIG. 92 shows an example of killing kinetics of UACC257 tumor cells expressing red fluorescent protein (RFP) (UACC257-RFP) by non-transduced cells (NT), TCR-only transduced cells (TCR), IL-15-only transduced cells (sIL15-only), and IL-15 and TCR-transduced cells (sIL15+TCR). Cells were challenged with UACC257 cells at about 0 hours, about 70 hours, about 140 hours, and about 240 hours at an effector:target ratio of 4:1. Fold tumor growth of UACC257-RFP cells alone is shown as a control. Data was grouped (n=3), expressed as the mean, and TCR+ normalized. Data was collected using an IncuCyte.

[0233] [Figure 93] Figure 93 shows an exemplary tumor proliferation index of UACC257-RFP cells cultured with TCR-only transduced cells (TCR) and TCR-transduced cells with IL-15 (IL15+TCR). Cells were cultured at an effector:target ratio of 4:1. Data were grouped (n=3) and TCR+ normalized. Results were expressed as the mean. The area under the integral curve (AUC) normalized to the tumor-only condition is shown.

[0234] [Figure 94]Figure 94 shows, by way of example, the percentage of CD8+TCR+ cells positive for each of 2B4, 4-1BB, LAG-3, PD-1, TIGIT, TIM-3, CD39 and CD69 before the cells were exposed to antigen-bearing tumor cells. Expression percentages are shown for non-transduced cells (NT), as well as cells transduced with TCR only (TCR), cells transduced with IL-18 only (IL18), cells transduced with IL-18 and TCR (IL18+TCR), cells transduced with IL-12 only (IL12), and cells transduced with IL-12 and TCR (IL12+TCR). Data were grouped (n=4) and expressed as the mean.

[0235] [Figure 95] Figure 95 shows, as an example, the percentage of CD8+TCR+ cells positive for 2B4, 4-1BB, LAG-3, PD-1, TIGIT, TIM-3, CD39 and CD69, respectively, after the fifth challenge of effector cells with UACC257 antigen-bearing tumor cells. The expression percentages are shown for non-transduced cells (NT), as well as cells transduced with TCR only (TCR), cells transduced with IL-18 only (IL18), cells transduced with IL-18 and TCR (IL18+TCR), cells transduced with IL-12 only (IL12), and cells transduced with IL-12 and TCR (IL12+TCR). Data are grouped (n=4) and expressed as the mean.

[0236] [Figure 96]Figure 96 shows, as an example, the percentage of CD8+TCR+ cells positive for 2B4, 4-1BB, LAG-3, PD-1, TIGIT, TIM-3, CD39 and CD69, respectively, after the fifth challenge of effector cells with A375 antigen-bearing tumor cells. The percentages of expression are shown for non-transduced cells (NT), as well as cells transduced with TCR only (TCR), cells transduced with IL-18 only (IL18), cells transduced with IL-18 and TCR (IL18+TCR), cells transduced with IL-12 only (IL12), and cells transduced with IL-12 and TCR (IL12+TCR). Data are grouped (n=4) and expressed as the mean.

[0237] [Figure 97-1] FIG. 97A shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR only transduced cells (TCR), IL-18 only transduced cells (IL18), IL-18 and TCR transduced cells (IL18+TCR), IL-12 only transduced cells (IL12), and IL-12 and TCR transduced cells (IL12+TCR) approximately 16-22 hours after a single challenge with UACC257-RFP cells.

[0238] FIG. 97B shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after a second challenge with UACC257-RFP cells.

[0239] [Figure 97-2]FIG. 97C shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after a third challenge with UACC257-RFP cells.

[0240] FIG. 97D shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after the fourth challenge with UACC257-RFP cells.

[0241] [Figure 97-3] FIG. 97E shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after the fifth challenge with UACC257-RFP cells. Data were grouped (n=4) and presented as the mean. IFNγ concentrations in the culture medium are shown in pg / mL.

[0242] [Figure 98-1]FIG. 98A shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after a single challenge with A375-RFP cells.

[0243] FIG. 98B shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after a second challenge with A375-RFP cells.

[0244] [Figure 98-2] FIG. 98C shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after a third challenge with A375-RFP cells.

[0245] FIG. 98D shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after the fourth challenge with A375-RFP cells.

[0246] [Figure 98-3] FIG. 98E shows exemplary IFNγ production by non-transduced cells (NT), as well as TCR-only transduced cells (TCR), IL-18-only transduced cells (IL18), IL-18 and TCR-transduced cells (IL18+TCR), IL-12-only transduced cells (IL12), and IL-12 and TCR-transduced cells (IL12+TCR) approximately 16-22 hours after the fifth challenge with A375-RFP cells. Data were grouped (n=4) and presented as the mean. IFNγ concentrations in the culture medium are shown in pg / mL.

[0247] [Figure 99-1] FIG. 99A shows exemplary IFNγ production by non-transduced cells (NT), as well as cells transduced with TCR only (TCR), cells transduced with secreted IL-15 only (sIL15), and cells transduced with secreted IL-15 and TCR (sIL15+TCR) approximately 16-22 hours after a single challenge with UACC257-RFP cells.

[0248] FIG. 99B shows exemplary IFNγ production by non-transduced cells (NT), as well as cells transduced with TCR only (TCR), cells transduced with IL-15 only (sIL15), and cells transduced with IL-15 and TCR (sIL15+TCR), approximately 16-22 hours after a second challenge with UACC257-RFP cells.

[0249] [Figure 99-2] Figure 99C shows exemplary IFNγ production by non-transduced cells (NT), as well as cells transduced with TCR only (TCR), cells transduced with IL-15 only (sIL15), and cells transduced with IL-15 and TCR (sIL15+TCR) approximately 16-22 hours after the fourth challenge with UACC257-RFP cells. Data were grouped (n=3) and presented as the mean. IFNγ concentrations in the culture medium are shown in pg / mL.

[0250] [Figure 100-1] Figures 100A-100F show the results of an exemplary intracellular staining panel. Data was acquired for non-transduced cells (NT), cells transduced with TCR only (TCR), cells transduced with IL-18 and TCT (IL18+TCR), and cells transduced with IL-12 and TCR (IL12+TCR) 13 hours after cells were co-cultured with UACC257 tumor cells in the presence of protein transport inhibitors. Data was grouped (n=4) and expressed as the mean. Figure 100A shows, in this example, the percentage of CD107a-positive CD8+TCR+ cells. Figure 100B shows, in this example, the percentage of Granzyme B-positive CD8+TCR+ cells. Figure 96C shows, in this example, the percentage of IFNγ-positive CD8+TCR+ cells. Figure 100D shows, in this example, the percentage of IL-2-positive CD8+TCR+ cells. Figure 100E shows the percentage of MIP1β positive CD8+TCR+ cells in this example. Figure 100F shows the percentage of TNFα positive CD8+TCR+ cells in this example. [Figure 100-2] Same as above. [Figure 100-3] Same as above.

[0251] [Figure 101]Figures 101A-101C show the results of an exemplary intracellular staining panel. Data was obtained 13 hours after cells were co-cultured with UACC257 tumor cells in the presence of protein transport inhibitors. Data was grouped (n=4) and presented as the mean. Figure 101A shows the percentage of TCR-only transduced cells (TCR) that expressed 0-1, 2-4, or 5-6 of the following: CD107a, Granzyme B, IFNγ, IL-2, MIP1β, and TNFα. Figure 101B shows the percentage of IL-18 and TCR transduced cells (IL18+TCR) that expressed 0-1, 2-4, or 5-6 of the following: CD107a, Granzyme B, IFNγ, IL-2, MIP1β, and TNFα. Figure 101C shows the percentage of IL-12 and TCR transduced cells (IL12+TCR) that expressed 0-1, 2-4, or 5-6 of the following: CD107a, granzyme B, IFNγ, IL-2, MIP1β, and TNFα.

[0252] [Figure 102] Figure 102 shows the percentage of TemRA, Tem, T naive / scm, and Tcm cells, graphed individually for four cell donors transduced with TCR only (TCR), IL-18 and TCR (IL18+TCR), or IL-12 and TCR (IL12+TCR) as examples. This Tmem panel was run on cells that were not exposed to antigen-presenting tumor cells. The flow cytometer was gated on CD8+TCR+ cells. Data were expressed as the mean.

[0253] [Figure 103] Figure 103 shows, as an example, the percentage of TemRA, Tem, T naive / scm, and Tcm cells transduced with TCR only (TCR), TCR with IL-18 (IL18+TCR), or TCR with IL-12 (IL12+TCR). Data were grouped (n=4) from four cell donors and presented as the mean. This Tmem panel was performed on cells that were not exposed to antigen-presenting tumor cells. The flow cytometer was gated on CD8+TCR+ cells.

[0254] [Figure 104] Figure 104 shows the percentage of CD27+CD28-, CD27-CD28-, CD27+CD28+, and CD27-CD28+ cells, graphed individually for four cell donors transduced with TCR only (TCR), IL-18 and TCR (IL18+TCR), or IL-12 and TCR (IL12+TCR) as examples. This Tmem panel was performed on cells that were not exposed to antigen-presenting tumor cells. The flow cytometer was gated on CD8+TCR+ cells. Data were expressed as the mean.

[0255] [Figure 105] Figure 105 shows, as an example, the percentage of CD27+CD28-, CD27-CD28-, CD27+CD28+, and CD27-CD28+ cells transduced with TCR only (TCR), TCR with IL-18 (IL18+TCR), or TCR with IL-12 (IL12+TCR). Data were grouped from four cell donors (n=4) and presented as the mean. This Tmem panel was performed on cells that were not exposed to antigen-presenting tumor cells. The flow cytometer was gated on CD8+TCR+ cells.

[0256] [Fig. 106] Figure 106 shows the percentage of TemRA, Tem, T naive / scm, and Tcm cells, graphed individually for three donors transduced with TCR only (TCR), IL-15 only (sIL15 only), or IL-15 and TCR (sIL15+TCR) as examples. Non-transduced cells (NT) were assayed as a control. This Tmem panel was performed on cells that were not exposed to antigen-presenting tumor cells. The flow cytometer was gated on CD3+CD8+ cells. Data were expressed as the mean.

[0257] [Figure 107A]Figures 107A-107D show fold amplification (Figure 107A), TCR frequency (Figure 107B), total number of TCR+ cells (Figure 107C), and secreted IL-15 concentration as measured by IL-15 ELISA (Figure 107D) for cells transduced with TCR only (TCR), IL-15 and TCR (sIL15.TCR), and IL-15 and CD8 TCR (sIL15.CD8βαTCR). Non-transduced cells (NT) were assayed as a control. [Figure 107B] Same as above. [Figure 107C] Same as above. [Fig. 107D] Same as above.

[0258] [Figure 108A] Figures 108A-108B show examples of cytolytic activity against UAC257-RFP tumor cells co-cultured with cells transduced with TCR alone (TCR), IL-15 and TCR (sIL15.TCR), and IL-15 and CD8 TCR (sIL15.CD8βαTCR). [Figure 108B] Same as above.

[0259] [Figure 108C] Figure 108C shows IFNγ production by transduced products 16-22 hours after initial tumor challenge with UACC257-RFP cells. Non-transduced cells (NT) were assayed as a control. Cells were cultured at an effector:target ratio of 1:1. Data were grouped (n=4) and TCR+ normalized. Results are presented as the mean. The integral area under the curve (AUC) normalized to the tumor-only condition is shown.

[0260] [Figure 109A] Figures 109A-109B show examples of cytolytic activity against hs695T-RFP tumor cells co-cultured with cells transduced with TCR alone (TCR), IL-15 and TCR (sIL15.TCR), and IL-15 and CD8 TCR (sIL15.CD8βαTCR). [Figure 109B] Same as above.

[0261] [Figure 109C] Figure 109C shows IFNγ production by transduced products 16-22 hours after initial tumor challenge with hs695T-RFP cells. Non-transduced cells (NT) were assayed as a control. Cells were cultured at an effector:target ratio of 2:1. Data were grouped (n=4) and TCR+ normalized. Results are presented as the mean. The integral area under the curve (AUC) normalized to the tumor-only condition is shown.

[0262] [Figure 110A] Figures 110A-110D show the percentage of TemRA, Tem, T naive / scm, and Tcm cells, as examples, graphed for four donors transduced with TCR only (TCR), IL-15 and TCR (sIL15+TCR), or IL-15 and CD8βαTCR (sIL15.CD8βαTCR). Non-transduced cells (NT) were assayed as controls. Panels were performed on cells that were not exposed to antigen-presenting tumor cells ("Pre-Ag", Figure 110A), or after four antigen challenges over 9-10 days with antigen-presenting UACC257 tumor cells (Figure 110B), hs695T tumor cells (Figure 110C), and A375 tumor cells (Figure 110D). Flow cytometer gated on CD8+ cells. Data were grouped (n=4) and presented as the mean. [Figure 110B] Same as above. [Figure 110C] Same as above. [Figure 110D] Same as above.

[0263] [Fig. 111A]Figures 111A-111D show the percentage of cells expressing LAG3, PD-1, TIGIT, TIM3, CD69 and CD39, as examples, graphed for four donors transduced with TCR only (TCR), IL-15 and TCR (sIL15+TCR), or IL-15 and CD8βα TCR (sIL15.CD8βαTCR). Non-transduced cells (NT) were assayed as controls. Panels were performed on cells that were not exposed to antigen-presenting tumor cells ("Pre-Ag", Figure 111A), or after four antigen challenges over 9-10 days with antigen-presenting UACC257 tumor cells (Figure 111B), hs695T tumor cells (Figure 111C) and A375 tumor cells (Figure 111D). Flow cytometer gated on CD8+ cells. Data were grouped (n=4) and expressed as the mean. [Fig. 111B] Same as above. [Figure 111C] Same as above. [Fig. 111D] Same as above.

[0264] [Fig. 112A] Figures 112A-112C show, by way of example, flow plots of cells transduced with TCR only (TCR), IL-15 and TCR (sIL15.TCR), or IL15 and CD8βα TCR (sIL15.CD8βαTCR). Non-transduced cells (NT) were assayed as controls. The X-axis shows staining for a cell viability marker (Helix NP) and the Y-axis shows staining for an apoptosis marker (ApoTracker™). Flow plots were performed on cells after four antigen challenges over a period of 9-10 days with antigen-presenting UACC257 tumor cells. [Fig. 112B] Same as above. [Figure 112C] Same as above.

[0265] [Fig. 112D]Figures 112D-112E show the frequency of live cells and dead apoptotic cells, respectively. The flow cytometer was gated on CD8+ cells. Data were grouped and expressed as the mean. [Figure 112E] Same as above.

[0266] [Fig. 113A] Figures 113A-113C show the proliferation index of cells transduced with TCR only (TCR), IL-15 and TCR (sIL15.TCR), or IL15 and CD8βα TCR (sIL15.CD8βαTCR) challenged twice over a six day period with UACC257 (Figure 113A), hs695T (Figure 113B), and A375 (Figure 113C) tumor cells. [Fig. 113B] Same as above. [Figure 113C] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0267] IL-12p35 / IL-12p40 fusion polypeptide In some embodiments, an IL-12p35 / IL-12p40 fusion polypeptide may be provided. In some embodiments, a nucleic acid encoding an IL-12p35 / IL-12p40 fusion polypeptide may be provided. In some embodiments, a vector comprising an IL-12p35 / IL-12p40 fusion polypeptide may be provided. In some embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide. In some embodiments, the IL-12p35 / IL-12p40 fusion polypeptide may comprise a fusion polypeptide of an IL-12α(p35) (which may be referred to as IL-12αp35, IL-12α, or IL-12p35) polypeptide and an IL-12β(p40) (which may be referred to as IL-12βp40, IL-12β, or IL-12p40) polypeptide.

[0268] In some embodiments, the IL-12p35 polypeptide may be located C-terminal to the IL-12p40 polypeptide in an IL-12p35 / IL-12p40 fusion polypeptide. (FIG. 67A). In another embodiment, the IL-12p35 polypeptide may be located N-terminal to the IL-12p40 polypeptide in an IL-12p35 / IL-12p40 fusion polypeptide. (FIG. 67B). In some embodiments in FIG. 67A and FIG. 67B, the linker is optional. In some embodiments in FIG. 67A and FIG. 67B, the line may represent a direct bond without an intervening sequence, or may represent an intervening sequence, such as, but not limited to, a linker, a Furin, a sequence encoding a 2A polypeptide, a factor Xa site, an untranslated sequence, a translated sequence, a sequence comprising one or more restriction endonuclease sites, or a combination thereof. The term "IL-12p35 / IL-12p40 fusion polypeptide" is not intended to imply a particular order of the polypeptides in the fusion polypeptide, unless otherwise specified. In some embodiments, the order of the IL-12p35 / IL-12p40 fusion polypeptide can be such that IL-12P40 is N-terminal to IL-12p35, as shown in Figure 67A.

[0269] In embodiments, the IL-12p35 / IL-12p40 fusion polypeptide may be soluble and / or secreted by cells transduced to express it.

[0270] In some embodiments, the IL-12p35 and IL-12p40 polypeptides may be linked by one or more linkers.

[0271] In embodiments, an IL-12p35 / IL-12p40 fusion polypeptide can include the entire mature IL-12p35 polypeptide, the entire mature IL-12-p40 polypeptide, or both.

[0272] In embodiments, IL-12p35 may be mutated and / or truncated, IL-12p40 may be mutated and / or truncated, or both may be mutated and / or truncated.

[0273] In embodiments, the IL-12p35 / IL-12p40 fusion polypeptide may include one or more signal peptides.

[0274] In some embodiments, the IL-12p35 / IL-12p40 fusion polypeptide may comprise the structure shown in Figure 67A or Figure 67B. In some embodiments, the signal peptide may be cleaved or otherwise removed from the IL-12p35 / IL-12p40 fusion polypeptide.

[0275] In some embodiments, the vectors described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0276] In some embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-12p35 / IL-12p40 fusion polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0277] In embodiments, such polypeptides, nucleic acids, vectors, and / or cells may be isolated, recombinant, and / or engineered.

[0278] In embodiments, expression of an IL-12p35 / IL-12p40 fusion polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or any combination thereof, of immune cells, such as, but not limited to, T cells and / or natural killer cells, in the tumor microenvironment, as compared to cells that do not express an IL-12p35 / IL-12p40 fusion polypeptide. In embodiments, expression of an IL-12p35 / IL-12p40 fusion polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or any combination thereof, of immune cells, such as, but not limited to, T cells and / or natural killer cells, in the tumor microenvironment, as compared to cells that do not express an IL-12p35 / IL-12p40 fusion polypeptide. In embodiments, expression of an IL-12p35 / IL-12p40 fusion polypeptide may increase the effectiveness of immune cells, such as, but not limited to, T cells and / or natural killer cells, in killing tumor cells, as compared to cells that do not express an IL-12p35 / IL-12p40 fusion polypeptide. In embodiments, expression of an IL-12p35 / IL-12p40 fusion polypeptide may increase the ability of immune cells, such as, but not limited to, T cells and / or natural killer cells, to survive in the tumor microenvironment, sustain tumor cell killing, or any combination thereof, compared to cells that do not express an IL-12p35 / IL-12p40 fusion polypeptide. In embodiments, expression of an IL-12p35 / IL-12p40 fusion polypeptide may increase the ability of immune cells, such as, but not limited to, T cells and / or natural killer cells, to maintain a naive phenotype.

[0279] Persistence may be assessed by, for example and without limitation, the length of time that cells are detectable in an individual (e.g., a patient) after infusion. For example and without limitation, persistence may be measured days, weeks, months, or years after infusion, for example and without limitation, about 1 week, about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, and / or about 30 months after infusion. Persistence may be assessed by, for example and without limitation, PCR of peripheral blood samples, flow cytometry of peripheral blood samples, and / or analysis of tumor biopsy samples. The persistence of cells expressing IL-12p35 / IL-12p40 fusion polypeptides may be compared, for example and without limitation, to the typical persistence of infused ACT cells, or to the persistence of similar cells that do not express IL-12p35 / IL-12p40 fusion polypeptides.

[0280] The continued ability to kill tumor cells can be measured, by way of non-limiting example, via (i) a serial killing assay using IncuCyte, in which the ability to kill / impair tumor growth is assessed as measured by fold expansion during repeated tumor stimulation over a period of time, and / or (ii) production of cytokines / effector molecules (IFNγ via ELISA and other pro-inflammatory cytokines via Luminex (cytokines measured include but are not limited to IFNγ, TNFα, granulocyte colony activator, and IgG1). The continued ability of cells expressing an IL-12p35 / IL-12p40 fusion polypeptide to kill tumor cells can be measured via, by way of non-limiting example, the continued ability of similar cells not expressing an IL-12p35 / IL-12p40 fusion polypeptide to kill tumor cells, or the continued ability of other control cells to kill tumor cells.

[0281] Phenotypic naivety can be assessed, as a non-limiting example, through Tmem panel assay by flow cytometry. Typically, flow cytometry gating is off CD8+TCR+ cells. Typically, a more naive phenotype can be indicated by a higher frequency of Tnaive / scm (CD45RA+CCR7+) and Tcm (CD45RA-CCR7+) T memory subsets, as well as an increase or retention of CD39-CD69- and CD27+CD28+ populations. Low CD57 expression may also be desirable.

[0282] When assessing the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naïveness, or other properties of cells expressing an IL-12p35 / IL-12p40 fusion polypeptide, cells such as, by way of non-limiting example, untransduced cells, cells transduced with TCR only, cells transduced with CD8 and TCR, or combinations thereof, can be utilized as control cells.

[0283] In embodiments, an IL-12p35 / IL-12p40 fusion polypeptide may act in cis (e.g., affecting cells in which it is expressed), in trans (e.g., affecting cells in which it is not expressed), or any combination thereof. In embodiments in which an IL-12p35 / IL-12p40 fusion polypeptide acts in trans, cells adjacent to or in the vicinity of cells expressing an IL-12p35 / IL-12p40 fusion polypeptide (e.g., within a tumor microenvironment) may exhibit any or a combination of improvements that are the same or comparable to those described for cells expressing an IL-12p35 / IL-12p40 fusion polypeptide, compared to cells that are not adjacent to or in the vicinity of cells expressing an IL-12p35 / IL-12p40 fusion polypeptide.

[0284] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0285] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0286] In certain aspects, the cells described herein can be isolated and / or recombinant cells.

[0287] In embodiments, an IL-12p35 polypeptide may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 305. In embodiments, a function of IL-12p35, including but not limited to one or more signaling functions of IL-12p35, is preserved and / or enhanced in the mutant IL-12p35 polypeptide.

[0288] In embodiments, the IL-12p35 polypeptide may comprise SEQ ID NO:305, which comprises one, two, three, four, or five amino acid substitutions. In embodiments, the amino acid substitutions may be conservative or non-conservative. In embodiments, the amino acid substitutions may be conservative amino acid substitutions. In embodiments, a function of IL-12p35, including but not limited to one or more signaling functions of IL-12p35, is preserved and / or enhanced in the mutant IL-12p35 polypeptide.

[0289] In embodiments, an IL-12p35 polypeptide may be encoded by a nucleic acid sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 306. In embodiments, a function of IL-12p35, including but not limited to one or more signaling functions of IL-12p35, is preserved and / or enhanced in an IL-12p35 polypeptide encoded by a variant nucleic acid sequence.

[0290] In embodiments, an IL-12p35 polypeptide may be encoded by a nucleic acid sequence comprising SEQ ID NO:306, comprising one, two, three, four, or five nucleic acid substitutions. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid, or may result in a codon encoding a different amino acid. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding a conservative amino acid substitution. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid. In embodiments, a function of IL-12p35, including but not limited to one or more signaling functions of IL-12p35, is preserved and / or enhanced in an IL-12p35 polypeptide encoded by a variant nucleic acid sequence.

[0291] In embodiments, an IL-12p40 polypeptide may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 307. In embodiments, a function of IL-12p40, including but not limited to one or more signaling functions of IL-12p40, is preserved and / or enhanced in a variant IL-12p40 polypeptide.

[0292] In embodiments, the IL-12p40 polypeptide may comprise SEQ ID NO:307, which comprises one, two, three, four, or five amino acid substitutions. In embodiments, the amino acid substitutions may be conservative or non-conservative. In embodiments, the amino acid substitutions may be conservative amino acid substitutions. In embodiments, a function of IL-12p40, including but not limited to one or more signaling functions of IL-12p40, is preserved and / or enhanced in the mutant IL-12p40 polypeptide.

[0293] In embodiments, an IL-12p40 polypeptide may be encoded by a nucleic acid sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 308. In embodiments, a function of IL-12p40, including but not limited to one or more signaling functions of IL-12p40, is preserved and / or enhanced in an IL-12p40 polypeptide encoded by a variant nucleic acid sequence.

[0294] In embodiments, an IL-12p40 polypeptide may be encoded by a nucleic acid sequence comprising SEQ ID NO:308, comprising one, two, three, four, or five nucleic acid substitutions. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid, or may result in a codon encoding a different amino acid. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding a conservative amino acid substitution. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid. In embodiments, a function of IL-12p40, including but not limited to one or more signaling functions of IL-12p40, is preserved and / or enhanced in an IL-12p40 polypeptide encoded by a variant nucleic acid sequence.

[0295] In some embodiments, the linker may be a peptide linker. In some embodiments, the peptide linker may be rigid or flexible. In some embodiments, the linker may be cleavable. In some embodiments, the linker may promote the stability or proper folding of the fusion polypeptide, increase the expression of the fusion polypeptide, improve the biological activity of the fusion polypeptide, promote the targeting of the fusion polypeptide, change the PK of the fusion polypeptide, or any combination thereof.

[0296] In embodiments, linkers include, but are not limited to, GSG, LE, SGSG (SEQ ID NO: 266), or a linker set forth in SEQ ID NOs: 331, 333, 335, 337, 339, 341, or 343-381, or a linker encoded by SEQ ID NO: 332, 334, 336, 338, 340, or 342.

[0297] In several embodiments, the linker may contain about 2 to 40 amino acids, about 4 to 38 amino acids, about 6 to 34 amino acids, about 8 to 32 amino acids, about 10 to 30 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 12 to 28 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 14 to 26 amino acids, about 12 to 24 amino acids, about 10 to 22 amino acids, about 10 to 20 amino acids, about 12 to 18 amino acids, about 14 to 16 amino acids, about 8 to 22 amino acids, about 6 to 24 amino acids, about 4 to 26 amino acids, or about 2 to 28 amino acids.

[0298] In some embodiments, the linker may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 331. In some embodiments, the linker may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 337. In some embodiments, the linker may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 339. In embodiments, the linker may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 331. In embodiments, one or more of the functions of the linker, including but not limited to, the flexibility, rigidity, cleavability, ability to promote stability or proper folding of a fusion polypeptide, ability to increase expression of a fusion polypeptide, ability to improve biological activity of a fusion polypeptide, ability to promote targeting of a fusion polypeptide, ability to alter the PK of a fusion polypeptide, or any combination thereof, are preserved and / or enhanced in the variant linker.

[0299] In some embodiments, the linker may comprise (a) SEQ ID NO: 331, which comprises 1, 2, 3, 4, or 5 amino acid substitutions; (b) SEQ ID NO: 337, which comprises 1, 2, 3, 4, or 5 amino acid substitutions; or (c) SEQ ID NO: 339, which comprises 1, 2, 3, 4, or 5 amino acid substitutions. In some embodiments, the amino acid substitutions may be conservative or non-conservative. In some embodiments, the amino acid substitutions may be conservative amino acid substitutions. In some embodiments, one or more of the functions of the linker, including but not limited to the flexibility, rigidity, cleavability, ability to promote stability or proper folding of the fusion polypeptide, ability to increase expression of the fusion polypeptide, ability to improve biological activity of the fusion polypeptide, ability to promote targeting of the fusion polypeptide, ability to alter the PK of the fusion polypeptide, or any combination thereof, are preserved and / or enhanced in the variant linker.

[0300] In embodiments, the linker may be encoded by a nucleic acid sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 332. In embodiments, the linker may be encoded by a nucleic acid sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 338. In embodiments, the linker may be encoded by a nucleic acid sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 340. In some embodiments, one or more of the functions of the linker, including but not limited to, the flexibility, rigidity, cleavability of the linker, the ability to promote stability or proper folding of a fusion polypeptide, the ability to increase expression of a fusion polypeptide, the ability to improve biological activity of a fusion polypeptide, the ability to promote targeting of a fusion polypeptide, the ability to alter the PK of a fusion polypeptide, or any combination thereof, are preserved and / or enhanced in the linker encoded by the variant nucleic acid sequence.

[0301] In some embodiments, the linker may be encoded by a nucleic acid sequence comprising: (a) SEQ ID NO: 332, comprising 1, 2, 3, 4, or 5 nucleic acid substitutions; (b) SEQ ID NO: 338, comprising 1, 2, 3, 4, or 5 nucleic acid substitutions; or (c) SEQ ID NO: 340, comprising 1, 2, 3, 4, or 5 nucleic acid substitutions. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid, or may result in a codon encoding a different amino acid. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more of the functions of the linker, including but not limited to the flexibility, rigidity, cleavability, ability to promote stability or proper folding of a fusion polypeptide, ability to increase expression of a fusion polypeptide, ability to improve biological activity of a fusion polypeptide, ability to promote targeting of a fusion polypeptide, ability to alter the PK of a fusion polypeptide, or any combination thereof, are preserved and / or enhanced in the linker encoded by the variant nucleic acid sequence.

[0302] In embodiments, an IL-12p35 / IL-12p40 fusion polypeptide including a linker may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 309. In embodiments, (i) a function of IL-12p35, such as but not limited to, one or more signaling functions of IL-12p35, (ii) a function of IL-12p40, such as but not limited to, one or more signaling functions of IL-12p40, or both (i) and (ii) are preserved and / or enhanced in a variant IL-12p35 / IL-12p40 fusion polypeptide.

[0303] In embodiments, the IL-12p35 / IL-12p40 fusion polypeptide including a linker may comprise SEQ ID NO:309, which comprises one, two, three, four, or five amino acid substitutions. In embodiments, the amino acid substitutions may be conservative or non-conservative. In embodiments, the amino acid substitutions may be conservative amino acid substitutions. In embodiments, the IL-12p35 / IL-12p40 fusion polypeptide including a linker may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:309. In embodiments, (i) a function of IL-12p35, such as but not limited to, one or more signaling functions of IL-12p35, (ii) a function of IL-12p40, such as but not limited to, one or more signaling functions of IL-12p40, or both (i) and (ii) are preserved and / or enhanced in a mutant IL-12p35 / IL-15p40 fusion polypeptide.

[0304] In embodiments, a IL-12p35 / IL-12p40 fusion polypeptide comprising a linker may be encoded by a nucleic acid sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 310. In embodiments, a IL-12p35 / IL-12p40 fusion polypeptide comprising a linker may have a sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:309. In embodiments, (i) a function of IL-12p35, such as, but not limited to, one or more signaling functions of IL-12p35, (ii) a function of IL-12p40, such as, but not limited to, one or more signaling functions of IL-12p40, or both (i) and (ii) are preserved and / or enhanced in the IL-12p35 / IL-15p40 fusion polypeptide encoded by the variant nucleic acid sequence.

[0305] In some embodiments, the IL-12p35 / IL-12p40 fusion polypeptide including a linker may be encoded by a nucleic acid sequence including SEQ ID NO:310, including one, two, three, four, or five nucleic acid substitutions. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid, or may result in a codon encoding a different amino acid. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid. In some embodiments, the IL-12p35 / IL-12p40 fusion polypeptide including a linker may have a sequence including at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:309. In embodiments, (i) a function of IL-12p35, such as, but not limited to, one or more signaling functions of IL-12p35, (ii) a function of IL-12p40, such as, but not limited to, one or more signaling functions of IL-12p40, or both (i) and (ii) are preserved and / or enhanced in the IL-12p35 / IL-15p40 fusion polypeptide encoded by the variant nucleic acid sequence.

[0306] In embodiments, a nucleic acid encoding an IL-12p35 / IL-12p40 fusion polypeptide may contain one or more stop codons (e.g., TAA, TAG, or TGA) that are positioned, by way of non-limiting example, at the 3' end of the nucleotides encoding an IL-12p35 polypeptide, e.g., the encoded fusion polypeptide is in an N-terminus-IL-12p40-IL-12p35-C-terminus orientation, or at the 3' end of the nucleotides encoding an IL-12p40 polypeptide, e.g., the encoded fusion polypeptide is in an N-terminus-IL-12p35-IL-12p40-C-terminus orientation.

[0307] In some embodiments, the order of the IL-12p35 / IL-12p40 fusion polypeptide may be N-terminal to IL-12p35, as shown in FIG. 67A. In some embodiments, the IL-12p35 / IL-12p40 fusion polypeptide including the linker may be encoded by a nucleic acid that also includes and / or encodes one or more CNS2, one or more CNS1, one or more CD69 promoters, one or more post-transcriptional response elements (PRE), and one or more factor Xa sites. In some embodiments, such a construct may be encoded by SEQ ID NO:320 (FIG. 68, construct AH). In some embodiments, the vector may include SEQ ID NO:320. In some embodiments, T cells may be transduced with a vector including SEQ ID NO:320. In some embodiments, the linker-containing IL-12p35 / IL-12p40 fusion polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more MSCV promoters, one or more post-transcriptional response elements (PREs), and one or more factor Xa sites. In some embodiments, such a construct may be encoded by SEQ ID NO: 321 (FIG. 68, construct AI). In some embodiments, the vector may include SEQ ID NO: 321. In some embodiments, the T cells may be transduced with a vector that includes SEQ ID NO: 321. In some embodiments, the linker-containing IL-12p35 / IL-12p40 fusion polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more NFAT promoters, one or more minimal IL2 promoters, one or more post-transcriptional response elements (PREs), and one or more factor Xa sites. In some embodiments, such a construct may be encoded by SEQ ID NO: 322 (FIG. 68, construct AJ). In some embodiments, the vector may include SEQ ID NO: 322. In embodiments, T cells may be transduced with a vector comprising SEQ ID NO:322.

[0308] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0309] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0310] In certain aspects, the cells described herein can be isolated and / or recombinant cells. IL-15 Polypeptides

[0311] In embodiments, an IL-15 polypeptide may be provided. In embodiments, a nucleic acid encoding an IL-15 polypeptide may be provided. In embodiments, a vector comprising an IL-15 polypeptide may be provided. In embodiments, a cell described herein may comprise an IL-15 polypeptide. In embodiments, a cell described herein may comprise an I5 / polypeptide, a CD8 polypeptide, a cellular receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In embodiments, the cell may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof. In embodiments, the IL-15 polypeptide may comprise the entire mature IL-15 polypeptide. In embodiments, the IL-15 may be mutated and / or truncated. In embodiments, such polypeptides, nucleic acids, vectors, and / or cells may be isolated, recombinant, and / or engineered.

[0312] In embodiments, the IL-15 polypeptide may be soluble and / or secreted by cells transduced to express it.

[0313] In some embodiments, an IL-15 polypeptide may include one or more signal peptides, propeptides, or both. In some embodiments, a signal peptide, propeptide, or both may be cleaved or otherwise removed from an IL-15 polypeptide.

[0314] In some embodiments, the vectors described herein may include an IL-15 polypeptide and a CD8 polypeptide described herein. In some embodiments, the vectors described herein may include an IL-15 polypeptide, a CD8 polypeptide, a cellular receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0315] In some embodiments, the cells described herein may comprise an IL-15 polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-15 polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0316] In some embodiments, expression of an IL-15 polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or any combination thereof of immune cells, such as, but not limited to, T cells and / or natural killer cells, in a tumor microenvironment, compared to cells that do not express an IL-15 polypeptide. In some embodiments, expression of an IL-15 polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or any combination thereof of immune cells, such as, but not limited to, T cells and / or natural killer cells, in a tumor microenvironment, compared to cells that do not express an IL-15 polypeptide. In some embodiments, expression of an IL-15 polypeptide may increase the effectiveness of immune cells, such as, but not limited to, T cells and / or natural killer cells, in killing tumor cells, compared to cells that do not express an IL-15 polypeptide. In some embodiments, expression of an IL-15 polypeptide may increase the ability of immune cells, such as, but not limited to, T cells and / or natural killer cells, to survive in a tumor microenvironment, to sustain tumor cell killing, or any combination thereof, compared to cells that do not express an IL-15 polypeptide. In embodiments, expression of IL-15 may increase the ability of immune cells, such as, but not limited to, T cells and / or natural killer cells, to maintain a naive phenotype.

[0317] Persistence can be assessed by, for example and not by limitation, the length of time that the cells are detectable in an individual (e.g., a patient) after injection. For example and not by limitation, persistence can be measured days, weeks, months, or years after injection, for example and not by limitation, about 1 week, about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, and / or about 30 months after injection. Persistence can be assessed by, for example and not by limitation, PCR of peripheral blood samples, flow cytometry of peripheral blood samples, and / or analysis of tumor biopsy samples. The persistence of cells expressing IL-15 polypeptide from a transgene can be compared, for example and not by limitation, to the typical persistence of injected ACT cells, or to the persistence of similar cells that do not express IL-15 polypeptide from a transgene.

[0318] The continued ability to kill tumor cells may be measured, by way of non-limiting example, via (i) a serial killing assay using IncuCyte (wherein the ability to kill / impair tumor growth is assessed as measured by fold proliferation during repeated tumor stimulation over a period of time), and / or (ii) via production of cytokines / effector molecules (IFNγ via ELISA, and other pro-inflammatory cytokines via Luminex (cytokines measured include but are not limited to IFNγ, TNFα, Granzyme B, Perforin, IL-2, IL-6, MIP-1β, MIP-1α, GM-CSF, RANTES, IL-18, IL-4, IL-10, and IP10). The continued ability of cells expressing an IL-15 polypeptide from a transgene to kill tumor cells may be compared, by way of non-limiting example, to the continued ability of similar cells not expressing an IL-15 polypeptide from a transgene to kill tumor cells, or the continued ability of other control cells to kill tumor cells.

[0319] Phenotypic naivety can be assessed, as a non-limiting example, through Tmem panel assay by flow cytometry. Typically, flow cytometry gating is off CD8+TCR+ cells. Typically, a more naive phenotype can be indicated by a higher frequency of Tnaive / scm (CD45RA+CCR7+) and Tcm (CD45RA-CCR7+) T memory subsets, as well as an increase or retention of CD39-CD69- and CD27+CD28+ populations. Low CD57 expression may also be desirable.

[0320] When assessing the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naïveness, or other properties of cells expressing an IL-15 polypeptide from a transgene, cells such as, by way of non-limiting example, untransduced cells, cells transduced with TCR only, cells transduced with CD8 and TCR, or combinations thereof, can be utilized as control cells.

[0321] In embodiments, an IL-15 polypeptide may act in cis (e.g., affecting cells in which it is expressed), in trans (e.g., affecting cells in which it is not expressed), or any combination thereof. In embodiments in which an IL-15 polypeptide acts in trans, cells adjacent to or in the vicinity of cells expressing an IL-15 polypeptide (e.g., within a tumor microenvironment) may exhibit any or a combination of improvements that are the same or comparable to those described for cells expressing an IL-15 polypeptide, compared to cells that are not adjacent to or in the vicinity of cells expressing an IL-15 polypeptide.

[0322] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0323] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0324] In embodiments, an IL-15 polypeptide may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 311. In embodiments, an IL-15 polypeptide may have a sequence that comprises at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 313.

[0325] In embodiments, the IL-15 polypeptide may comprise (a) SEQ ID NO: 311, which comprises one, two, three, four, or five amino acid substitutions, or (b) SEQ ID NO: 313, which comprises one, two, three, four, or five amino acid substitutions. In embodiments, the amino acid substitutions may be conservative or non-conservative. In embodiments, the amino acid substitutions may be conservative amino acid substitutions. In embodiments, a function of IL-15, including but not limited to one or more signaling functions of IL-15, is preserved and / or enhanced in the mutant IL-15 polypeptide.

[0326] In embodiments, the IL-15 polypeptide may be encoded by a nucleic acid sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 312. In embodiments, the IL-15 polypeptide may be encoded by a nucleic acid sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 314. In embodiments, the function of IL-15, including but not limited to, one or more signaling functions of IL-15, is preserved and / or enhanced in the variant IL-15 polypeptide. In embodiments, a function of IL-15, including but not limited to, one or more signaling functions of IL-15, is preserved and / or enhanced in an IL-15 polypeptide encoded by a variant nucleic acid sequence.

[0327] In embodiments, the IL-15 polypeptide may be encoded by a nucleic acid sequence comprising (a) SEQ ID NO: 312, comprising one, two, three, four or five nucleic acid substitutions, or (b) SEQ ID NO: 314, comprising one, two, three, four or five nucleic acid substitutions. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding a conservative amino acid substitution. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid. In embodiments, a function of IL-15, including but not limited to one or more signaling functions of IL-15, is preserved and / or enhanced in the IL-15 polypeptide encoded by the variant nucleic acid sequence.

[0328] In some embodiments, a nucleic acid encoding an IL-15 polypeptide can include one or more stop codons (e.g., TAA, TAG, or TGA), which, by way of non-limiting example, are located at the 3' end of the nucleotide sequence encoding the IL-15 polypeptide.

[0329] In embodiments, the IL-15 polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more MSCV promoters, one or more post-transcriptional response elements (PRE), and one or more factor Xa sites. The IL-15 polypeptide may include a signal peptide (SP) and a propeptide (PP). In embodiments, such a construct may be encoded by SEQ ID NO:323 (Figure 68, construct AK). In embodiments, the vector may include SEQ ID NO:323. In embodiments, T cells may be transduced with a vector including SEQ ID NO:323.

[0330] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0331] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0332] In certain aspects, the cells described herein can be isolated and / or recombinant cells. IL-18 Polypeptides

[0333] In embodiments, an IL-18 polypeptide may be provided. In embodiments, a nucleic acid encoding an IL-18 polypeptide may be provided. In embodiments, a vector comprising an IL-18 polypeptide may be provided. In embodiments, the cells described herein may comprise an IL-18 polypeptide. In embodiments, the cells described herein may comprise an IL-18 polypeptide, a CD8 polypeptide, a cellular receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof. In embodiments, the IL-18 polypeptide may comprise the entire mature IL-18 polypeptide. In embodiments, the IL-18 may be mutated and / or truncated. In embodiments, such polypeptides, nucleic acids, vectors, and / or cells may be isolated, recombinant, and / or engineered.

[0334] In embodiments, the IL-15 polypeptide may be soluble and / or secreted by cells transduced to express it.

[0335] In embodiments, an IL-18 polypeptide can include one or more signal peptides, propeptides, or both.

[0336] In some embodiments, the vectors described herein may include an IL-18 polypeptide and a CD8 polypeptide described herein. In some embodiments, the vectors described herein may include an IL-18 polypeptide, a CD8 polypeptide, a cellular receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0337] In some embodiments, the cells described herein may comprise an IL-18 polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise an IL-18 polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0338] In some embodiments, expression of an IL-18 polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or any combination thereof of immune cells, such as, but not limited to, T cells and / or natural killer cells, in a tumor microenvironment, compared to cells that do not express an IL-18 polypeptide. In some embodiments, expression of an IL-18 polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or any combination thereof of immune cells, such as, but not limited to, T cells and / or natural killer cells, in a tumor microenvironment, compared to cells that do not express an IL-18 polypeptide. In some embodiments, expression of an IL-18 polypeptide may increase the effectiveness of immune cells, such as, but not limited to, T cells and / or natural killer cells, in killing tumor cells, compared to cells that do not express an IL-18 polypeptide. In some embodiments, expression of an IL-18 polypeptide may increase the ability of immune cells, such as, but not limited to, T cells and / or natural killer cells, to survive in a tumor microenvironment, to sustain tumor cell killing, or any combination thereof, compared to cells that do not express an IL-18 polypeptide. In embodiments, expression of IL-18 may increase the ability of immune cells, such as, but not limited to, T cells and / or natural killer cells, to maintain a naive phenotype.

[0339] Persistence can be assessed by, for example and not by way of limitation, the length of time that the cells are detectable in an individual (e.g., a patient) after injection. For example and not by way of limitation, persistence can be measured days, weeks, months, or years after injection, for example and not by way of limitation, about 1 week, about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, and / or about 30 months after injection. Persistence can be assessed by, for example and not by way of limitation, PCR of peripheral blood samples, flow cytometry of peripheral blood samples, and / or analysis of tumor biopsy samples. The persistence of cells expressing IL-18 polypeptide from a transgene can be compared, for example and not by way of limitation, to the typical persistence of injected ACT cells, or to the persistence of similar cells that do not express IL-18 polypeptide from a transgene.

[0340] The continued ability to kill tumor cells may be measured, by way of non-limiting example, via (i) a serial killing assay using IncuCyte (wherein the ability to kill / impair tumor growth is assessed as measured by fold proliferation during repeated tumor stimulation over a period of time), and / or (ii) via production of cytokines / effector molecules (IFNγ via ELISA, and other pro-inflammatory cytokines via Luminex (cytokines measured include but are not limited to IFNγ, TNFα, Granzyme B, Perforin, IL-2, IL-6, MIP-1β, MIP-1α, GM-CSF, RANTES, IL-18, IL-4, IL-10, and IP10). The continued ability of cells expressing an IL-18 polypeptide from a transgene to kill tumor cells may be compared, by way of non-limiting example, to the continued ability of similar cells not expressing an IL-18 polypeptide from a transgene to kill tumor cells, or the continued ability of other control cells to kill tumor cells.

[0341] Phenotypic naivety can be assessed, as a non-limiting example, through Tmem panel assay by flow cytometry. Typically, flow cytometry gating is off CD8+TCR+ cells. Typically, a more naive phenotype can be indicated by a higher frequency of Tnaive / scm (CD45RA+CCR7+) and Tcm (CD45RA-CCR7+) T memory subsets, as well as an increase or retention of CD39-CD69- and CD27+CD28+ populations. Low CD57 expression may also be desirable.

[0342] When assessing the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naïveness, or other properties of cells expressing an IL-18 polypeptide from a transgene, cells such as, by way of non-limiting example, untransduced cells, cells transduced with TCR only, cells transduced with CD8 and TCR, or combinations thereof, can be utilized as control cells.

[0343] In embodiments, an IL-18 polypeptide may act in cis (e.g., affecting cells in which it is expressed), in trans (e.g., affecting cells in which it is not expressed), or any combination thereof. In embodiments in which an IL-18 polypeptide acts in trans, cells adjacent to or in the vicinity of cells expressing an IL-18 polypeptide (e.g., within a tumor microenvironment) may exhibit any or a combination of improvements that are the same or comparable to those described for cells expressing an IL-18 polypeptide, compared to cells that are not adjacent to or in the vicinity of cells expressing an IL-18 polypeptide.

[0344] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0345] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0346] In embodiments, an IL-18 polypeptide may have a sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 315. In embodiments, a function of IL-18, including but not limited to, one or more signaling functions of IL-18, is preserved and / or enhanced in the mutant IL-18 polypeptide.

[0347] In embodiments, the IL-18 polypeptide may comprise SEQ ID NO:315, which comprises one, two, three, four, or five amino acid substitutions. In embodiments, the amino acid substitutions may be conservative or non-conservative. In embodiments, the amino acid substitutions may be conservative amino acid substitutions. In embodiments, the function of IL-18, including but not limited to, one or more signaling functions of IL-18, is preserved and / or enhanced in the mutant IL-18 polypeptide.

[0348] In embodiments, an IL-18 polypeptide may be encoded by a nucleic acid sequence comprising at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 316. In embodiments, a function of IL-18, including but not limited to, one or more signaling functions of IL-18, is preserved and / or enhanced in an IL-18 polypeptide encoded by a variant nucleic acid sequence.

[0349] In embodiments, an IL-18 polypeptide may be encoded by a nucleic acid sequence comprising SEQ ID NO:316, comprising one, two, three, four, or five nucleic acid substitutions. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid, or may result in a codon encoding a different amino acid. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding a conservative amino acid substitution. In embodiments, one or more nucleic acid substitutions in a codon may result in a codon encoding the same amino acid. In embodiments, a function of IL-18, including but not limited to one or more signaling functions of IL-18, is preserved and / or enhanced in an IL-18 polypeptide encoded by a variant nucleic acid sequence.

[0350] In some embodiments, a nucleic acid encoding an IL-18 polypeptide can include one or more stop codons (e.g., TAA, TAG, or TGA), which, by way of non-limiting example, are located at the 3' end of the nucleotide sequence encoding the IL-18 polypeptide.

[0351] In some embodiments, the IL-18 polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more NFAT promoters, one or more minimal IL2 promoters, one or more post-transcriptional response elements (PREs), and one or more factor Xa sites. In some embodiments, such a construct may be encoded by SEQ ID NO: 324 (FIG. 68, construct AL). In some embodiments, the vector may comprise SEQ ID NO: 324. In some embodiments, the T cells may be transduced with a vector comprising SEQ ID NO: 324. In some embodiments, the IL-18 polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more CNS2, one or more CNS1, one or more CD69 promoters, one or more post-transcriptional response elements (PREs), and one or more factor Xa sites. In some embodiments, such a construct may be encoded by SEQ ID NO: 325 (FIG. 68, construct AM). In some embodiments, the vector may comprise SEQ ID NO: 325. In some embodiments, the T cells may be transduced with a vector comprising SEQ ID NO: 325. In embodiments, the IL-18 polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more MSCV promoters, one or more post-transcriptional response elements (PREs), and one or more factor Xa sites. In embodiments, such a construct may be encoded by SEQ ID NO:326 (Figure 68, Construct AN). In embodiments, the vector may include SEQ ID NO:326. In embodiments, T cells may be transduced with a vector including SEQ ID NO:326.

[0352] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0353] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0354] In certain aspects, the cells described herein can be isolated and / or recombinant cells.

[0355] In some embodiments, the IL-12p35 / IL-12p40 fusion polypeptide, IL-15 polypeptide, and / or IL-18 polypeptide may include one or more signal peptides. In some embodiments, the signal peptide may be cleaved or otherwise removed from the mature polypeptide. In some embodiments, the signal peptide may include one or more signal peptides derived from IL-12p35, IL-12p40, IL-15, or IL-18. In some embodiments, the polypeptide or fusion polypeptide may include one or more heterologous signal peptides, i.e., all or part of a signal peptide derived from a molecule other than IL-12p35, IL-12p40, IL-15, or IL-18. In some embodiments, the heterologous signal peptide may be derived from IL-2, CD33, IgVκ, or IgE.

[0356] In embodiments, the signal peptide may increase or promote transcription, translation, translocation, or a combination thereof of a polypeptide or fusion polypeptide compared to the native IL-12p35 signal peptide, IL-12p40 signal peptide, IL-15 signal peptide, IL-18 signal peptide, or any combination thereof. In embodiments, the signal peptide may be fused to the N-terminus or C-terminus of an IL-12p35 / IL-12p40 fusion polypeptide, IL-15 polypeptide, or IL-18 polypeptide.

[0357] In some embodiments, the vectors described herein may include any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. In some embodiments, the vectors described herein may include IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, CD8 polypeptides, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof.

[0358] In some embodiments, the cells described herein may include any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. In some embodiments, the cells described herein may include IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, CD8 polypeptides, a cell receptor (TCR) comprising an alpha and beta chain, a TCR comprising a gamma and delta chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may include an alpha beta T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+ / CD8+ cell, or any combination thereof. Modified CD8 Polypeptides

[0359] In embodiments, the CD8 polypeptides described herein may comprise the general structure of an N-terminal signal peptide (optional), a CD8α immunoglobulin (Ig)-like domain, a CD8β stalk region (domain), a CD8α transmembrane domain, and a CD8α cytoplasmic domain. The modified CD8 polypeptides described herein showed unexpected improvements in the functionality of T cells co-transduced with vectors expressing a TCR and a CD8 polypeptide.

[0360] In embodiments, the CD8 polypeptides described herein may include the general structure of an N-terminal signal peptide (optional), a CD8α immunoglobulin (Ig)-like domain, a stalk region or domain, a CD8α transmembrane domain, and a CD8α cytoplasmic domain.

[0361] In embodiments, the CD8 polypeptides described herein comprise: (a) an immunoglobulin (Ig)-like domain that comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) an Ig-like domain that comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:2; The CD8 polypeptide may comprise a region having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:3, (c) a transmembrane domain having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:4. The CD8 polypeptide described herein may be expressed in T cells with T cell receptors or CAR-T and used in methods of adoptive cell therapy (ACT). The T cells may be αβ T cells or γδ T cells.

[0362] In another embodiment, the CD8 polypeptide described herein may comprise: (a) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:1; (b) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:2; (c) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:3; and (d) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:4. The CD8 polypeptides described herein can be expressed in T cells with a T cell receptor or CAR-T and used in methods of adoptive cell therapy (ACT). The T cells can be αβ T cells or γδ T cells.

[0363] In another embodiment, the CD8 polypeptide described herein may comprise (a) SEQ ID NO:1, comprising 1, 2, 3, 4 or 5 amino acid substitutions, (b) SEQ ID NO:2, comprising 1, 2, 3, 4 or 5 amino acid substitutions, (c) SEQ ID NO:3, comprising 1, 2, 3, 4 or 5 amino acid substitutions, and (d) SEQ ID NO:4, comprising 1, 2, 3, 4 or 5 amino acid substitutions. In some embodiments, the substitutions are conservative amino acid substitutions. The CD8 polypeptide described herein may be expressed in T cells with T cell receptors or CAR-T and used in methods of adoptive cell therapy (ACT). The T cells may be γδ T cells or γδ T cells.

[0364] CD8 is a membrane-anchored glycoprotein that functions as a coreceptor for antigen recognition of peptide / MHC class I complexes by the T cell receptor (TCR) and plays a key role in T cell development in the thymus and in T cell activation in the periphery. Functional CD8 is a dimeric protein composed of either two α chains (CD8αα) or an α chain and a β chain (CD8αβ), and surface expression of the β chain may be required to associate with the co-expressed α chain to form the CD8αβ heterodimer. CD8αα and CD8αβ can be differentially expressed on various lymphocytes. CD8αβ is predominantly expressed by the αβTCR. + Expressed on the surface of T cells and thymocytes, CD8αα binds the αβTCR + A subset of γδTCR + Intestinal intraepithelial lymphocytes, NK cells, dendritic cells, and CD4 + It is expressed on a subset of T cells.

[0365] For example, the human CD8 gene can express a protein of 235 amino acids. Figure 1 shows the CD8 protein (CD8α1, SEQ ID NO: 258), which in one embodiment is divided into the following domains (starting at the amino terminus of the polypeptide and ending at the carboxy terminus): (1) the signal peptide (amino acids -21 to -1), which may be cleaved off in human cells during transport of the receptor to the cell surface and therefore may not constitute the mature active receptor portion; (2) the immunoglobulin (Ig)-like domain (in this embodiment, amino acids 1 to 115), which, like the immunoglobulin family of proteins of many other molecules involved in the regulation of the immune system, may assume a structure called the immunoglobulin fold. The crystal structure of the CD8αα receptor in complex with the human MHC molecule HLA-A2 demonstrates how the Ig domain of the CD8αα receptor binds to the ligand; (3) the juxtamembrane region (in this embodiment, amino acids 116 to 160). This may be an extended linker region, which allows the CD8αα receptor to reach from the surface of the T cell over the MHC to the a3 domain of the MHC where it binds. The stalk region may be glycosylated and may be inflexible; (4) a transmembrane domain (in this embodiment, amino acids 161-188), which may anchor the CD8α receptor in the cell membrane and thus is not part of the soluble recombinant protein; and (5) a cytoplasmic domain (in this embodiment, amino acids 189-214), which may bind to itself and p56. lck Through the association of IL-1 and IL-2, it can mediate signaling functions in T cells and can participate in the T cell activation cascade through phosphorylation.

[0366] The sequence of CD8α will generally have a sufficient portion of the immunoglobulin domain so that it can bind to MHC. Generally, the CD8α molecule may contain all or a substantial portion of the immunoglobulin domain of CD8α, e.g., SEQ ID NO: 258, but in certain embodiments may contain at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 amino acids of the immunoglobulin domain. The CD8α molecule of the present disclosure may be a dimer (e.g., CD8αα or CD8αβ), and CD8α monomers may be included within the scope of the present disclosure. In certain embodiments, the CD8α of the present disclosure may include CD8α1 (SEQ ID NO: 258) and CD8α2 (SEQ ID NO: 259). In one aspect, the disclosure may include CD8α1 (SEQ ID NO: 258) encoded by SEQ ID NO: 318.

[0367] The α and β subunits of CD8 may have similar structural motifs, including an Ig-like domain, a stalk region of 30–40 amino acids, a transmembrane region, and a short cytoplasmic domain of approximately 20 amino acids. The α and β chains of CD8 have two and one N-linked glycosylation sites, respectively, in the Ig-like domain, and they share less than 20% identity in amino acid sequence. The CD8 β stalk region is 10–13 amino acids shorter than the CD8 α stalk region and is highly glycosylated with O-linked carbohydrates. These carbohydrates on the β stalk region (but not on the α stalk region) appear to be highly heterogeneous with complex sialylation and may be differentially regulated during thymic developmental stages and upon T cell activation. Glycan addition has been shown to play a regulatory role in glycoprotein function and immune responses. Glycans near the transmembrane domain can affect the orientation of adjacent motifs. The unique biochemical properties of the stalk region of the CD8 β chain may present a promising candidate for the regulation of coreceptor function.

[0368] A CD8α polypeptide may be modified by replacing the stalk region of CD8α with the stalk region of CD8β to generate a modified CD8α polypeptide. In embodiments, a modified CD8α polypeptide described herein may have a CD8β stalk region that comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2. The modified CD8 alpha polypeptides described herein may have an immunoglobulin (Ig)-like domain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. The modified CD8 polypeptides may have a transmembrane domain comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3. The modified CD8 polypeptides described herein may have a cytoplasmic tail that comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. The CD8 polypeptides described herein may have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5.The CD8 polypeptides described herein may include one or more signal peptides that comprise at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:294, fused directly or indirectly to the N-terminus or C-terminus of the mCD8α polypeptide. The CD8 polypeptides described herein may have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:7. T cells

[0369] The T cells may express one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. As a non-limiting example, the T cells may co-express a T cell receptor (TCR) and one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, and / or IL-18 polypeptides. As another non-limiting example, the T cells may co-express a T cell receptor (TCR) and a CD8 polypeptide described herein. As another non-limiting example, the T cells may co-express (i) a T cell receptor (TCR), (ii) one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, and / or IL-18 polypeptides, and (iii) a CD8 polypeptide described herein. The T cells may also express a chimeric antigen receptor (CAR), a CAR analog, or a CAR derivative. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0370] The T cells, when a population, can be αβ T cells, γδ T cells, natural killer T cells, or a combination thereof. The T cells can be CD4+ T cells, CD8+ T cells, or CD4+ / CD8+ T cells. In embodiments, the cells can include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0371] The T cells can be αβ T cells and can express a CD8 polypeptide as described herein. The CD8 polypeptide can be modified or unmodified. The T cells can be αβ T cells and can express a modified CD8 polypeptide as described herein, e.g., a modified CD8α polypeptide, or a modified CD8α polypeptide comprising a CD8β stalk region, e.g., m1CD8α of constructs #11 and #12 (FIG. 4), or CD8α* (FIG. 55B). The T cells can be αβ T cells and can express one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide, a CD8 polypeptide, and / or a CAR. In some embodiments, the CD8 polypeptide can include a CD8α chain and / or a CD8β chain, and the CD8α chain and / or the CD8β chain can be independently modified or unmodified.

[0372] The T cells may be γδ T cells and may express a CD8 polypeptide as described herein. In some embodiments, the CD8 polypeptide may include a CD8α chain and / or a CD8β chain, which may be independently modified or unmodified. In some embodiments, the T cells may be γδ T cells and may express a CD8 polypeptide as described herein, e.g., a modified CD8α polypeptide, or a modified CD8α polypeptide comprising a CD8β stalk region, e.g., m1CD8α of constructs #11 and #12 (FIG. 4) and CD8α* (FIG. 55B). The T cells may be γδ T cells and may express one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, and / or a CAR. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0373] T cells can be provided that include one or more nucleic acids encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide can include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain can independently be modified or unmodified.

[0374] T cells may be provided that include one or more nucleic acids encoding one or any combination of a TCR comprising an α chain and a β chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. T cells may be provided that include one or more nucleic acids encoding one or any combination of a TCR comprising a γ chain and a δ chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. Cells may be provided that include one or more nucleic acids encoding one or any combination of a CAR, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0375] A T cell may be provided that comprises one or more nucleic acids encoding (i) a TCR comprising an α chain and a β chain, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. A cell may be provided that comprises one or more nucleic acids encoding (i) a TCR comprising a γ chain and a δ chain, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. A cell may be provided that comprises one or more nucleic acids encoding (i) a CAR, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0376] T cells may be provided that include one or more nucleic acids encoding (i) a TCR that includes an α chain and a β chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. Cells may be provided that include one or more nucleic acids encoding (i) a TCR that includes a γ chain and a δ chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. Cells may be provided that include one or more nucleic acids encoding (i) a CAR, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0377] A T cell may be provided that includes a TCR comprising an α chain and a β chain, and one or more nucleic acids encoding a CD8 polypeptide. A cell may be provided that includes a TCR comprising a γ chain and a δ chain, and one or more nucleic acids encoding a CD8 polypeptide. A cell may be provided that includes a CAR, and one or more nucleic acids encoding a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified. Natural killer (NK) cells

[0378] Natural killer (NK) cells can be engineered and used in adoptive cell therapy (ACT). See, e.g., Morton LT, et al., "T cell receptor engineering of primary NK cells to therapeutically target tumors and tumor immune evasion", J Immunother Cancer, March 14, 2022; 10: e003715, which is incorporated herein by reference in its entirety. In some embodiments, engineered NK cells are provided.

[0379] The NK cells may express one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. As a non-limiting example, the NK cells may co-express a T cell receptor (TCR) and one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, and / or IL-18 polypeptides. As another non-limiting example, the NK cells may co-express a T cell receptor (TCR) and a CD8 polypeptide described herein. As another non-limiting example, the NK cells may co-express (i) a T cell receptor (TCR), (ii) one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, and / or IL-18 polypeptides, and (iii) a CD8 polypeptide described herein. The NK cells may also express a chimeric antigen receptor (CAR), a CAR analog, or a CAR derivative. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0380] The NK cells may express a CD8 polypeptide as described herein. The CD8 polypeptide may be modified or unmodified. The NK cells may express a modified CD8 polypeptide as described herein, e.g., a modified CD8α polypeptide, or a modified CD8α polypeptide comprising a CD8β stalk region, e.g., m1CD8α of constructs #11 and #12 (FIG. 4), or CD8α* (FIG. 55B). The NK cells may express one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide, a CD8 polypeptide, and / or a CAR. In some embodiments, the CD8 polypeptide may comprise a CD8α chain and / or a CD8β chain, and the CD8α chain and / or the CD8β chain may be independently modified or unmodified.

[0381] NK cells can be provided that include one or more nucleic acids encoding one or any combination of an IL-12p35 polypeptide, an IL-12p40 polypeptide, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide can include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain can independently be modified or unmodified.

[0382] NK cells may be provided that include one or more nucleic acids encoding one or any combination of a TCR comprising an α chain and a β chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. NK cells may be provided that include one or more nucleic acids encoding one or any combination of a TCR comprising a γ chain and a δ chain, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. NK cells may be provided that include one or more nucleic acids encoding one or any combination of a CAR, an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0383] A NK cell may be provided that comprises one or more nucleic acids encoding (i) a TCR comprising an α chain and a β chain, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. A cell may be provided that comprises one or more nucleic acids encoding (i) a TCR comprising a γ chain and a δ chain, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. A cell may be provided that comprises one or more nucleic acids encoding (i) a CAR, (ii) a CD8 polypeptide, and (iii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0384] NK cells may be provided that include one or more nucleic acids encoding (i) a TCR comprising an α chain and a β chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. NK cells may be provided that include one or more nucleic acids encoding (i) a TCR comprising a γ chain and a δ chain, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. NK cells may be provided that include one or more nucleic acids encoding (i) a CAR, and (ii) one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0385] NK cells may be provided that include a TCR comprising an α chain and a β chain, and one or more nucleic acids encoding a CD8 polypeptide. Cells may be provided that include a TCR comprising a γ chain and a δ chain, and one or more nucleic acids encoding a CD8 polypeptide. Cells may be provided that include a CAR, and one or more nucleic acids encoding a CD8 polypeptide. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified. T cell receptor

[0386] The T cells may co-express a T cell receptor (TCR), an antigen binding protein, or both, along with one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or a CD8 polypeptide described herein, including, but not limited to, those listed in Table 3 (SEQ ID NOs: 15-92). Additionally, the T cells may express one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide described herein. In embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.US Patent Application Publication No. 2017 / 0267738;US Patent Application Publication No. 2017 / 0312350;US Patent Application Publication No. 2018 / 0051080;US Patent Application Publication No. 2018 / 0164315;US Patent Application Publication No. 2018 / 0161396;US Patent Application Publication No. 2018 / 0162922;US Patent Application Publication No. 2018 / 0273602;US Patent Application Publication No. 2019 / 0016801;US Patent Application Publication No. 2019 / 0002556;US Patent Application Publication No. 2019 / 0135914;US US Patent 10,538,573; US Patent 10,626,160; US Patent Application Publication 2019 / 0321478; US Patent Application Publication 2019 / 0256572; US Patent 10,550,182; US Patent 10,526,407; US Patent Application Publication 2019 / 0284276; US Patent Application Publication 2019 / 0016802; US Patent Application Publication 2019 / 0016803; US Patent Application Publication 2019 / 0016804; US Patent 10,583,573; US Patent Application Publication 2020 / 0339652;US Patent 10,537,624;US Patent 10,596,242;US Patent Application Publication 2020 / 0188497;US Patent 10,800,845;US Patent Application Publication 2020 / 0385468;US Patent 10,527,623;US Patent 10,725,044;US Patent Application Publication 2020 / 0249233;US Patent 10,702,609;US Patent Application Publication 2020 / 0254106;US Patent 10,800,832;US Patent Application Publication 2020 / 0123221; US ​​Patent 10,590,194; US Patent 10,723,796; US Patent Application Publication No. 2020 / 0140540; US Patent 10,618,956; US Patent Application Publication No. 2020 / 0207849; US Patent Application Publication No. 2020 / 0088726; and US Patent Application Publication No. 2020 / 0384028, the contents of each of which are incorporated herein by reference in their entirety. The cells may be T cells or natural killer cells, or any combination thereof. The T cells may be CD4+ cells, CD8+ cells, CD4+ / CD8+ cells, αβ T cells, γδ T cells, or natural killer T cells. In some embodiments, the TCRs described herein may be single chain TCRs or soluble TCRs.

[0387] Additionally, a TCR that may be co-expressed in a T cell with one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide described herein may be a TCR comprised of an alpha chain (TCRα) and a beta chain (TCRβ). In embodiments, the CD8 polypeptide may comprise a CD8α chain and / or a CD8β chain, which may be independently modified or unmodified.The TCR alpha and beta chains that may be used in the TCR are R11KEA (SEQ ID NOs: 15 and 16), R20P1H7 (SEQ ID NOs: 17 and 18), R7P1D5 (SEQ ID NOs: 19 and 20), R10P2G12 (SEQ ID NOs: 21 and 22), R10P1A7 (SEQ ID NOs: 23 and 24), R4P1D10 (SEQ ID NOs: 25 and 26), R4P3F9 (SEQ ID NOs: 27 and 28), R4P3H3 (SEQ ID NOs: 29 and 30), R36P3F9 (SEQ ID NOs: 31 and 32), R52 P2G11 (SEQ ID NOs: 33 and 34), R53P2A9 (SEQ ID NOs: 35 and 36), R26P1A9 (SEQ ID NOs: 37 and 38), R26P2A6 (SEQ ID NOs: 39 and 40), R26P3H1 (SEQ ID NOs: 41 and 42), R35P3A4 (SEQ ID NOs: 43 and 44), R37P1C9 (SEQ ID NOs: 45 and 46), R37P1H1 (SEQ ID NOs: 47 and 48), R42P3A9 (SEQ ID NOs: 49 and 50), R43P3F2 (SEQ ID NOs: 51 and 52), R43P3G5 (SEQ ID NOs: 53 and 54), R59P2E7 (SEQ ID NOs: 55 and 56), R11P3D3 (SEQ ID NOs: 57 and 58), R16P1C10 (SEQ ID NOs: 59 and 60), R16P1E8 (SEQ ID NOs: 61 and 62), R17P1A9 (SEQ ID NOs: 63 and 64), R17P1D7 (SEQ ID NOs: 65 and 66), R17P1G3 (SEQ ID NOs: 67 and 68), R17P2B6 (SEQ ID NOs: 69 and 70), R11P3D3KE (SEQ ID NOs: 71 and 303), R39P1C12 (SEQ ID NOs: 304 and 74), R39P1F5 (SEQ ID NOs: 75 and 76), R40P1C2 (SEQ ID NOs: 77 and 78), R41P3E6 (SEQ ID NOs: 79 and 80), R43P3G4 (SEQ ID NOs: 81 and 82), R44P3B3 (SEQ ID NOs: 83 and 84), R44P3E7 (SEQ ID NOs: 85 and 86), R49P2B7 (SEQ ID NOs: 87 and 88), R55P1G7 (SEQ ID NOs: 89 and 90), or R59P2A7 (SEQ ID NOs: 91 and 92). The cells can be T cells, natural killer cells, or any combination thereof. The T cells can be αβ T cells, γδ T cells, or natural killer T cells.

[0388] Table 1 shows examples of peptides that are bound by the TCR when the peptide is in a complex with an MHC molecule (in humans, MHC molecules are sometimes called HLA, human leukocyte antigens). Table 1: T cell receptors and peptides [Table 1]

[0389] Tumor-associated antigens (TAA) Tumor-associated antigen (TAA) peptides may be used with the IL-12p35 / IL-12p40 fusion polypeptide constructs, IL-15 polypeptide constructs, IL-18 polypeptide constructs, and / or CD8 polypeptide constructs, methods, and embodiments described herein. For example, the T cell receptors (TCRs) described herein may specifically bind to TAA peptides when bound to human leukocyte antigens (HLA), also known as major histocompatibility complex (MHC) molecules. Human MHC molecules are also designated human leukocyte antigens (HLA).

[0390] Tumor associated antigen (TAA) peptides that may be used with the IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein include, but are not limited to, those listed in Table 3, as well as those listed in U.S. Patent Application Publication No. 2016 / 0187351; U.S. Patent Application Publication No. 2017 / 0165335; U.S. Patent Application Publication No. 2017 / 0035807; U.S. Patent Application Publication No. 2016 / 0280759; U.S. Patent Application Publication No. 2016 / 0287687;US Patent Application Publication No. 2016 / 0346371;US Patent Application Publication No. 2016 / 0368965;US Patent Application Publication No. 2017 / 0022251;US Patent Application Publication No. 2017 / 0002055;US Patent Application Publication No. 2017 / 0029486;US Patent Application Publication No. 2017 / 0037089;US Patent Application Publication No. 2017 / 0136108;US Patent Application Publication No. 2017 / 0101473;US Patent Application Publication No. 2017 / 0096461;US Patent Application Publication No. 2017 / 0165 337;US Patent Application Publication No. 2017 / 0189505;US Patent Application Publication No. 2017 / 0173132;US Patent Application Publication No. 2017 / 0296640;US Patent Application Publication No. 2017 / 0253633;US Patent Application Publication No. 2017 / 0260249;US Patent Application Publication No. 2018 / 0051080;US Patent Application Publication No. 2018 / 0164315;US Patent Application Publication No. 2018 / 0291082;US Patent Application Publication No. 2018 / 0291083;US Patent Application Publication No. 2019 / 0255110;US Patent 9, 717,774; US Patent 9,895,415; US Patent Application Publication No. 2019 / 0247433; US Patent Application Publication No. 2019 / 0292520; US Patent Application Publication No. 2020 / 0085930; US Patent 10,336,809; US Patent 10,131,703; US Patent 10,081,664; US Patent 10,081,664; US Patent 10,093,715; US Patent 10,583,573; and US Patent Application Publication No. 2020 / 00085930. The contents of each of these publications, sequences described therein, and sequence listings are incorporated herein by reference in their entirety. The tumor associated antigen (TAA) peptides described herein can be bound to HLA (MHC molecules).HLA-bound tumor-associated antigen (TAA) peptides can be recognized by the TCRs described herein, optionally co-expressed with the CD8 polypeptides described herein.

[0391] T cells can be engineered to express chimeric antigen receptors (CARs) that contain ligand binding domains derived from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26, NKRP1, NKp30, NKp44, NKp46, CD244 (2B4), DNAM-1, and NKp80, or anti-tumor antibodies such as anti-Her2neu or anti-EGFR, as well as signaling domains obtained from CD3-zeta, Dap 10, CD28, 4-IBB, and CD40L. In some examples, the chimeric receptor is selected from the group consisting of MICA, MICB, Her2neu, EGFR, mesothelin, CD38, CD20, CD19, PSA, RON, CD30, CD22, CD37, CD38, CD56, CD33, CD30, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, 5T4, PLIF, Binds to Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipid F77, fibroblast activation protein, PSMA, STEAP-1, STEAP-2, c-met, CSPG4, Nectin-4, VEGFR2, PSCA, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3R, SLTRK6, gp100, MART1, tyrosinase, SSX2, SSX4, NYESO-1, epithelial tumor antigen (ETA), MAGEA family genes (e.g., MAGE3A.MAGE4A), KKLC1, mutant ras, βraf, p53, MHC class I chain-related molecule A (MICA) or MHC class I chain-related molecule B (MICB), HPV, or CMV. The cell can be a T cell, a natural killer cell, or any combination thereof. The T cells can be αβ T cells, γδ T cells, or natural killer T cells. T cell culture

[0392] Described herein are methods for activating, transducing, and / or expanding T cells, such as, for example, tumor infiltrating lymphocytes, CD8+ T cells, CD4+ T cells, and T cell expression that can be used. T cells can be activated, transduced, and expanded while depleting α- and / or β-TCR positive cells. T cells can be αβ T cells, γδ T cells, or natural killer T cells.

[0393] Described herein is a method for ex vivo expansion of a population of engineered γδ T cells for adoptive transfer therapy. The engineered γδ T cells of the present disclosure can be expanded ex vivo. The engineered T cells described herein can be expanded in vitro without activation by APC or without co-culture with APC and aminophosphate. Methods for transducing T cells are described in U.S. Patent Application No. 2019 / 0175650, published June 13, 2019, the contents of which are incorporated by reference in their entirety. Other methods for transducing and culturing T cells may also be used.

[0394] T cells, including γδ T cells, can be isolated from complex samples cultured in vitro. In embodiments, the entire PBMC population can be activated and expanded without prior depletion of specific cell populations, such as monocytes, αβ T cells, B cells, and NK cells. In embodiments, an enriched T cell population is generated, followed by specific activation and expansion. In embodiments, activation and expansion of γδ T cells can be performed in the presence or absence of natural or engineered antigen presenting cells (APCs). In embodiments, isolation and expansion of T cells from tumor specimens can be performed using immobilized T cell mitogens, including antibodies specific for γδ TCR, and other γδ TCR activators, including lectins. In embodiments, isolation and expansion of γδ T cells from tumor specimens can be performed in the absence of γδ T cell mitogens, including antibodies specific for γδ TCR, and other γδ TCR activators, including lectins.

[0395] T cells, including γδ T cells, can be isolated from a subject, e.g., leukapheresis of a human subject. In some embodiments, γδ T cells are not isolated from peripheral blood mononuclear cells (PBMCs). T cells can be isolated using anti-CD3 and anti-CD28 antibodies, optionally with recombinant human interleukin-2 (rhIL-2), e.g., about 50-150 U / mL rhIL-2.

[0396] Isolated T cells can be rapidly expanded in response to contact with one or more antigens. For example, some γδ T cells, such as Vγ9Vδ2+ T cells, can be rapidly expanded in vitro in response to contact with several antigens, such as prenyl-pyrophosphate, alkylamines, and metabolites or microbial extracts, during tissue culture. Stimulated T cells can present multiple antigen-presenting molecules, costimulatory molecules, and adhesion molecules, which facilitate the isolation of T cells from a complex sample. T cells in a complex sample can be stimulated in vitro with at least one antigen for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or another suitable period. Stimulation of T cells with a suitable antigen can expand the T cell population in vitro.

[0397] Activation and expansion of γδ T cells can be performed using the activating and costimulatory agents described herein to induce proliferation and sustained populations of specific γδ T cells. In some embodiments, activation and expansion of γδ T cells from different cultures can be performed to obtain different clonal population subsets or mixed polyclonal population subsets. In some embodiments, different agonist agents can be used to identify agents that provide specific γδ activation signals. In some embodiments, agents that provide specific γδ activation signals can be different monoclonal antibodies (MAbs) directed against the γδ TCR. In some embodiments, companion costimulatory agents can be used that help induce proliferation of specific γδ T cells without inducing cellular energetics and apoptosis. These costimulatory agents include, for example, ligands that bind to receptors expressed on γδ cells, such as NKG2D, CD161, CD70, JAML, DNAX accessory molecule-1 (DNAM-1), ICOS, CD27, CD137, CD30, HVEM, SLAM, CD122, DAP, and CD28. In some embodiments, the costimulatory agent may be an antibody specific for a unique epitope of the CD2 and CD3 molecules. CD2 and CD3 may have different conformations when expressed on αβ or γδ T cells. In some embodiments, antibodies specific for CD3 and CD2 may result in distinct activation of γδ T cells.

[0398] Non-limiting examples of antigens that can be used to stimulate in vitro expansion of T cells, including γδ T cells, from complex samples include phenyl-pyrophosphates, such as isopentenyl pyrophosphate (IPP), alkyl-amines, metabolites of human microbial pathogens, metabolites of commensal bacteria, methyl-3-butenyl-1-pyrophosphate (2M3B1PP), (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), ethyl pyrophosphate, and the like. (EPP), farnesyl pyrophosphate (FPP), dimethylallyl phosphate (DMAP), dimethylallyl pyrophosphate (DMAPP), ethyl-adenosine triphosphate (EPPPA), geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP), isopentenyl-adenosine triphosphate (IPPPA), monoethyl phosphate (MEP), monoethyl pyrophosphate (MEPP), 3-formyl-1-butyl-pyrophosphate (TUBAg 1), X-pyrophosphate (TUBAg 2), 3-formyl-1-butyl-uridine triphosphate (TUBAg 3), 3-formyl-1-butyl-deoxythymidine triphosphate (TUBAg 4), monoethylalkylamines, allyl pyrophosphate, crotoyl pyrophosphate, dimethylallyl-γ-uridine triphosphate, crotoyl-γ-uridine triphosphate, allyl-γ-uridine triphosphate, ethylamine, isobutylamine, sec-butylamine, iso-amylamine and nitrogen-containing bisphosphonates.

[0399] Prior to engineering the T cells, a T cell population comprising γδ T cells can be expanded in vivo. Non-limiting examples of reagents that may be used to promote expansion of T cell populations in vitro may include anti-CD3 antibodies, anti-CD2 antibodies, anti-CD27 antibodies, anti-CD30 antibodies, anti-CD70 antibodies, anti-OX40 antibodies, IL-2, IL-15, IL-12, IL-9, IL-33, IL-18 or IL-21, CD70 (CD27 ligand), Phytohemagglutinin (PHA), Concavalin A (ConA), Phytolacca maculatus (PWM), Protein Peanut Agglutinin (PNA), Soybean Agglutinin (SBA), Les Culinaris Agglutinin (LCA), Pisum Sativum Agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea Lectin (VGA), or another suitable mitogen capable of stimulating T cell proliferation. Additionally, T cells may be expanded using MCSF, IL-6, eotaxin, IFN-alpha, IL-7, gamma-induced protein 10, IFN-gamma, IL-1RA, IL-12, MIP-1 alpha, IL-2, IL-13, MIP-1 beta, IL-2R, IL-15, and any combination thereof.

[0400] The ability of γδ T cells to recognize a wide range of antigens can be enhanced by genetically engineering γδ T cells. γδ T cells can be engineered to provide universal allogeneic therapy that recognizes selected antigens in vivo. Genetic engineering of γδ T cells can include stable integration of constructs expressing tumor recognition moieties, such as αβ TCR, γδ TCR, chimeric antigen receptors (CARs) that combine both antigen binding and T cell activation functions in one receptor, antigen-binding fragments thereof, or lymphocyte activation domains, into the genome of isolated γδ T cells, stable integration of constructs expressing cytokines (e.g., IL-15, IL-12, IL-2, IL-7, IL-21, IL-18, IL-19, IL-33, IL-4, IL-9, IL-23, or IL1β) to enhance T cell proliferation, survival, and function in vitro and in vivo. Genetic engineering of the isolated γδ T-cell may also involve deleting or disrupting gene expression from one or more endogenous genes in the genome of the isolated γδ T-cell, such as, for example, an MHC locus.

[0401] Engineered (or transduced) T cells, including γδ T cells, can be expanded in vitro without stimulation with antigen-presenting cells or aminobisphosphonates. Antigen-reactive engineered T cells of the present disclosure can be expanded in vitro and in vivo. In embodiments, active populations of engineered T cells can be expanded in vitro without antigenic stimulation with antigen-presenting cells, antigenic peptides, non-peptide molecules, or small molecule compounds, such as aminobisphosphonates, but using specific antibodies, cytokines, mitogens, or fusion proteins, such as IL-17 Fc fusions, MICA Fc fusions, and CD70 Fc fusions. Examples of antibodies that can be used to expand the γδ T cell population include anti-CD3, anti-CD27, anti-CD30, anti-CD70, anti-OX40, anti-NKG2D or anti-CD2 antibodies, examples of cytokines can include IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7 and / or IL-33, examples of mitogens include CD70, which is the ligand for human CD27, phytohemagglutinin (PHA), concavalin A (ConA), pokeweed mitogen (PWM), protein peanut agglutinin (PNA), soybean agglutinin (SBA), Les Culinaris Agglutinin (LCA), Pisum Sativum Agglutinin (PSA), Helix pomatia agglutinin (HPA), Vicia graminea agglutinin (VAG), and the like. Lectin (VGA), or another suitable mitogen capable of stimulating T cell proliferation.

[0402] A population of engineered T cells, including γδ T cells, can be expanded in less than 60 days, less than 48 days, less than 36 days, less than 24 days, less than 12 days, or less than 6 days. In embodiments, a population of engineered T cells can be expanded for about 7 days to about 49 days, about 7 days to about 42 days, about 7 days to about 35 days, about 7 days to about 28 days, about 7 days to about 21 days, or about 7 days to about 14 days. T cells can be expanded for about 1 to 21 days. For example, T cells can be expanded for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.

[0403] In embodiments, the same methodology can be used to isolate, activate, and expand αβ T cells.

[0404] In embodiments, the same methodology can be used to isolate, activate, and expand γδ T cells. vector

[0405] Engineered cells can be generated using a variety of methods, including those recognized in the literature, for example, by injecting a polynucleotide encoding an expression cassette with a tumor recognition moiety or another type of recognition moiety into the cell via a transposon / transposase system, or via a viral-based gene transfer system, such as a lentiviral or retroviral system, or via other methods, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO 4), for example, nanoengineered materials such as Ormosil, adenovirus, retrovirus, lentivirus, adeno-associated virus, or by another suitable method. Many viral methods have been used in human gene therapy, for example, those described in WO 1993 / 020221, the contents of which are incorporated herein in their entirety. Non-limiting examples of viral methods that can be used to engineer cells can include gamma-retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia virus, poxvirus, or adenovirus-associated virus methods. Cells can include αβ T cells, γδ T cells, natural killer cells, natural killer T cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or any combination thereof.

[0406] Viruses used for transfection of cells include natural viruses as well as artificial viruses. Viruses can be either enveloped or non-enveloped viruses. Parvoviruses (such as AAV) are examples of non-enveloped viruses. Viruses can be enveloped viruses. Viruses used for transfection of T cells can be retroviruses, particularly lentiviruses. Viral envelope proteins that can facilitate viral infection of eukaryotic cells can include vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR) (SEQ ID NO: 97), and lentiviral vectors derived from HIV-1 pseudotyped with envelope glycoproteins (GP) from modified gibbon ape leukemia virus (GALVTR). These envelope proteins can efficiently facilitate the entry of other viruses, such as parvoviruses, including adeno-associated virus (AAV), and have demonstrated broad efficacy. For example, Moloney murine leukemia virus (MLV) 4070 env (described, e.g., in Merten et al., J. Virol. 79:834-840, 2005, the contents of which are incorporated herein by reference), RD114 ev, the chimeric envelope protein RD114pro or RDpro (which is an RD114-HIV chimera constructed by replacing the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, described, e.g., in Bell et al. Experimental Biology and Medicine 2010;235:1269-1276, the contents of which are incorporated herein by reference), baculovirus GP64 env (described, e.g., in Wang et al. J. Virol. 81:10869-10878, 2007, the contents of which are incorporated herein by reference), or GALV env (e.g., as described in Merten et al., J. Virol. 79:834-840, 2005).Other viral envelope proteins, including those disclosed herein by reference, or derivatives thereof, may be used.

[0407] A single lentiviral cassette can be used to create a single lentiviral vector, which expresses at least four individual monomeric proteins of two different dimers from a single multicistronic mRNA, thereby co-expressing the dimers on the cell surface.For example, the integration of one copy of the lentiviral vector is sufficient to transform T cells to co-express TCRαβ and CD8αβ, optionally αβ T cells or γδ T cells.

[0408] The vector may contain multicistronic cassettes in a single vector capable of expressing two or more, three or more, four or more, five or more, six or more, or seven or more genes, whose encoded polypeptides may interact with each other or form dimers. The dimers may be homodimers, e.g., two identical proteins form a dimer, or heterodimers, e.g., two structurally different proteins form a dimer.

[0409] Additionally, multiple vectors may be used to transfect cells with the constructs and sequences described herein. One or more vectors may contain one or any combination of a TCR transgene, an IL-12p35 / IL-12p40 fusion polypeptide transgene, an IL-15 polypeptide transgene, an IL-18 polypeptide transgene, and / or a CD8 transgene, in any order. As a non-limiting example, a first vector may contain a transgene encoding a TCR, a second vector may contain a transgene encoding an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide, and a third vector may contain a transgene encoding a CD8 polypeptide as described herein, and the vectors may be transfected into cells simultaneously or sequentially in any order using known methods. As another non-limiting example, one vector may encode two transgenes in any order, or one vector may encode three or more transgenes in any order. As another non-limiting example, a cell line stably transfected with one or more transgenes may in turn be transfected with one or more other transgenes. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, which may independently be modified or unmodified.

[0410] The one or more vectors may comprise one or more nucleic acids encoding one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, and / or an IL-18 polypeptide. The one or more vectors may comprise one or more nucleic acids encoding a CD8 polypeptide. The one or more vectors may comprise one or more nucleic acids encoding a CD8 α polypeptide. The one or more vectors may comprise one or more nucleic acids encoding a CD8 β polypeptide. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, which may be independently modified or unmodified.

[0411] The one or more vectors may comprise one or more nucleic acids encoding a T cell receptor (TCR) comprising an α chain and a β chain. The one or more vectors may comprise one or more nucleic acids encoding a T cell receptor (TCR) comprising a γ chain and a δ chain. The one or more vectors may comprise one or more nucleic acids encoding a chimeric antigen receptor (CAR).

[0412] The two or more vectors may comprise nucleic acids encoding one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain may independently be modified or unmodified.

[0413] A vector may comprise nucleic acids encoding one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, and / or a CAR. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, which may be independently modified or unmodified.

[0414] As used herein, the term "cistronic" refers to a portion of a nucleic acid molecule that provides for the formation of one polypeptide chain, i.e., that encodes one polypeptide chain. For example, "monocistron" refers to one portion of a nucleic acid molecule that provides for the formation of one polypeptide chain, i.e., that encodes one polypeptide chain. "bicistron" refers to two portions of a nucleic acid molecule that provides for the formation of two polypeptide chains, i.e., that encodes two polypeptide chains. "tricistron" refers to three portions of a nucleic acid molecule that provides for the formation of three polypeptide chains, i.e., that encodes three polypeptide chains. "multicistronic" refers to two or more portions of a nucleic acid molecule that provides for the formation of more than one polypeptide chain, i.e., that encodes more than one polypeptide chain.

[0415] As used herein, the term "arranged in tandem" refers to the arrangement of genes one after the other in a single tandem on a nucleic acid sequence. The genes are ligated together contiguously on the nucleic acid sequence and the coding strand (sense strand) of each gene is ligated together on the nucleic acid sequence.

[0416] The transgene may further comprise one or more multicistronic elements, which may be located between, by way of non-limiting example, any one, part, or each of the nucleic acid sequences encoding TCRα or a portion thereof, TCRβ or a portion thereof, CD8α or a portion thereof, CD8β or a portion thereof, IL-12p35 / IL-12p40 fusion polypeptide or a portion thereof, IL-15 polypeptide or a portion thereof, and / or IL-18 polypeptide or a portion thereof. The multicistronic element may be located between any two nucleic acid sequences encoding, by way of non-limiting example, TCRα, TCRβ, CD8α, CD8β, IL-12p35 / IL-12p40 fusion polypeptide, IL-15 polypeptide, and / or IL-18 polypeptide, and these coding sequences may be in any order. The multicistronic element may include a sequence encoding a ribosomal skip element selected from T2A, P2A, E2A or F2A, or an internal ribosome entry site (IRES).

[0417] As used herein, the term "self-cleaving 2A peptide" refers to a relatively short peptide (approximately 20 amino acids long, depending on the virus of origin) that acts co-translationally by preventing the formation of a normal peptide bond between glycine and the last proline, causing ribosome skipping to the next codon, and creating a new peptide cleavage between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the "upstream" protein, while a proline is added to the N-terminus of the "downstream" protein. The self-cleaving 2A peptide may be selected from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot and mouth disease virus (F2A), or any combination thereof (see, e.g., Kim et al., PLOS One 6:e18556, 2011, the contents of which, including the nucleic acid and amino acid sequences of 2A, are incorporated herein by reference in their entirety). The addition of one or more linker sequences (such as, but not limited to, GSG, LE, SGSG (SEQ ID NO: 266), or a linker set forth in SEQ ID NOs: 331, 333, 335, 337, 339, 341 or 343-381, or a linker encoded by SEQ ID NOs: 332, 334, 336, 338, 340 or 342) before the self-cleaving 2A sequence allows for efficient synthesis of biologically active proteins such as TCRs.

[0418] As used herein, the term "internal ribosome entry site (IRES)" refers to a nucleotide sequence located within a messenger RNA (mRNA) sequence that is capable of initiating translation without the aid of a 5' cap structure. Typically, IRESs are located in the 5' untranslated region (5'UTR), but may be located elsewhere in the mRNA. IRES can be derived from viruses, from cellular mRNAs, in particular picornaviruses, e.g. polio, EMCV and FMDV, flaviviruses, e.g. hepatitis C virus (HCV), pestiviruses, e.g. classical swine fever virus (CSFV), retroviruses, e.g. murine leukemia virus (MLV), lentiviruses, e.g. simian immunodeficiency virus (SIV), and insect RNA viruses, e.g. cricket paralysis virus, as well as from cellular mRNAs, translation initiation factors, e.g. eIF4G and DAP5, transcription factors, e.g. c-Myc and NF-κB inhibitors (NRF), growth factors, e.g. vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), platelet derived growth factor B (PDGF-B), homeotic genes, e.g. antennapedia, e.g. X-linked inhibitor of The IRES may be selected from survival proteins such as apoptosis (XIAP) and Apaf-1, as well as IRESs derived from other cellular mRNAs, such as, for example, BiP.

[0419] The constructs and vectors described herein may be used with the methods described in U.S. Patent Application Publication No. 2019 / 0175650, published June 13, 2019, the contents of which are incorporated herein by reference in their entirety.

[0420] In some embodiments, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence. In some embodiments, the post-transcriptional regulatory element (PRE) sequence may be selected from a Woodchuck Hepatitis Virus PRE (WPRE) (e.g., but not limited to, a wild-type WPRE, such as SEQ ID NO: 264, or a mutant WPRE, such as, but not limited to, WPREmut1 (SEQ ID NO: 256) or WPREmut2 (SEQ ID NO: 257)), or a Hepatitis B Virus (HBV) PRE (HPRE) (SEQ ID NO: 385), a variant thereof, or any combination thereof.

[0421] In some embodiments, the vector may further comprise one or more promoters, which may be selected from a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, a myelin basic protein (MBP) promoter, a glial fibrillary acidic protein (GFAP) promoter, a modified MoMuLV LTR containing myeloproliferative sarcoma virus enhancer (MNDU3), a ubiquitin C promoter, an EF-1 alpha promoter, a murine stem cell virus (MSCV) promoter, a promoter from CD69, a nuclear factor of activated T-cells (NFAT) promoter, an IL-2 promoter, a minimal IL-2 promoter, or a combination thereof.

[0422] In embodiments, the vector may include one or more Kozak sequences. In embodiments, the Kozak sequence may initiate, increase, or enhance translation, or a combination thereof. In embodiments, the Kozak sequence may be GCCACC (SEQ ID NO: 380). In embodiments, the Kozak sequence may be ACCATGG (SEQ ID NO: 381). In embodiments, the Kozak sequence may be GCCNCCATGG, where N is a purine (A or G) (SEQ ID NO: 384).

[0423] In embodiments, the vector may contain one or more factor Xa sites.

[0424] In some embodiments, the vector may include one or more enhancers. In some embodiments, the enhancer may include Conserved Non-Coding Sequence (CNS) 0, CNS1, CNS2, CNS3, CNS4, or a portion thereof, or any combination thereof.

[0425] In embodiments, the vector may be a viral vector or a non-viral vector.

[0426] In embodiments, the vector may be selected from an adenovirus, a poxvirus, an alphavirus, an arenavirus, a flavivirus, a rhabdovirus, a retrovirus, a lentivirus, a herpesvirus, a paramyxovirus, a picornavirus, or a combination thereof.

[0427] In embodiments, the vector may be pseudotyped with an envelope protein of a virus selected from native feline endogenous virus (RD114), chimeric RD114 (RD114TR), gibbon ape leukemia virus (GALV), chimeric GALV (GALV-TR), amphotropic murine leukemia virus (MLV 4070A), baculovirus (GP64), vesicular stomatitis virus (VSV-G), fowl plague virus (FPV), Ebola virus (EboV), or baboon retrovirus envelope glycoprotein (BaEV), and lymphocytic choriomeningitis virus (LCMV), or a combination thereof.

[0428] Non-viral vectors can also be used in conjunction with the sequences, constructs, and cells described herein.

[0429] Cells may be transfected by other means known in the art, including lipofection (liposome-based transfection), electroporation, calcium phosphate transfection, biolistic particle delivery (e.g., gene gun), microinjection, or any combination thereof. A variety of methods for transfecting cells are known in the art. See, for example, Sambrook & Russell (eds.) Molecular Cloning: A Laboratory Manual (3rd ed.) Volumes 1-3 (2001) Cold Spring Harbor Laboratory Press; Ramamoorth & Narvekar "Non Viral Vectors in Gene Therapy- An Overview." J Clin Diagn Res. (2015) 9(1): GE01-GE06. Gene editing

[0430] In embodiments, transgenes (e.g., transgenes encoding the alpha and / or beta chains of CD8, transgenes encoding the alpha and / or beta chains of TCR, transgenes encoding IL-12 fusion polypeptides, transgenes encoding IL-15 polypeptides, and / or transgenes encoding IL-18 polypeptides) can be inserted into cells using gene addition, gene editing, gene replacement, and / or gene transfer techniques, including but not limited to knock-in techniques, including but not limited to targeted knock-in techniques. The cells can be, by way of non-limiting examples, T cells or natural killer cells or combinations thereof. The T cells can be, by way of non-limiting examples, alpha beta T cells, gamma delta T cells, natural killer T cells, CD4+ cells, CD8+ cells, CD4+ / CD8+ cells, or combinations thereof. Non-limiting examples include techniques such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (including, but not limited to, using Cas9, Cas12, Cas12a, Cas12a2 and / or Cas13), transcription-activator-like effector nuclease (TALEN) systems, and / or transposon-based systems (see, e.g., U.S. Patent Application Publication No. 2019 / 0169637, which is incorporated herein in its entirety). Non-limiting examples of transposon-based systems include Sleeping Beauty (see, e.g., U.S. Pat. Nos. 7,985,739, 6,613,752, and 9,228,180 and U.S. Patent Application Publication Nos. 2005 / 0003542, 2004 / 0092471, 2002 / 0103152, 2016 / 0264949, 2018 / 0135032, 2011 / 0117072, 2019 / 0169638, 2005 / 0112764, 2017 / 0029774, 2021 / 0139583).Nos. 10,287,559, 11,186,847, 10,131,885, 9,546,382, 8,399,643, 8,592,211, 6,962,810, 7,105,343, and 6,551,825, and U.S. Patent Application Publication No. 2018 / 0142219. , 2017 / 0166874, 2016 / 0160235, 2020 / 0087635, 2018 / 0195086, 2013 / 0160152, 2010 / 0287633, 2022 / 0064610, 2009 / 0042297, 2002 / 0173634, and 2017 / 0226531, each of which is incorporated herein in its entirety), and / or TcBuster systems (see, e.g., U.S. Pat. Nos. 11,278,570, 11,162,084, and 11,111,483, and U.S. Patent Application Publication Nos. 2021 / 0277366, 2020 / 0339965, and 2020 / 0323902, each of which is incorporated herein in its entirety). composition

[0431] The composition can comprise one or any combination of the IL-12p35 / IL-12p40 fusion polypeptide, IL-15 polypeptide, IL-18 polypeptide, and / or CD8 polypeptide described herein, and / or the TCR polypeptide described herein. Further, the composition described herein can comprise T cells and / or natural killer (NK) cells that express one or any combination of the IL-12p35 / IL-12p40 fusion polypeptide, IL-15 polypeptide, IL-18 polypeptide, and / or CD8 polypeptide described herein. The composition described herein can comprise T cells and / or natural killer cells that express one or any combination of the IL-12p35 / IL-12p40 fusion polypeptide, IL-15 polypeptide, IL-18 polypeptide, and / or CD8 polypeptide described herein, and a T cell receptor (TCR), optionally, a TCR that specifically binds to one of the TAAs described herein that is complexed with an antigen-presenting protein, such as, for example, MHC, also referred to as HLA in humans with respect to human leukocyte antigens. In a plurality of embodiments, the CD8 polypeptide can comprise the CD8α chain and / or the CD8β chain, and the CD8α chain and / or the CD8β chain can be independently modified or unmodified.

[0432] For ease of administration, the T cells and / or natural killer cells described herein can be made within a pharmaceutical composition or, using a pharmaceutically acceptable carrier or diluent, within an implant suitable for in vivo administration. Means for making such a composition or implant are described in the art. See, for example, Remington’s Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980).

[0433] The T cells and / or natural killer cells described herein may be formulated into a preparation in semi-solid or liquid form, such as in the form of a capsule, solution, infusion, or injection. Means known in the art may be utilized to prevent or minimize the release and absorption of the composition until it reaches the target tissue or organ, or to ensure the sustained release of the composition. However, it is desirable to employ a pharma- ceutical acceptable form that does not prevent the cells from expressing the CAR or TCR. Therefore, it is desirable that the T cells and / or natural killer cells described herein may be made into a pharmaceutical composition that includes a carrier. The T cells and / or natural killer cells described herein may be formulated with a physiologically acceptable carrier or excipient for preparing a pharmaceutical composition. The carrier and composition may be sterilized. Carriers include, for example, balanced salt solutions such as Hank's balanced salt solution, or normal saline. The formulation must be compatible with the mode of administration. Suitable pharma- ceutical acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, and any combination thereof. Pharmaceutical preparations can be mixed, if desired, with auxiliary agents that do not deleteriously react with T cells and / or natural killer cells, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers. The cells can be αβ T cells, γδ T cells, and / or natural killer cells expressing one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, CD8 polypeptides, optionally a TCR as described herein. In embodiments, the CD8 polypeptide can include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or CD8 β chain can be independently modified or unmodified.

[0434] The compositions described herein may be provided in unit dosage form, where each dosage unit, e.g., an injectable solution, contains a predetermined amount of the composition, alone or in combination with other active agents as appropriate.

[0435] The composition described herein may be a pharmaceutical composition.The pharmaceutical composition described herein may further comprise an adjuvant selected from the group consisting of colony stimulating factor, including but not limited to granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, interferon alpha, or combinations thereof.

[0436] The pharmaceutical compositions described herein may include an adjuvant selected from the group consisting of colony stimulating factors, such as granulocyte-macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod.

[0437] Adjuvants include, but are not limited to, cyclophosphamide, imiquimod, or resiquimod. Other non-limiting examples of adjuvants include Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or any combination thereof.

[0438] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), dsRNA analogs such as Poly(I:C) and its derivatives (e.g., AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, ... Immune checkpoint inhibitors including sufamide, sunitinib, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab and cemiplimab, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF These include Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies that target important structures of the immune system (e.g., anti-CD40, anti-TGF beta, anti-TNF alpha receptor) and SC58175, which may act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present disclosure can be easily determined by those skilled in the art without undue experimentation.

[0439] Other adjuvants include, but are not limited to, anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon alpha, interferon beta, CpG oligonucleotides and derivatives thereof, poly(I:C) and derivatives thereof, RNA, sildenafil, and particle formulations including poly(lactide-co-glycolide) (PLG), polyinosinic-polycytidylic-poly-I-lysine carboxymethylcellulose (poly-ICLC), virosomes, and / or interleukin-1 (IL-1), IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-18, IL-21, and IL-23. See, e.g., Narayanan et al. J. Med. Chem. (2003) 46(23):5031-5044; Pohar et al. Scientific Reports 7 14598 (2017); Grajkowski et al. Nucleic Acids Research (2005) 33(11):3550-3560; Martins et al. Expert Rev Vaccines (2015) 14(3):447-59.

[0440] The compositions described herein may include one or more adjuvants. Adjuvants are substances that non-specifically enhance or strengthen immune responses (e.g., immune responses to antigens mediated by CD8 positive T cells and helper T (TH) cells) and are therefore considered useful in the medicaments of the present disclosure. Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or a TLR5 ligand derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, imiquimod (ALDARA®), resiquimod, ImuFact IMP321, interleukins such as IL-2, IL-13, IL-21, interferon alpha or beta or pegylated derivatives thereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactide-co-glycolide) [PLG] system and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin, acid-fast bacillus extracts and synthetic bacterial cell wall mimics, and other commercial adjuvants such as Ribi's Detox, Quil or Superfos. Exemplary adjuvants include Freund's or GM-CSF. Several immune adjuvants (e.g., MF59) specific for dendritic cells and their preparations have been previously reported. Cytokines may also be used.Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha, IFN-beta).

[0441] CpG immunostimulatory oligonucleotides have also been reported to improve the efficacy of adjuvants in vaccination. Without being bound by theory, CpG oligonucleotides act by activating the innate immune system (non-adaptive immune system) through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular immunity against a variety of antigens, including peptide and protein antigens, live and killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, by enhancing dendritic cell maturation and differentiation, TH1 cell activation is enhanced and potent cytotoxic T lymphocytes (CTLs) are generated, even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained, even in the presence of vaccine adjuvants, such as ammonium sulfate (alum) and incomplete Freund's adjuvant (IFA), which typically promote a TH2 bias. CpG oligonucleotides show stronger adjuvant activity when formulated or administered simultaneously with other adjuvants, or when formulated or administered simultaneously in formulations such as microparticles, nanoparticles, liquid emulsions, and similar formulations. This is especially necessary to induce a strong response when the antigen is relatively weak. It is also possible to accelerate the immune response and reduce the antigen dose by about two orders of magnitude while, in some experiments, generating an antibody response equivalent to a full dose vaccine without CpG (Krieg, 2006). US 6,406,705 B1 reports that the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens induced antigen-specific immune responses. The CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) from Mologen (Berlin, Germany). In some embodiments, dSLIM can be a component of the pharmaceutical composition described herein. Other TLR binding molecules may also be used, such as, for example, RNA that binds to TLR7, TLR8, and / or TLR9. Treatment and preparation methods

[0442] The engineered T cells and / or engineered natural killer cells may express one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein. Additionally, the engineered T cells and / or engineered natural killer cells may express a TCR as described herein. The TCR expressed by the engineered T cells and / or engineered natural killer cells may recognize a TAA bound to an HLA as described herein. The engineered T cells and / or engineered natural killer cells of the present disclosure may be used to treat a subject in need of treatment for a disease, such as, for example, cancer, as described herein. The T cells may be αβ T cells, or γδ T cells, expressing IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides, and optionally a TCR as described herein. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0443] A method of treating a disease (e.g., a disorder) in a subject using the T cells and / or natural killer cells described herein may include administering to the subject a therapeutically effective amount of the engineered T cells and / or engineered natural killer cells described herein, optionally γδ T cells. The T cells and / or natural killer cells described herein may be administered in a variety of regimens (e.g., timing, concentration, dose, treatment interval, and / or formulation). The subject may be pretreated, for example, with chemotherapy, radiation, or a combination of both, prior to administration of the engineered T cells and / or engineered natural killer cells of the present disclosure. The population of engineered T cells and / or engineered natural killer cells may be frozen or cryopreserved prior to administration to the subject. The population of engineered T cells and / or engineered natural killer cells may include two or more cells expressing the same tumor recognition moiety, different tumor recognition moieties, or a combination of the same tumor recognition moiety and different tumor recognition moieties. For example, a population of engineered T cells and / or engineered natural killer cells can include several distinct engineered T cells and / or natural killer cells designed to recognize different antigens or different epitopes of the same antigen. The cells can be αβ T cells, γδ T cells, or combinations thereof, expressing one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide described herein, and optionally a TCR as described herein. In embodiments, the CD8 polypeptide can include a CD8 α chain and / or a CD8 β chain, which can be independently modified or unmodified.

[0444] The T cells and / or natural killer cells described herein, including αβ T cells and γδ T cells, may be used to treat a variety of conditions. The cells may be αβ T cells, γδ T cells, and / or natural killer cells expressing one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide, and optionally a TCR as described herein. In embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, which may be independently modified or unmodified. The T cells and / or natural killer cells described herein may be used to treat cancer, including solid tumors and hematological malignancies. Non-limiting examples of cancer include non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, renal cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, gastric cancer, and prostate cancer.

[0445] The T cells and / or natural killer cells described herein may be used to treat infectious diseases. The T cells and / or natural killer cells described herein may be used to treat infectious diseases that may be caused by viruses. The T cells and / or natural killer cells described herein may be used to treat immune diseases, such as autoimmune diseases. The cells may be αβ T cells, γδ T cells, and / or natural killer cells expressing one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide, and optionally a TCR as described herein. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0446] Treatment with T cells and / or natural killer cells, optionally γδ T cells, as described herein may be provided to a subject before, during, and after clinical manifestation of a condition. Treatment may be provided to a subject one day, one week, six months, twelve months, or two years after clinical manifestation of a disease. Treatment may be provided to a subject for multiple days, one week, one month, six months, twelve months, two years, three years, four years, five years, six years, seven years, eight years, nine years, ten years, or more after clinical manifestation of a disease. Treatment may be provided to a subject less than one day, one week, one month, six months, twelve months, or two years after clinical manifestation of a disease. Treatment may also include treating humans in clinical trials. Treatment may include administering to a subject a pharmaceutical composition comprising the engineered T cells and / or natural killer cells as described herein. The cell may be an αβ T cell, a γδ T cell, and / or a natural killer cell expressing one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide, and optionally a TCR as described herein. In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, which may independently be modified or unmodified.

[0447] In embodiments, the activity of endogenous lymphocytes in the subject's body may be modulated by administering the engineered T cells and / or natural killer cells of the present disclosure to a subject. In embodiments, the endogenous T cells may be provided with antigen and the immune response may be boosted by administering the engineered T cells and / or natural killer cells of the present disclosure to a subject. In embodiments, the memory T cells may be CD4+ T cells. In embodiments, the memory T cells may be CD8+ T cells. In embodiments, the cytotoxicity of another immune cell may be activated by administering the engineered T cells and / or natural killer cells of the present disclosure to a subject. In embodiments, the other immune cell may be CD8+ T cells. In embodiments, the other immune cell may be a natural killer T cell. In embodiments, the regulatory T cells may be suppressed by administering the engineered γδ T cells of the present disclosure to a subject. In embodiments, the regulatory T cells may be FOX3+ Treg cells. In embodiments, the regulatory T cells may be FOX3- Treg cells. Non-limiting examples of cells whose activity may be modulated by the engineered T cells and / or engineered natural killer cells of the present disclosure may include hematopoietic stem cells, B cells, CD4, CD8, red blood cells, white blood cells, dendritic cells including dendritic antigen presenting cells, white blood cells, macrophages, memory B cells, memory T cells, monocytes, natural killer cells, neutrophils, granulocytes, T helper cells, and T killer cells. The cells may be αβ T cells, γδ T cells, natural killer cells, or any combination thereof expressing one or any combination of an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide, and optionally a TCR as described herein. In embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified.

[0448] In most bone marrow transplants, a combination of cyclophosphamide and total body irradiation may be routinely employed to prevent rejection of hematopoietic stem cells (HSCs) in the graft by the subject's immune system. In some embodiments, ex vivo incubation of donor bone marrow with interleukin-2 (IL-2) may be performed to enhance the generation of killer lymphocytes in the donor bone marrow. Interleukin-2 (IL-2) is a cytokine that may be required for the growth, proliferation, and differentiation of wild-type lymphocytes. Current research on adoptive transfer of γδ T cells into humans may require co-administration of γδ T cells and interleukin-2. However, both low and high doses of IL-2 may have highly toxic side effects. The toxicity of IL-2 may manifest in multiple organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In embodiments, the present disclosure provides methods for administering engineered T cells and / or engineered natural killer cells to a subject without co-administration of native cytokines or modified versions thereof, such as, for example, IL-2, IL-15, IL-12, IL-21, etc. In embodiments, the engineered T cells and / or engineered natural killer cells may be administered to a subject without co-administration with IL-2. In embodiments, the engineered T cells and / or engineered natural killer cells may be administered to a subject without co-administration with IL-2, for example, during a procedure such as a bone marrow transplant.

[0449] In some embodiments, the method may further include administering a chemotherapeutic agent. The dose of the chemotherapeutic agent may be sufficient to deplete the patient's T cell population. The chemotherapy may be administered about 5-7 days prior to administration of the T cells and / or natural killer cells. The chemotherapeutic agent may be cyclophosphamide, fludarabine, or a combination thereof. The chemotherapeutic agent may be administered at a dose of about 400-600 mg / m 2 The chemotherapy agent may include a dose of cyclophosphamide at about 10-30 mg / m 2 per day of fludarabine.

[0450] In some embodiments, the method may further include pretreating the patient with low dose radiation prior to administration of the composition comprising the T cells and / or natural killer cells. The low dose radiation may include about 1.4 Gy for 1-6 days, e.g., about 5 days, prior to administration of the composition comprising the T cells.

[0451] In embodiments, the patient may be HLA-A*02.

[0452] In embodiments, the patient may be HLA-A*06.

[0453] In some embodiments, the method may further include administering an anti-PD1 antibody. The anti-PD1 antibody may be a humanized antibody. The anti-PD1 antibody may be pembrolizumab. The dose of the anti-PD1 antibody may be about 200 mg. The anti-PD1 antibody may be administered every 3 weeks after administration of the T cells.

[0454] In some embodiments, the dose of T cells and / or natural killer cells is about 0.8-1.2×10 9 The dose of T cells and / or natural killer cells may be about 0.5×10 8 ~About 10×10 9 The dose of T cells and / or natural killer cells may be about 1.2-3×10 9 T cells and / or natural killer cells, approximately 3-6 x 10 9 T cells and / or natural killer cells, approximately 10 x 10 9 T cells and / or natural killer cells, approximately 5 x 10 9 T cells and / or natural killer cells, approximately 0.1 x 10 9 T cells and / or natural killer cells, approximately 1 x 10 8 T cells and / or natural killer cells, approximately 5 x 10 8 T cells and / or natural killer cells, approximately 1.2-6 x 10 9T cells and / or natural killer cells, approximately 1-6 x 10 9 T cells and / or natural killer cells, or approximately 1-8 x 10 9 The cells may be T cells and / or natural killer cells.

[0455] In some embodiments, the T cells and / or natural killer cells may be administered in three doses. The dose of the T cells and / or natural killer cells may be escalated with each administration. The T cells and / or natural killer cells may be administered by intravenous infusion.

[0456] In some embodiments, the TCR, CD8, IL-12, IL-15 and / or IL-18 sequences and related products and compositions described herein may be used in autologous or allogeneic adoptive cell therapy. In another embodiment, the TCR, CD8, IL-12, IL-15 and / or IL-18 sequences, T cells and / or natural killer cells thereof, and compositions may be used, for example, in U.S. Patent Application Publication No. 2019 / 0175650, U.S. Patent Application Publication No. 2019 / 0216852, U.S. Patent Application Publication No. 2019 / 024743, and U.S. Provisional Patent Application No. 62 / 980,844, which are incorporated herein by reference in their entirety.

[0457] The present disclosure also provides a population of modified T cells and / or natural killer cells expressing one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, and / or IL-18 polypeptides, and / or displaying exogenous CD8 polypeptides and T cell receptors as described herein, where the modified population of T cells can be activated and expanded with a combination of IL-2 and / or IL-15. In another embodiment, the modified population of T cells and / or natural killer cells can be expanded and / or activated with a combination of IL-2, IL-15 and / or zoledronate. In yet another embodiment, the modified population of T cells and / or natural killer cells can be expanded with a combination of IL-2, IL-15, and / or zoledronate without zoledronate, while being activated with a combination of IL-2, IL-15 and / or zoledronate. The present disclosure further provides for the use of other interleukins during activation and / or amplification, such as, for example, IL-12, IL-18, IL-21, and any combination thereof.

[0458] In some aspects, IL-21, histone deacetylase inhibitors (HDACi) or any combination thereof may be utilized in the field of cancer therapy with the methods described herein and / or with the ACT process described herein. In some embodiments, the present disclosure provides a method of reprogramming effector T cells to a central memory phenotype, comprising culturing the effector T cells with at least one HDACi together with IL-21. Representative HDACi include, for example, trichostatin A, trapoxin B, phenyl butyrate, valproic acid, vorinostat (suberanilohydroxamic acid), belinostat, panobinostat, dacinostat, entinostat, tacedinaline, and mocetinostat.

[0459] Compositions comprising engineered T cells and / or natural killer cells described herein may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the pharmaceutical compositions may be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. The engineered T cells and / or natural killer cells may also be administered to reduce the likelihood of developing, acquiring, or worsening the condition. For therapeutic applications, the effective amount of the engineered T cell and / or natural killer cell population may vary based on the severity and course of the disease or condition, previous treatments, the subject's health, weight, and / or responsiveness to drugs, and / or the judgment of the treating physician. The cells may be αβ T cells, γδ T cells, and / or natural killer cells engineered to express one or any combination of IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein, and optionally a TCR as described herein. In some embodiments, the CD8 polypeptide may include a CD8 α chain and / or a CD8 β chain, and the CD8 α chain and / or the CD8 β chain may be independently modified or unmodified. T cell therapy has been successful in treating various cancers. Li et al. Signal Transduction and Targeted Therapy 4(35):(2019). The contents of which are incorporated herein by reference. Method of administration

[0460] One or more of the engineered T cell populations and / or natural killer cell populations described herein may be administered to a subject in any order or simultaneously. If simultaneously, the engineered T cells and / or natural killer cells may be provided in one unified form, such as an intravenous injection, or in multiple forms, such as multiple intravenous infusions, subcutaneous injections, or pills. The engineered T cells and / or natural killer cells may be packaged together or separately, in one package, or in multiple packages. One or all of the engineered T cells and / or natural killer cells may be administered in multiple doses. If not simultaneously, the timing between multiple doses may vary from about one week, one month, two months, three months, four months, five months, six months, or about one year. In some embodiments, the engineered T cells and / or natural killer cells may be expanded in vivo in the subject's body after administration to the subject. The engineered T cells and / or natural killer cells may be frozen to provide cells for multiple treatments with the same cell preparation. The engineered T cells and / or natural killer cells of the present disclosure, and pharmaceutical compositions comprising same, can be packaged as kits. The kits can include instructions (e.g., written instructions) for use of the engineered T cells and / or engineered natural killer cells, and compositions comprising same.

[0461] A method of treating cancer may include administering to a subject a therapeutically effective amount of engineered T cells and / or natural killer cells, whereby the cancer is treated. In some embodiments, the therapeutically effective amount of engineered γδ T cells and / or engineered natural killer cells may be administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In some embodiments, the therapeutically effective amount of engineered T cells and / or engineered natural killer cells may be administered for at least 1 week. In some embodiments, the therapeutically effective amount of engineered T cells and / or engineered natural killer cells may be administered for at least 2 weeks.

[0462] The engineered T cells and / or engineered natural killer cells, optionally γδ T cells, described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administering the pharmaceutical composition comprising the engineered T cells and / or natural killer cells can vary. For example, the engineered T cells and / or engineered natural killer cells can be used as a prophylactic and can be administered continuously to a subject prone to a condition or disease to reduce the likelihood of the onset of the disease or condition. The engineered T cells and / or engineered natural killer cells can be administered to a subject during the onset of symptoms or as soon as possible after the onset of symptoms. Administration of the engineered T cells and / or engineered natural killer cells can begin immediately after the onset of symptoms, within the first 3 hours of the onset of symptoms, within about the first 6 hours of the onset of symptoms, within about the first 24 hours of the onset of symptoms, within about 48 hours of the onset of symptoms, or within any time period from the onset of symptoms. The initial administration can be via any practical route, such as any route described herein, using any formulation described herein. In some embodiments, administration of the engineered T cells and / or engineered natural killer cells of the present disclosure may be intravenous. One or more doses of engineered T cells and / or engineered natural killer cells may be administered as soon as possible after the onset of cancer, infectious disease, immune disease, sepsis, or at the time of bone marrow transplantation, for the period of time required to treat the immune disease, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. With respect to the treatment of cancer, one or more doses of engineered T cells and / or engineered natural killer cells may be administered several years after the onset of cancer, and before or after other treatments.In embodiments, the engineered γδ T cells and / or engineered natural killer cells can be administered for at least about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, or at least about 5 years. The length of treatment can vary from subject to subject. The cell may be an IL-12p35 / IL-12p40 fusion polypeptide, an IL-15 polypeptide, an IL-18 polypeptide, and / or a CD8 polypeptide described herein, optionally an αβ T cell, a γδ T cell, and / or a natural killer cell expressing a TCR described herein.

[0463] The engineered T cells and / or natural killer cells, optionally αβ T cells and / or γδ T cells, expressing one or any combination of the IL-12p35 / IL-12p40 fusion polypeptides, IL-15 polypeptides, IL-18 polypeptides, and / or CD8 polypeptides described herein, are at least about 1×10 3 Cells / ml, at least about 2 x 10 3 Cells / ml, at least about 3 x 10 3 Cells / ml, at least about 4 x 10 3 Cells / ml, at least about 5 x 10 3 Cells / ml, at least about 6 x 10 3 Cells / ml, at least about 7 x 10 3 Cells / ml, at least about 8 x 10 3 Cells / ml, at least about 9 x 10 3 Cells / ml, at least about 1 x 10 4Cells / ml, at least about 2 x 10 4 Cells / ml, at least about 3 x 10 4 Cells / ml, at least about 4 x 10 4 Cells / ml, at least about 5 x 10 4 Cells / ml, at least about 6 x 10 4 Cells / ml, at least about 7 x 10 4 Cells / ml, at least about 8 x 10 4 Cells / ml, at least about 9 x 10 4 Cells / ml, at least about 1 x 10 5 Cells / ml, at least about 2 x 10 5 Cells / ml, at least about 3 x 10 5 Cells / ml, at least about 4 x 10 5 Cells / ml, at least about 5 x 10 5 Cells / ml, at least about 6 x 10 5 Cells / ml, at least about 7 x 10 5 Cells / ml, at least about 8 x 10 5 Cells / ml, at least about 9 x 10 5 Cells / ml, at least about 1 x 10 6 Cells / ml, at least about 2 x 10 6 Cells / ml, at least about 3 x 10 6 Cells / ml, at least about 4 x 10 6 Cells / ml, at least about 5 x 10 6 Cells / ml, at least about 6 x 10 6 Cells / ml, at least about 7 x 10 6 Cells / ml, at least about 8 x 10 6 Cells / ml, at least about 9 x 10 6 Cells / ml, at least about 1 x 10 7 Cells / ml, at least about 2 x 10 7 Cells / ml, at least about 3 x 10 7 Cells / ml, at least about 4 x 10 7 Cells / ml, at least about 5 x 10 7 Cells / ml, at least about 6 x 10 7 Cells / ml, at least about 7 x 10 7 Cells / ml, at least about 8 x 10 7Cells / ml, at least about 9 x 10 7 Cells / ml, at least about 1 x 10 8 Cells / ml, at least about 2 x 10 8 Cells / ml, at least about 3 x 10 8 Cells / ml, at least about 4 x 10 8 Cells / ml, at least about 5 x 10 8 Cells / ml, at least about 6 x 10 8 Cells / ml, at least about 7 x 10 8 Cells / ml, at least about 8 x 10 8 Cells / ml, at least about 9 x 10 8 Cells / ml, at least about 1 x 10 9 Cells / ml or more, approximately 1×10 3 Cells / ml ~ at least about 1 x 10 8 cells / ml, approximately 1×10 5 Cells / ml ~ at least about 1 x 10 8 cells / ml, or approximately 1 x 10 6 Cells / ml ~ at least about 1 x 10 8 It may be present in the composition in the amount of cells / ml.

[0464] use The T cells, natural killer (NK) cells, and pharmaceutical compositions described herein may be used in methods of treatment, particularly cancer treatment. Thus, the present disclosure also provides the use of the T cells, natural killer (NK) cells, and pharmaceutical compositions described herein in methods of treatment, particularly cancer treatment. Furthermore, the present disclosure also provides the use of the T cells, natural killer (NK) cells, and pharmaceutical compositions described herein in the manufacture of a medicament, particularly a medicament for the treatment of cancer. The cancer may be selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, gastric cancer, and prostate cancer. The features and aspects described in relation to the above-mentioned methods of treatment, preparation, and administration are applicable mutatis mutandis to the uses described herein.

[0465] array The sequences described herein may comprise about 80%, about 85%, about 90%, about 85%, about 96%, about 97%, about 98%, or about 99%, or 100% identity to any of the sequences of SEQ ID NOs: 1-97, 256-266, 293, 294, or 305-384. The sequences described herein may comprise at least 80%, at least 85%, at least 90%, at least 85%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of the sequences of SEQ ID NOs: 1-97, 256-266, or 305-384. A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity over its entire length to the entire length of the reference sequence.

[0466] In another embodiment, the disclosure provides a sequence with at least about 80%, at least about 85%, at least about 90%, at least about 85%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% identity to WPREmut1 (SEQ ID NO: 256) or WPRE version 2, e.g., WPREmut2 (SEQ ID NO: 257). In another aspect, the disclosure provides a sequence with at least 1, 2, 3, 4, 5, 10, 15 or 20 amino acid substitutions in WPREmut1 (SEQ ID NO: 256) or WPRE version 2, e.g., WPREmut2 (SEQ ID NO: 257). In yet another aspect, the disclosure provides a sequence with up to 1, 2, 3, 4, 5, 10, 15 or 20 amino acid substitutions in WPREmut1 (SEQ ID NO: 256) or WPRE version 2, e.g., WPREmut2 (SEQ ID NO: 257). In another aspect, the sequence substitutions are conservative substitutions.

[0467] Percentage of identity can be calculated using global pairwise alignment (e.g., two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are known in the art. For example, the "needle" program can be used, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48: 443-453) to find the optimal alignment (including gaps) of two sequences when considering their entire length. The needle program is available, for example, at the ebi.ac.uk World Wide Web site and is described in detail in the following publications (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percent identity between two polypeptides according to the present disclosure is calculated using the EMBOSS:needle (global) program with the "Gap Open" parameter set to 10.0, the "Gap Extend" parameter set to 0.5, and the Blosum62 matrix.

[0468] A protein comprising or consisting of an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical," "at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference sequence, or a similar enumeration, may contain mutations, such as, for example, deletions, insertions and / or substitutions, compared to the reference sequence. The reference sequence may be, as non-limiting examples, a wild-type sequence, a mature wild-type sequence, a native sequence, a truncated wild-type sequence, a truncated mature wild-type sequence, a truncated native sequence, or a sequence disclosed herein. The reference sequence may be, as non-limiting examples, a wild-type sequence, a mature wild-type sequence, or a native sequence. In the case of substitutions, a protein consisting of an amino acid sequence that is at least, or at least about, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a reference sequence may correspond to a homologous sequence from another species other than the reference sequence.

[0469] Amino acid substitutions can be conservative or non-conservative, in some embodiments, the substitutions may be conservative, in which one amino acid is replaced with another amino acid having similar structural and / or chemical properties.

[0470] Conservative substitutions may include...

Claims

1. nucleic acids, (i) The polypeptide of SEQ ID NO: 311, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 311, (ii) The polypeptide of Sequence ID No. 305 and the polypeptide of Sequence ID No. 307, The C-terminus of SEQ ID NO: 305 is linked to the N-terminus of SEQ ID NO: 307, or the C-terminus of SEQ ID NO: 307 is linked to the N-terminus of SEQ ID NO: 305, with or without a linker between the sequences, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, and a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, and is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or The C-terminus of the polypeptide that is 100% identical is linked to the N-terminus of the polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, or the C-terminus of the polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307 is linked to the N-terminus of the polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, with or without a linker between the sequences, or (iii) The polypeptide of SEQ ID NO: 309, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 309, (iv) The polypeptide of SEQ ID NO: 315, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315, (v) A nucleic acid that codes for one or more selected from the group consisting of (i), (ii), (iii), and (iv).

2. (i) Sequence ID 312, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID 312, (ii) Sequence IDs 306 and 308, wherein the 3' end of sequence ID 306 is ligated to the 5' end of sequence ID 308, or the 3' end of sequence ID 308 is ligated to the 5' end of sequence ID 306, and there may or may not be a sequence encoding a linker between them; or sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence ID 306, and sequences that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence ID 308, and are at least 95%, at least 96%, at least 97%, at least The 3' end of the sequence, which is 98%, at least 99%, or 100% identical, is ligated to the 5' end of the sequence, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence number 308, or the 3' end of the sequence, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence number 308, is ligated to the 5' end of the sequence, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence number 306, and there may or may not be a sequence encoding a linker between those sequences, or (iii) Sequence ID 310, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID 310, (iv) Sequence ID 316, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID 316, The nucleic acid according to claim 1, comprising one or more selected from the group consisting of (v), (i), (ii), (iii), and (iv).

3. (a) at least one TCR polypeptide comprising a TCRα chain and a TCRβ chain, (b) (i) CD8α chain, (ii) CD8 β chain, or (iii) At least one CD8 polypeptide comprising a CD8α chain and a CD8β chain, or (c) at least one TCR polypeptide comprising a TCRα chain and a TCRβ chain, and (i) CD8α chain, (ii) CD8 β chain, or (iii) The nucleic acid according to claim 1, further comprising a nucleic acid encoding at least one CD8 polypeptide, which includes a CD8α chain and a CD8β chain.

4. The aforementioned at least one TCR is sequence numbers 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 It comprises a TCRα chain and a TCRβ chain selected from sequence numbers 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92, and in particular the TCRα chain and the TCRβ chain are selected from sequence numbers 15 and 16, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, and 71 and 303. The CD8α chain includes SEQ ID NOs: 7, 258, 259, 262, or their variants, and The nucleic acid according to claim 3, wherein the CD8β chain includes sequence numbers 8, 9, 10, 11, 12, 13, or 14.

5. The nucleic acid is the at least one TCR polypeptide comprising a TCRα chain and a TCRβ chain, and The nucleic acid according to claim 3, comprising at least one CD8 polypeptide that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequences 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 306, 308, 310, 312, 314, 316-319, or 320-326.

6. A vector comprising the nucleic acid described in claim 1.

7. The vector according to claim 6, further comprising a post-transcriptional regulatory element (PRE) sequence selected from woodchuck PRE (WPRE) (SEQ ID NO: 264), woodchuck PRE (WPRE) variant 1 (SEQ ID NO: 256), woodchuck PRE (WPRE) variant 2 (SEQ ID NO: 257), and hepatitis B virus (HBV) PRE (HPRE) (SEQ ID NO: 385).

8. The vector according to claim 6, wherein the vector is a viral vector or a non-viral vector.

9. The vector according to claim 8, wherein the viral vector is selected from adenovirus, poxvirus, alphavirus, arenavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, picornavirus, and combinations thereof, and is in particular a lentiviral vector.

10. A vector comprising nucleic acids including N1, N2, N3, N4, N5, L1, L2, L3 and L4, N1 encodes the CD8β chain, is present, or is not present. N1 includes sequence numbers 8, 9, 10, 11, 12, 13 or 14, N2 encodes the CD8α chain, N2 includes sequence numbers 7, 258, 259, 262, or their variants. N3 encodes the TCRβ chain, N4 codes for the TCRα chain, N4 and N3 correspond to sequence numbers 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52 and 53. Code 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, or 91 and 92, N5 codes for at least one interleukin, N5 is, (i) The polypeptide of SEQ ID NO: 311, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

311. (ii) polypeptides of SEQ ID NO: 305 and SEQ ID NO: 307, wherein the C-terminus of SEQ ID NO: 305 is linked to the N-terminus of SEQ ID NO: 307, or the C-terminus of SEQ ID NO: 307 is linked to the N-terminus of SEQ ID NO: 305, with or without a linker between their sequences, or polypeptides that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, and polypeptides that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, The C-terminus of the polypeptide, which is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, is linked to the N-terminus of the polypeptide, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, or the C-terminus of the polypeptide, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, is linked to the N-terminus of the polypeptide, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, with or without a linker between these sequences. (iii) A polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 309, or SEQ ID NO: 311, (iv) A polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO: 309, or SEQ ID NO: 315, or (v) Code one or more selected from the group consisting of (i), (ii), (iii), and (iv), and L1 to L4 each code at least one linker. L1 to L4 are independent, identical, or different, and L1 to L4 may or may not exist independently. A vector in which L1 to L4 optionally contain the sequence of sequence number 337 or 339, or a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence number 337 or 339.

11. The following formula I or formula II: 5'-N1-L1-N2-L2-N3-L3-N4-L4-N5-3' [I] 5'-N5-L1-N1-L2-N2-L3-N3-L4-N4-3' [II], The vector according to claim 10, including

12. (i) A nucleic acid encoding a 2A peptide or an intra-sequence ribosome entry site (IRES), located at one or more positions selected from the group consisting of between N1 and L1, between L1 and N2, between N2 and L2, between L2 and N3, between N3 and L3, between L3 and N4, between N4 and L4, and between L4 and N5, (ii) The vector according to claim 10 or 11, further comprising: (ii) a 2A peptide or a nucleic acid encoding an intrasequence ribosome entry site (IRES) located at one or more positions selected from the group consisting of between N5 and L1, between L1 and N1, between N1 and L2, between L2 and N2, between N2 and L3, between L3 and N3, between N3 and L4, and between L4 and N4, wherein the 2A peptide is optionally selected from P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

13. The vector according to claim 6, further comprising a nucleic acid encoding a chimeric antigen receptor (CAR).

14. A polypeptide, fusion polypeptide, or plurality of polypeptides encoded by the nucleic acid described in claim 1.

15. A method for preparing T cells and / or natural killer cells for immunotherapy, To isolate T cells and / or natural killer cells from human blood samples, Activating the isolated T cells and / or natural killer cells, Transduction of the nucleic acid described in claim 1 into the activated T cells and / or natural killer cells, and A method comprising amplifying the transduced T cells and / or natural killer cells.

16. The method according to claim 15, further comprising isolating CD4+CD8+ T cells from the transduced T cells, and amplifying the isolated CD4+CD8+ transduced T cells.

17. The method according to claim 15, wherein the blood sample comprises peripheral blood mononuclear cells (PMBCs).

18. The method according to claim 16, wherein the activation includes contacting the T cells and / or natural killer cells with an anti-CD3 antibody and an anti-CD28 antibody.

19. The activation, amplification, or both are performed in combination of IL-2 and IL-15, The method according to claim 15, wherein the activation, amplification, or both are carried out using a zoledronate in the presence of a combination of IL-2 and IL-15.

20. A method for increasing the persistence, functionality, naivety, lifespan, antigen-presenting cell-killing ability, or interferon-gamma (IFNγ) secretion, or a combination thereof, of T cells and / or natural killer (NK) cells, To isolate T cells and / or natural killer (NK) cells from human blood samples, Activating the isolated T cells and / or natural killer (NK) cells, Transducing the nucleic acid described in claim 1 into the activated T cells and / or natural killer (NK) cells to obtain transduced T cells and / or natural killer (NK) cells, and This includes obtaining the transduced T cells and / or natural killer (NK) cells, A method wherein the persistence, lifespan, functionality, naivety, ability to kill antigen-presenting cells, interferon-gamma (IFNγ) secretion, or a combination thereof of the transduced T cells and / or natural killer (NK) cells are increased compared to control cells.

21. The method according to claim 20, further comprising amplifying the transduced T cells and / or natural killer (NK) cells obtained.

22. The method according to claim 20, wherein the control cells include untransduced T cells and / or natural killer (NK) cells, T cells and / or natural killer (NK) cells transduced with only TCR, T cells and / or natural killer (NK) cells transduced with only TCR and CD8, or a combination thereof.

23. The method according to claim 20, wherein the persistence, lifespan, functionality, naivety, ability to kill antigen-presenting cells, interferon-gamma (IFNγ) secretion, or combination thereof of the transduced T cells and / or natural killer (NK) cells and the control cells are determined after one challenge using antigen-presenting cells, two challenges using antigen-presenting cells, three challenges using antigen-presenting cells, four challenges using antigen-presenting cells, five challenges using antigen-presenting cells, six challenges using antigen-presenting cells, seven challenges using antigen-presenting cells, or eight or more challenges using antigen-presenting cells.

24. The method according to claim 20, wherein the antigen-presenting cells present an antigen on their cell surface, and the obtained transduced T cells and / or natural killer (NK) cells, as well as control cells, have the ability to kill the antigen-presenting cells.

25. The method according to claim 24, wherein the antigen comprises a peptide in a complex with an MHC molecule on the cell surface.

26. T cells and / or natural killer (NK) cells transduced with the nucleic acid described in Claim 1.

27. (a) (i) a T cell receptor (TCR) comprising the α chain and β chain of the TCR, and a CD8 polypeptide comprising the α chain and β chain of CD8, or (ii) TCRs containing the α chain and β chain of TCRs, and CD8 polypeptides containing the α chain of CD8 but not the β chain of CD8, (b) comprising at least one interleukin, The α chain and β chain of the TCR are selected from SEQ ID NOs: 15 and 16, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, and 71 and 303. The α chain of CD8 is sequence numbers 7, 258, 259, 262, or a variant thereof. If present, the β chain of CD8 is sequence numbers 8, 9, 10, 11, 12, 13, or 14, and The at least one interleukin, (i) The polypeptide of SEQ ID NO: 311, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

311. (ii) polypeptides of SEQ ID NO: 305 and SEQ ID NO: 307, wherein the C-terminus of SEQ ID NO: 305 is linked to the N-terminus of SEQ ID NO: 307, or the C-terminus of SEQ ID NO: 307 is linked to the N-terminus of SEQ ID NO: 305, with or without a linker between their sequences, or polypeptides that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, and polypeptides that are at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, The C-terminus of the polypeptide, which is at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, is linked to the N-terminus of the polypeptide, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, or the C-terminus of the polypeptide, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 307, is linked to the N-terminus of the polypeptide, which is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 305, with or without a linker between these sequences. (iii) The polypeptide of Sequence ID No. 309, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID No.

309. (iv) The polypeptide of SEQ ID NO: 315, or a polypeptide that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315, (v) The T cell and / or natural killer (NK) cell according to claim 26, comprising one or more selected from the group consisting of (i), (ii), (iii), and (iv).

28. The T cell and / or natural killer (NK) cell according to claim 27, wherein the cell is one or more combinations selected from the group consisting of αβ T cells, γδ T cells, natural killer T cells, and natural killer (NK) cells.

29. The T cell and / or natural killer (NK) cell according to claim 28, wherein the αβ T cell is a CD4+ or CD8+ T cell or a CD4+CD8+ T cell.

30. The T cell and / or natural killer (NK) cell according to claim 28, wherein the γδ T cell is a Vγ9Vδ2+ T cell.

31. The T cell and / or natural killer (NK) cell according to claim 29, wherein the T cell and / or natural killer (NK) cell is isolated, genetically engineered, or both isolated and genetically engineered.

32. A composition comprising T cells and / or natural killer (NK) cells as described in claim 26.

33. The composition according to claim 32, wherein the composition is a pharmaceutical composition.

34. The composition according to claim 32, wherein the composition further comprises an adjuvant, an excipient, a carrier, a diluent, a buffer, a stabilizer, or a combination thereof.

35. The adjuvant may include anti-CD40 antibody, imiquimod, reciquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon alpha, interferon beta, CpG oligonucleotides and their derivatives, poly(I:C) and its derivatives, RNA, sildenafil, poly(lactidocoglycol) (PLG), particle formulations, virosoms, interleukin 1 (IL-1), interleukin 2 (IL-2), and interleukin 4. The composition according to claim 34, wherein the adjuvant is one or more selected from the group consisting of (IL-4), interleukin 7 (IL-7), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 21 (IL-21), and interleukin 23 (IL-23), and in particular the adjuvant is one or more selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

36. A therapeutic pharmaceutical comprising T cells and / or natural killer (NK) cells according to any one of claims 26 to 31, or a composition according to any one of claims 32 to 35.

37. A pharmaceutical agent for cancer treatment comprising T cells and / or natural killer (NK) cells according to any one of claims 26 to 31, or a composition according to any one of claims 32 to 35.

38. A therapeutic agent comprising T cells and / or natural killer (NK) cells according to any one of claims 26 to 31, or a composition according to any one of claims 32 to 35.

39. A cancer treatment agent comprising T cells and / or natural killer (NK) cells according to any one of claims 26 to 31, or a composition according to any one of claims 32 to 35.

40. Use of T cells and / or natural killer (NK) cells according to any one of claims 26 to 31, or the composition according to any one of claims 32 to 35, in the manufacture of a pharmaceutical product for the treatment of cancer.

41. The use according to claim 40, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, stomach cancer, and prostate cancer.

42. A cancer treatment agent and / or an immune response inducer comprising the composition according to any one of claims 32 to 35, wherein the cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, melanoma, liver cancer, breast cancer, uterine cancer, Merkel cell carcinoma, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain tumor, gastric cancer, and prostate cancer.

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