Dominant-negative TGF-beta receptor polypeptides, CD8 polypeptides, cells, compositions, and methods of use thereof

Abstract

The present disclosure relates to T cells capable of co-expressing a T cell receptor ("TCR") with a dominant negative TGFβ receptor ("dnTGFβR") polypeptide and / or a CD8 polypeptide, and their use in adoptive cell therapy. The present disclosure further provides dnTGFβR polypeptides, vectors thereof, and related methods. The present disclosure also provides modified CD8 polypeptides, vectors thereof, and related methods of making and using.

Description

[Technical field]

[0001] Related Applications This application is an international application claiming priority to U.S. Provisional Patent Application No. 63 / 336,062, 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 the file name "3000011-031977_Sequence-Listing_ST26", created on April 26, 2023, and 521,214 bytes in size, and is 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 receptor (TCR), CD8 polypeptides, and / or dnTGFβR (dominant negative TGF beta receptor) polypeptides, and their use in adoptive cell therapy (ACT). The present disclosure further provides modified CD8 sequences, dnTGFβRII sequences, vectors, compositions, transformed cells, and related methods thereof. [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 contacts 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 leads to the association of its α-chain cytoplasmic tail with the tyrosine kinase p56lck, activating CTLs.

[0005] Transforming growth factor beta (TGFβ or TGF-β) is a cytokine that has an important role in immune cell function. Transforming growth factor beta receptor I (TGFβRI) and transforming growth factor beta receptor II (TGFβRII) are important receptors in the signal transduction of TGFβ. TGFβRII is a transmembrane serine / threonine kinase protein that binds to TGFβ in a complex with transforming growth factor beta receptor I (TGFβRI). After TGFβ binds, TGFβRII phosphorylates TGFβRI, which then further activates signal transduction. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Li et al.Signal Transduction and Targeted Therapy 4(35):(2019) Summary of the Invention [Problem to be solved by the invention]

[0007] 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. [Means for solving the problem]

[0008] In some embodiments, a dominant negative TGFβ receptor (dnTGFβR) polypeptide may be provided. In some embodiments, a dominant negative TGFβ receptor I (dnTGFβRI) polypeptide may be provided. In some embodiments, a dominant negative TGFβ receptor II (dnTGFβRII) polypeptide may be provided. In some embodiments, an isolated nucleic acid sequence encoding one or more dnTGFβRI polypeptides and / or dnTGFβRII polypeptides may be provided. In some embodiments, an isolated vector may be provided comprising one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more dnTGFβRI polypeptides and / or dnTGFβRII polypeptides. In some embodiments, a cell comprising or expressing one or more dnTGFβRI polypeptides and / or dnTGFβRII polypeptides may be provided. In some embodiments, a cell comprising or expressing one or more nucleic acid sequences encoding one or more dnTGFβRI polypeptides and / or dnTGFβRII polypeptides may be provided. In some embodiments, a cell may be provided that contains or expresses one or more vectors that contain one or more nucleic acid sequences encoding one or more dnTGFβRI and / or dnTGFβRII polypeptides. In some embodiments, the cells described herein may contain one or more dnTGFβRI and / or dnTGFβRII polypeptides, one or more CD8 polypeptides, a cellular receptor (TCR) that contains an alpha and beta chain, one or more TCRs that contain a gamma and delta chain, one or more chimeric antigen receptors (CARs), or any combination thereof. In some embodiments, the cells may include alpha beta T cells, gamma delta T cells, natural killer cells, natural killer T cells, CD4+ cells, CD8+ cells, CD4+ / CD8+ cells, or any combination thereof. In some embodiments, a composition may be provided that includes such polypeptides, nucleic acids, vectors, and / or cells. In some embodiments, such polypeptides, nucleic acids, vectors, and / or cells may be isolated, recombinant, and / or engineered.

[0009] 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.

[0010] In some embodiments, the dnTGFβRII polypeptide may comprise a mutated and / or truncated TGFβ receptor II (TGFβRII). In some embodiments, the dnTGFβRII polypeptide may comprise a truncated TGFβRII. In some embodiments, the dnTGFβRII polypeptide may comprise a C-terminal truncated TGFβRII. In some embodiments, the dnTGFβRII polypeptide may comprise a truncated TGFβRII to remove all or part of the intracellular signaling portion of TGFβRII. In some embodiments, the dnTGFβRII polypeptide may comprise a mutated TGFβRII to completely or partially disable the intracellular signaling portion of TGFβRII. Each of the TGFβRIIvar1 and TGFβRIIvar2 disclosed herein lacks a cytoplasmic domain required for downstream signaling. Without being bound by theory, in some embodiments, dnTGFβRII may function, for example, as follows: truncated TGFβRII retains the ability to bind TGF-β and form a heteromeric complex with TGFβRI, but the lack of a cytoplasmic domain prevents phosphorylation of TGFβRI and subsequent activation of downstream elements. Furthermore, the inclusion of a single truncated TGFβRII protein in a heteromeric TGF-β receptor complex is sufficient to abolish signal transduction, suggesting that the protein functions in a dominant-negative manner.

[0011] In some embodiments, the dnTGFβRII polypeptide may include an extracellular domain, a transmembrane domain, and / or a cytoplasmic domain. In some embodiments, the cytoplasmic domain may be truncated, mutated, or absent.

[0012] In some embodiments, dnTGFβRII variant 1 (dnTGFβRIIvar1) and / or dnTGFβRII variant 2 (dnTGFβRIIvar2) are provided, which are examples of dnTGFβRII polypeptides. dnTGFβRIIvar1 may comprise or consist of SEQ ID NO: 305, and may be encoded by a nucleic acid comprising or consisting of SEQ ID NO: 306. dnTGFβRIIvar1 may comprise or consist of SEQ ID NO: 307, and may be encoded by a nucleic acid comprising or consisting of SEQ ID NO: 308. TGFβRIIvar1 and TGFβRIIvar2 disclosed herein each lack the cytoplasmic domain required for downstream signaling, and in each, the remaining transmembrane and extracellular regions contain minor differences in size / sequence.

[0013] In some embodiments, the cells described herein may comprise a dnTGFβRII polypeptide and a CD8 polypeptide described herein. In some embodiments, the cells described herein may comprise a dnTGFβRII polypeptide, a CD8 polypeptide, a T cell receptor (TCR) comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments, the cells may comprise an αβ T cell, a γδ 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.

[0014] 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 some embodiments, the CD8β region may be a CD8β stalk region or domain.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 amino acid substitutions may be conservative or non-conservative. In some embodiments, the amino acid substitutions may be conservative amino acid substitutions.

[0020] In embodiments, the CD8 polypeptide described herein can be a CD8α polypeptide or a modified CD8α polypeptide.

[0021] In embodiments, the CD8 polypeptide described herein can be a CD8αβ polypeptide or a modified CD8α polypeptide.

[0022] 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.

[0023] 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 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, and 91 and 92.

[0024] In some embodiments, the isolated nucleic acid may include a nucleic acid encoding a T cell receptor comprising an alpha chain and a beta chain, and a CD8 polypeptide comprising an alpha chain and a beta chain. In some embodiments, the CD8 polypeptide may be modified or unmodified. The isolated nucleic acid may include a nucleic acid at least about 80% identical to the nucleic acid 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 of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301.

[0025] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0026] In embodiments, the isolated nucleic acid comprises the nucleic acid of SEQ ID NO:267.

[0027] In embodiments, the isolated nucleic acid comprises the nucleic acid of SEQ ID NO:279.

[0028] 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.

[0029] 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.

[0030] In embodiments, the isolated polypeptide may comprise the amino acid sequence of SEQ ID NO:268.

[0031] In embodiments, the isolated polypeptide may comprise the amino acid sequence of SEQ ID NO:280.

[0032] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein.

[0033] In embodiments, the disclosure provides a vector comprising a nucleic acid encoding a polypeptide described herein.

[0034] In embodiments, one or more vectors may contain a nucleic acid encoding a dnTGFβRII polypeptide.

[0035] In embodiments, one or more vectors may contain a nucleic acid encoding a CD8 polypeptide.

[0036] In embodiments, one or more vectors may contain a nucleic acid encoding a CD8α polypeptide.

[0037] In embodiments, one or more vectors may contain a nucleic acid encoding a CD8 β polypeptide.

[0038] In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0039] 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 some embodiments, the one or more vectors may comprise one or more nucleic acids encoding a chimeric antigen receptor (CAR).

[0040] 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 α chain and a β chain, a TCR comprising a γ chain and a δ chain, a CAR, a dnTGFβRII 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.

[0041] 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 α chain and a β chain, a dnTGFβRII 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 γ chain and a δ chain, a dnTGFβRII 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, a dnTGFβRII 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.

[0042] 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, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding a CAR, a dnTGFβRII polypeptide, 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.

[0043] In some embodiments, a vector may be provided that includes a TCR comprising an α chain and a β chain, and one or more nucleic acids encoding a dnTGFβRII polypeptide. In some embodiments, a vector may be provided that includes a TCR comprising a γ chain and a δ chain, and one or more nucleic acids encoding a dnTGFβRII polypeptide. In some embodiments, a vector may be provided that includes a CAR and one or more nucleic acids encoding a dnTGFβRII polypeptide.

[0044] In some embodiments, a vector 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. In some embodiments, a vector 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. 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 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.

[0045] In embodiments, nucleic acids may be provided that encode a polypeptide comprising: (i) SEQ ID NO:305, 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:305; (ii) SEQ ID NO:307, 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:307; or (iii) both (i) and (ii).

[0046] In embodiments, a nucleic acid may be provided that comprises: (i) SEQ ID NO:306, 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:306; (ii) SEQ ID NO:308, 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:308; or (iii) both (i) and (ii).

[0047] In embodiments, a nucleic acid comprising: (i) 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; (ii) SEQ ID NO:313, 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:313; or (iii) both (i) and (ii) may be provided.

[0048] In embodiments, the nucleic acids described herein may further comprise a nucleic acid sequence encoding at least one TCR polypeptide, at least one CD8 polypeptide, or at least one TCR polypeptide and at least one CD8 polypeptide.

[0049] In embodiments, a nucleic acid may be provided that includes: (a) a nucleic acid sequence encoding (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 but not a β chain; and (b) at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptide, In this case, the TCR alpha chain and the TCR beta 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 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, 90, 91 and 92; the CD8 α chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof; the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14; and at least one of the at least one dnTGFβRII polypeptides is selected from (i) SEQ ID NO: SEQ ID NO: 305, 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: 305, or (ii) SEQ ID NO: 307, 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: 307. In some embodiments, the nucleic acid may include a nucleic acid sequence encoding dnTGFβRIIvar1, and a nucleic acid sequence encoding dnTGFβRIIvar2.

[0050] In embodiments, a nucleic acid may be provided that comprises: (a) a nucleic acid sequence encoding (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) a nucleic acid sequence encoding at least one dominant negative TGF beta receptor II (dnTGF beta RII) polypeptide, wherein the TCR alpha chain and 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 SEQ ID NO: 7, 258, 259, 262 or a variant thereof, and the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, and the at least one dnTGFβRII polypeptide is selected from (i) SEQ ID NO: 305 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: 305, or (ii) SEQ ID NO: 307 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: 307. In some embodiments, the nucleic acid may comprise a nucleic acid sequence encoding dnTGFβRIIvar1, and a nucleic acid sequence encoding dnTGFβRIIvar2.

[0051] In embodiments, a nucleic acid may be provided that includes (a) a nucleic acid sequence that is 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, and (b) a nucleic acid sequence or sequences encoding at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide.

[0052] In embodiments, a nucleic acid may be provided that includes: (a) a nucleic acid sequence that is 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; and (b) a nucleic acid sequence or sequences encoding at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide.

[0053] In embodiments, the nucleic acid sequence or sequence encoding at least one of the at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptides may be selected from (i) SEQ ID NO:306, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:306, or (ii) SEQ ID NO:308, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:308.

[0054] In several embodiments, a vector may be provided that comprises N1, N2, N3, N4, N5, L1, L2, L3 and L4 in any order, where N1 comprises a nucleic acid sequence encoding a CD8 β chain and is present or absent, N2 comprises a nucleic acid sequence encoding a CD8 α chain, N3 comprises a nucleic acid sequence encoding a TCR β chain, N4 comprises a nucleic acid sequence encoding a TCR α chain, N5 comprises a nucleic acid sequence encoding at least one dominant negative TGF β receptor II (dnTGFβRII) polypeptide, L1-L4 each comprise a nucleic acid sequence encoding at least about one linker, L1-L4 are each independently the same or different, and L1-L4 are each independently present or absent.

[0055] In some embodiments, the compound of formula I or II is 5'-N1-L1-N2-L2-N3-L3-N4-L4-N5-3' [I] 5'-N5-L1-N1-L2-N2-L3-N3-L4-N4-3' [II] A vector comprising the

[0056] In embodiments, N1 may comprise a nucleic acid sequence encoding SEQ ID NO:8, 9, 10, 11, 12, 13, or 14.

[0057] In embodiments, N2 may comprise a nucleic acid sequence encoding SEQ ID NO: 7, 258, 259, 262, or a variant thereof.

[0058] 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, 62 and 63, 63 and 64, 64 and 65, 65 and 66, 66 and 67, 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, 85 and 86, 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 103, 104 and 105, 106 and 107, 108 and 109, 109 and 109, 109 and 102, 109 and 104, 109 and 105 In one embodiment, the nucleic acid sequence may include a nucleic acid sequence encoding 3 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.

[0059] In embodiments, N5 may comprise a nucleic acid sequence encoding (i) SEQ ID NO:305, 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:305, or (ii) SEQ ID NO:307, 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:307.

[0060] In embodiments, the vector 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.

[0061] In some embodiments, the vector 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.

[0062] In some 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 dominant negative TGF beta receptor II (dnTGF beta RII) polypeptide, The TCR α chain and the TCR β chain are 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, 31 and 304, 31 and 305, 31 and 306, 31 and 307, 31 and 309, 32 and 321, 32 and 322, 32 and 323, 32 and 324, 32 and 325, 32 and 326, 32 and 327, 32 and 328, 32 and 329, 33 and 330, 33 and 331, 33 and 332, 33 and 333, 33 and 334, 33 and 335, 33 and 336, 33 and 337, 33 and 338, 33 and 339, 34 and 341, 34 and 342, 34 and 343, 34 and 344, 34 and 3 04 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, 91 and 92, wherein 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 wherein at least one of the at least one dominant-negative TGF beta receptor II (dnTGF beta RII) polypeptides is selected from the group consisting of is selected from (i) SEQ ID NO: 305, 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: 305, or (ii) SEQ ID NO: 307, 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: 307. In embodiments, the T cells and / or natural killer cells may comprise dnTGFβRIIvar1 and dnTGFβRIIvar2.

[0063] 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 but not a β chain; and (b) at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptide, wherein the TCR α chain and 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; the CD8 α chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof; and the CD8 β chain, if present, is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. In some embodiments, the T cells and / or natural killer cells may comprise dnTGFβRIIvar1 and dnTGFβRIIvar2. In some embodiments, at least one of the at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptides may comprise SEQ ID NO:305, 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:305. In some embodiments, at least one of the at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptides may comprise SEQ ID NO:307, 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:307.

[0064] In embodiments, a nucleic acid may be provided that includes: (a) a nucleic acid sequence encoding (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 but not a β chain; and (b) at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptide, wherein the TCR α chain and the TCR β chain are selected from the group consisting of SEQ ID NO: 15, 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, 91 and 92, the CD8 α chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, at least one of the at least one dnTGFβRII polypeptides is encoded by a nucleic acid sequence that also comprises an MSCV promoter and a WPRE sequence, and (i) a sequence similar to SEQ ID NO: 312, or a sequence similar to SEQ ID NO: 312, or (ii) 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: 313, 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: 313. In some embodiments, the nucleic acid may include a nucleic acid sequence encoding dnTGFβRIIvar1 and a nucleic acid sequence encoding dnTGFβRIIvar2.

[0065] In embodiments, a nucleic acid may be provided that includes: (a) a nucleic acid sequence encoding (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 but not a β chain; and (b) a nucleic acid sequence encoding at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptide, 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, 71 and 303, the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, and the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or a variant thereof. is 14, and at least one of the at least one dnTGFβRII polypeptides is encoded by a nucleic acid sequence that also includes an MSCV promoter and a WPRE sequence, and is selected from (i) 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, or (ii) SEQ ID NO: 313, 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: 313. In some embodiments, the nucleic acid may include a nucleic acid sequence encoding dnTGFβRIIvar1, and a nucleic acid sequence encoding dnTGFβRIIvar2.

[0066] In embodiments, a nucleic acid may be provided that includes (a) a nucleic acid sequence that is 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, and (b) a nucleic acid sequence or sequences encoding at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide.

[0067] In embodiments, a nucleic acid may be provided that includes: (a) a nucleic acid sequence that is 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; and (b) a nucleic acid sequence or sequences encoding at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide. In some embodiments, the nucleic acid sequence encoding at least one of the at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptides may also include an MSCV promoter and a WPRE sequence, and may be selected from (i) 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, or (ii) SEQ ID NO:313, 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:313.

[0068] In embodiments, a T cell and / or natural killer (NK) cell may be provided that comprises: (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 but not a β chain, and (b) at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptide, wherein the TCRα chain and the TCRβ The strands are 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, 91 and 92, wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof, and the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14, and is encoded by a nucleic acid sequence that also comprises an MSCV promoter and a WPRE sequence, and (i) a sequence similar to SEQ ID NO: 312, or a sequence similar to SEQ ID NO: 312, or (ii) a sequence that is 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: 313, 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: 313. In embodiments, the T cells and / or natural killer cells may comprise dnTGFβRIIvar1 and dnTGFβRIIvar2.

[0069] 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 but not a β chain; and (b) at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptide, wherein the TCR α chain and 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; the CD8 α chain is SEQ ID NO: 7, 258, 259, 262 or a variant thereof; and the CD8 β chain, if present, is SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. In embodiments, the T cells and / or natural killer cells can include dnTGFβRIIvar1 and dnTGFβRIIvar2. In embodiments, at least one of the at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptides can also include an MSCV promoter and a WPRE sequence and can be encoded by a nucleic acid 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 several embodiments, at least one of the at least one dominant negative TGFβ receptor II (dnTGFβRII) polypeptides may be encoded by a nucleic acid sequence selected from SEQ ID NO:313, 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:313.

[0070] 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 CD4+CD8+ 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 may be γδ T cells or αβ T cells. In embodiments, activation and / or expansion may be performed in the presence of a combination of IL-2 and IL-15, optionally with zoledronate.

[0071] 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 or natural killer (NK) cells, wherein the persistence, longevity, functionality, naivety, 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 some 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 more challenges with antigen-presenting cells. In some 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 two challenges with antigen-presenting cells, three challenges with antigen-presenting cells, or more challenges with antigen-presenting cells. In some embodiments, the transduced T cells and / or natural killer (NK) cells and control cells may be cultured in the presence of exogenous TGF-β, optionally TGF-β1. In embodiments, exogenous TGF-β, optionally TGF-β1, may be added to the cell culture daily. In embodiments, exogenous TGF-β, optionally TGF-β1, may be added to the cell culture at the same time that tumor cells may be added to the cell culture.

[0072] 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 a transduced T cell and / or natural killer (NK) cell, and obtaining the transduced T cell or natural killer (NK) cell, wherein IFNγ secretion of the transduced T cell and / or natural killer (NK) cell is increased compared to that of a control cell. In embodiments, the method may further comprise expanding the transduced T cell and / or natural killer (NK) cell. 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 more challenges with antigen presenting cells.In some embodiments, IFNγ secretion by the transduced T cells and / or natural killer (NK) cells and control cells can be determined after two challenges with antigen-presenting cells, three challenges with antigen-presenting cells, or more challenges with antigen-presenting cells. In some embodiments, the transduced T cells and / or natural killer (NK) cells and control cells can be cultured in the presence of exogenous TGF-β, optionally TGF-β1. In some embodiments, exogenous TGF-β, optionally TGF-β1, can be added to the cell culture daily. In some embodiments, exogenous TGF-β, optionally TGF-β1, can be added to the cell culture at the same time that the tumor cells are added to the cell culture.

[0073] 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 and control cells can have the ability to kill the antigen-presenting cells. In some embodiments, the antigens can include peptides. In some embodiments, the antigens that include peptides can be in a complex with MHC molecules on the cell surface.

[0074] 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, independently, a first polypeptide and a second polypeptide, PL is a linker, and PL comprises SEQ ID NO:320 or 322, 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:320 or 322.

[0075] 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:321 or 323, 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:321 or 323.

[0076] In embodiments, a polypeptide, multiple polypeptides, or a fusion polypeptide encoded by the nucleic acids described herein may be provided.

[0077] In embodiments, the polypeptide, polypeptides, or fusion polypeptides described herein may be isolated, recombinant, or both isolated and recombinant.

[0078] 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 or 307, 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, as well as a T cell and / or a natural killer (NK) cell. In some embodiments, the T cell may be an αβ T cell, a γδ T cell, and / or a natural killer T cell. In some embodiments, the αβ T cell may be a CD4+ T cell. In some embodiments, the αβ T cell may be a CD8+ T cell. In some embodiments, the γδ T cell may be a Vγ9Vδ2+ T cell.

[0079] In embodiments, the nucleic acids described herein may be isolated, recombinant, or both isolated and recombinant.

[0080] In embodiments, the vectors described herein may be isolated, recombinant, or both isolated and recombinant.

[0081] In embodiments, the T cells and / or natural killer (NK) cells described herein may be isolated, recombinant, engineered, or any combination thereof.

[0082] 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 dominant negative TGF beta RII. 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).

[0083] 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.

[0084] 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.

[0085] 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.

[0086] In some embodiments, expression of a dnTGFβRII polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or a 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 a dnTGFβRII polypeptide. In some embodiments, expression of a dnTGFβRII polypeptide may improve the persistence, functionality, proliferation, survival, expansion, or a 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 a dnTGFβRII polypeptide. In some embodiments, expression of a dnTGFβRII 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 a dnTGFβRII polypeptide. In embodiments, expression of a dnTGFβRII 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 a combination thereof, compared to cells that do not express a dnTGFβRII polypeptide. In embodiments, expression of a dnTGFβRII 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.

[0087] Persistence can be assessed by, for example and not by way of limitation, the length of time that 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, and 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. Persistence of cells expressing dnTGFβRII polypeptides 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 dnTGFβRII polypeptides.

[0088] 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 a dnTGFβRII polypeptide to kill tumor cells may be compared, by way of non-limiting example, to the continued ability of similar cells not expressing a dnTGFβRII polypeptide to kill tumor cells, or to the continued ability of other control cells to kill tumor cells.

[0089] 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.

[0090] When assessing the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naivety, or other properties of cells expressing dnTGFβRII, 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, may be utilized as control cells. Because dnTGFβRII may act to reduce or eliminate TGFβ signaling, assessment of the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naivety, or other properties of cells expressing dnTGFβRII may be performed in the presence of exogenous TGFβ, such as, for example, TGF-β1.

[0091] In some embodiments, the dnTGFβRII 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 in a combination thereof. In embodiments in which the dnTGFβRII polypeptide acts in trans, cells adjacent to or in the vicinity of cells expressing the dnTGFβRII polypeptide (e.g., in the tumor microenvironment) may exhibit any or a combination of the same or comparable improvements as described for cells expressing the dnTGFβRII polypeptide, compared to cells not adjacent to or in the vicinity of cells expressing the dnTGFβRII polypeptide. Without being bound by theory, the dnTGFβRII may act to reduce the amount of TGF-β in the tumor microenvironment. Also, cells expressing dnTGFβRII may exhibit improved ability to secrete cytokines in response to target antigens in the presence of TGF-β, compared to cells not expressing dnTGFβRII.

[0092] In embodiments, the disclosure provides a nucleic acid encoding a polypeptide described herein. In embodiments, the disclosure provides a vector comprising a nucleic acid encoding a polypeptide described herein. In embodiments, one or more vectors may comprise a nucleic acid encoding a dnTGFβRII polypeptide. In embodiments, one or more vectors may comprise a nucleic acid encoding a CD8 polypeptide. In embodiments, one or more vectors may comprise a nucleic acid encoding a CD8α polypeptide. In embodiments, one or more vectors may comprise a nucleic acid encoding a CD8β polypeptide.

[0093] 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 some embodiments, the one or more vectors may comprise one or more nucleic acids encoding a chimeric antigen receptor (CAR).

[0094] 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 α chain and a β chain, a TCR comprising a γ chain and a δ chain, a CAR, a dnTGFβRII 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.

[0095] 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 α chain and a β chain, a dnTGFβRII 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 γ chain and a δ chain, a dnTGFβRII 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, a dnTGFβRII 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.

[0096] 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, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a vector may be provided that includes one or more nucleic acids encoding a CAR, a dnTGFβRII polypeptide, 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.

[0097] In some embodiments, a vector may be provided that includes a TCR comprising an α chain and a β chain, and one or more nucleic acids encoding a dnTGFβRII polypeptide. In some embodiments, a vector may be provided that includes a TCR comprising a γ chain and a δ chain, and one or more nucleic acids encoding a dnTGFβRII polypeptide. In some embodiments, a vector may be provided that includes a CAR and one or more nucleic acids encoding a dnTGFβRII polypeptide.

[0098] In some embodiments, a vector 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. In some embodiments, a vector 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. 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 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.

[0099] In embodiments, two or more vectors may be co-transduced into one or more cells, co-expressed in one or more cells, or combinations 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 combinations thereof.

[0100] In embodiments, two or more vectors can comprise nucleic acids encoding one or any combination of a dnTGFβRII 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.

[0101] In embodiments, a vector may contain nucleic acids encoding one or any combination of a dnTGFβRII 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 the CD8 β chain may be independently modified or unmodified.

[0102] In some embodiments, the nucleic acid may be polycistronic and one or more polycistronic nucleic acids may be utilized. Expressing 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 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), or other mechanisms. 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.

[0103] 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).

[0104] 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.

[0105] 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.

[0106] In embodiments, the vector may include a nucleic acid encoding a 2A peptide or an IRES located between the nucleic acid encoding the TCR alpha chain and the nucleic acid encoding the TCR beta chain.

[0107] In some embodiments, the vector may include a nucleic acid encoding a 2A peptide or an IRES positioned between the nucleic acid encoding the TCR alpha chain or the nucleic acid encoding the TCR beta chain and the nucleic acid encoding the dnTGF beta RII polypeptide.

[0108] In embodiments, a vector may include a nucleic acid encoding one or any combination of a dnTGFβRII 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, and the vector may include either a nucleic acid encoding a polypeptide or a fusion polypeptide, or a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) disposed between each of 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 the CD8 β chain may be independently modified or unmodified.

[0109] 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: 366), or a variant thereof, or any combination thereof.

[0110] 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.

[0111] In embodiments, the vector may be a viral vector or a non-viral vector.

[0112] 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.

[0113] 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.

[0114] 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: 365).

[0115] In embodiments, the vector may contain one or more factor Xa sites.

[0116] 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 portions thereof, or combinations thereof.

[0117] 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.

[0118] 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.

[0119] In some embodiments, the T cells may be CD4+ T cells. In some embodiments, the T cells may be CD8+ T cells. In some embodiments, the T cells may be CD4+ / CD8+ T cells. In some embodiments, the T cells may be αβ T cells. In some 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 described herein. In some embodiments, the T cells may be αβ T cells and may express a modified CD8 polypeptide 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 a dnTGFβRII polypeptide, a modified CD8 polypeptide, and / or a CAR.

[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 T cells may be γδ 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 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 a dnTGFβRII polypeptide, a modified CD8 polypeptide, and / or a CAR.

[0122] 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 α chain and a β chain, a TCR comprising a γ chain and a δ chain, a CAR, a dnTGFβRII 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 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 α chain and a β chain, a dnTGFβRII 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 γ chain and a δ chain, a dnTGFβRII, 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, a dnTGFβRII 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.

[0124] In some embodiments, a cell may be provided that includes one or more nucleic acids encoding a TCR comprising an α chain and a β chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a cell may be provided that includes one or more nucleic acids encoding a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a cell may be provided that includes one or more nucleic acids encoding a CAR, a dnTGFβRII polypeptide, 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.

[0125] In some embodiments, a cell may be provided that includes a TCR comprising an α chain and a β chain, and one or more nucleic acids encoding a dnTGFβRII polypeptide. In some embodiments, 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 dnTGFβRII polypeptide. In some embodiments, a cell may be provided that includes a CAR, and one or more nucleic acids encoding a dnTGFβRII 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 a 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 a population of modified cells comprising one or more nucleic acids encoding one or any combination of a polypeptide as described herein, e.g., an exogenous CD8 coreceptor comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, 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, a dnTGFβRII polypeptide, e.g., an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or about 100% to SEQ ID NO:305 or SEQ ID NO:307, and / or a T cell receptor. In some embodiments, the population of cells may include αβ T cells, γδ T cells, natural killer cells, CD4+ T cells, CD8+ T cells, CD4+ / CD8+ cells, or a 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 a 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 a 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 combinations 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 a combination thereof. [Brief description of the drawings]

[0135] [Figure 1] Figure 1 shows a representative CD8α subunit, e.g., SEQ ID NO: 258 (CD8α1). 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) that contains an lck-binding motif. [Diagram 2] FIG. 2 shows a sequence alignment between CD8α1 (SEQ ID NO: 258) and m1CD8α (SEQ ID NO: 7). [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. [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, 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. [Diagram 5] Figure 5A shows the titer of the viral vector shown in Figure 4. Figure 5B shows the titer of additional viral vectors according to 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). [Figure 6] FIG. 6 shows the production of T cells. [Figure 7-1] FIG. 7A shows the expression of activation markers in CD3+CD8+ cells before and after activation. [Figure 7-2] FIG. 7B shows the expression of activation markers before and after activation in CD3+CD4+ cells. [Figure 8]FIG. 8A shows the fold amplification 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). FIG. 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.WPREwt (TCR with wild-type WPRE) (Construct #8); NT=non-transduced T cells (negative control). [Figure 9-1] FIG. 9A shows a flow plot of cells transduced with construct #9. [Figure 9-2] FIG. 9B shows a flow plot of cells transduced with construct #10 according to the present disclosure. [Figure 9-3] FIG. 9C shows a flow plot of cells transduced with construct #11. [Figure 9-4] FIG. 9D shows a flow plot of cells transduced with construct #12. [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). [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). [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). [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). [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). [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). [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). [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). [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). [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). [Figure 20-1] 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 20-2] Same as above [Figure 20-3] Same as above [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. [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. [Diagram 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. [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. [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. [Figure 26] FIG. 26 shows T cell production according to the present disclosure. [Figure 27] Figure 27A shows expression of activation markers in CD3+CD8+ cells before and after activation. Figure 27B shows expression of activation markers in CD3+CD4+ cells before and after activation according to the present disclosure. [Figure 28] FIG. 28 shows the fold amplification of cells transduced with the various constructs. [Figure 29-1] 29A-29B show the % CD8+CD4+ cells transduced with various constructs according to the present disclosure. [Figure 29-2] Same as above [Figure 30-1] 30A-30B show the % Tet of CD8+CD4+ cells transduced with various constructs according to the present disclosure. [Figure 30-2] Same as above [Figure 31-1] 31A-31B show the Tet MFI (CD8+CD4+Tet+) of cells transduced with various constructs according to the present disclosure. [Figure 31-2] Same as above [Figure 32-1] 32A-32B show the percentage of CD8+CD4- (of CD3+) cells transduced with various constructs according to the present disclosure. [Figure 32-2] Same as above [Figure 33-1]33A-33B show the % CD8+Tet+ (of CD3+) cells transduced with various constructs according to the present disclosure. [Figure 33-2] Same as above [Figure 34-1] 34A-34B show the Tet MFI (CD8+Tet+) of cells transduced with various constructs according to the present disclosure. [Figure 34-2] Same as above [Figure 35-1] 35A-35B show the % Tet+ (of CD3+) cells transduced with various constructs according to the present disclosure. [Figure 35-2] Same as above [Figure 36-1] 36A-36B show the VCN of cells transduced with various constructs according to the present disclosure. [Figure 36-2] Same as above [Figure 37] FIG. 37 shows T cell production according to the present disclosure. [Figure 38] FIG. 38 shows the % Tet CD8+CD4+ among cells transduced with the various constructs. [Figure 39] FIG. 39 shows the Tet MFI of CD8+CD4+Tet+ among cells transduced with various constructs. [Diagram 40] FIG. 40 shows the Tet MFI of CD8+Tet+ cells transduced with various constructs. [Diagram 41] FIG. 41 shows the % Tet+ CD3+ cells transduced with various constructs. [Diagram 42] FIG. 42 shows the vector copy number (VCN) of cells transduced with the various constructs. [Diagram 43] Figure 43 shows the % proportion of T cell subsets in cells transduced with various constructs. FACS analysis was gated on CD3+TCR+. [Figure 44-1]Figures 44A-44B show the % proportion of T cell subsets in cells transduced with various constructs. FACS analysis was gated on CD4+CD8+ for Figure 44A and on CD4-CD8+TCR+ for Figure 44B. [Figure 44-2] Same as above [Figure 45-1] 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 45-2] Same as above [Figure 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. [Figure 47] Figure 47 shows ICI marker frequencies (2B4, 41BB, LAG3, PD-1, TIGIT, TIM3, CD39+CD69+, and CD39-CD69-). [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 [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 [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 [Figure 51] 51A-51C show the results of FACS analysis gated on CD4+CD8+ cells for A375, CD4:1 E:T. [Figure 52] 52A-52C show the results of FACS analysis gated on CD4-CD8+ cells for A375, CD4:1 E:T. [Diagram 53] 53A-53C show the results of FACS analysis gated on CD3+TCR+ cells for A375, CD4:1 E:T. [Figure 54] FIG. 54 shows T cell production according to the present disclosure. [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). [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 the present disclosure. [Figure 57] FIG. 57 shows the levels 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 the present disclosure. [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 the present disclosure. [Figure 59] FIG. 59 shows a scheme for determining the levels of cytokine secretion by dendritic cells (DCs) in the presence of PBMCs and target cells transduced with various constructs according to the present disclosure. [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 the present disclosure. [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 the present disclosure. [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 the present disclosure. [Figure 63-1] 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 the present disclosure. [Figure 63-2] Same as above [Figure 63-3] Same as above [Figure 63-4] Figure 63D shows the EC50 values ​​(ng / ml) in Figures 63A to 63C. [Figure 64-1] Figures 64A-64C show IFNγ production from transduced PBMCs obtained from donor #4 (Figure 64A), donor #1 (Figure 64B), and donor #3 (Figure 64C) according to the present disclosure, and their respective EC50 values ​​(mg / ml). [Figure 64-2] Same as above [Figure 64-3] Same as above [Figure 64-4] Figure 64D shows a comparison of EC50 values ​​(ng / ml) between the various donors in Figures 64A-64C. [Figure 65-1] 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) in accordance with the present disclosure. [Figure 65-2] Same as above [Figure 65-3] Same as above [Figure 66] Figure 66 shows a schematic example of a dnTGFβRII polypeptide bound to the membrane of a T cell, as may be provided in multiple embodiments. As a non-limiting example, dnTGFβRII may bind to TGFβ but prevent signaling in response to TGFβ binding. [Figure 67]Figure 67A shows an example of a nucleic acid comprising an MSCV promoter, a sequence encoding a dnTGFβRIIvar1 polypeptide, and a sequence encoding a WPRE, which may be provided in multiple embodiments. Figure 67B shows an example of a nucleic acid comprising an MSCV promoter, a sequence encoding a dnTGFβRIIvar2 polypeptide, and a sequence encoding a WPRE, which may be provided in multiple embodiments. In multiple embodiments of Figures 67A and 67B, the lines linking the MSCV promoter to the dnTGFβRII and linking the dnTGFβRII to the WPRE may represent direct bonds without intervening sequences, or may represent intervening sequences, such as, but not limited to, linkers, untranslated sequences, translated sequences, sequences including one or more restriction endonuclease sites, or combinations thereof. [Figure 68] Figure 68 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 68, 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. [Figure 69] Figure 69 shows vector copy number in cells transduced with vectors encoding dnTGFβRIIvar1 (var1) or dnTGFβRIIvar2 (var2) in exemplary transductions. Vector copy number (copies per cell) (Y axis) is plotted against the volume of lentiviral vector used to transduce cells (in μL per 1×106 cells) (X axis). [Figure 70]FIG. 70 shows vector copy numbers in non-transduced cells (NT) and in exemplary transductions: (i) cells transduced with only a vector encoding a TCR at 2.5 μL per million cells (TCR only, both bars); (ii) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 2.5 μL per million cells (TCR / DNR(2.5), white bars); (iii) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 5.0 μL per million cells (TCR / DNR(5.0), white bars); (iv) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIv at 10.0 μL per million cells (TCR / DNR(5.0), white bars). Vector copy numbers are shown in cells transduced with a vector encoding ar1 (TCR / DNR(10.0), white bars), (v) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 2.5 μL per million cells (TCR / DNR(2.5), black bars), (vi) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 5.0 μL per million cells (TCR / DNR(5.0), black bars), or (vii) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 10.0 μL per million cells (TCR / DNR(10.0), black bars). Vector copy numbers (copies per cell) (Y-axis) are shown. [Figure 71-1]FIG. 71A shows the percentage of TCR-positive CD3+ cells (black bars) or dnTGFβRIIvar1-positive CD3+ cells (white bars) in exemplary transductions. Cells were either not transduced (NT) or (i) transduced with only a vector encoding the TCR at 2.5 μL per million cells (TCR only), (ii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 2.5 μL per million cells (TCR / DNR(2.5)), (iii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 5.0 μL per million cells (TCR / DNR(5.0)), or (iv) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 10.0 μL per million cells (TCR / DNR(10.0)). [Figure 71-2] FIG. 71B shows the percentage of TCR-positive CD3+ cells (black bars) or dnTGFβRIIvar2-positive CD3+ cells (white bars) in exemplary transductions. Cells were either not transduced (NT) or (i) transduced with only a vector encoding the TCR at 2.5 μL per million cells (TCR only), (ii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 2.5 μL per million cells (TCR / DNR(2.5)), (iii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 5.0 μL per million cells (TCR / DNR(5.0)), or (iv) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 10.0 μL per million cells (TCR / DNR(10.0)). [Figure 72]FIG. 72 shows the percentage of CD3+ cells double positive for TCR and dnTGFβRIIvar1 (white bars) or TCR and dnTGFβRIIvar2 (black bars) in exemplary transductions. Cells were either not transduced (NT) or (i) transduced with only a vector encoding the TCR at 2.5 μL per million cells (TCR only, both bars), (ii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 2.5 μL per million cells (TCR / DNR(2.5), white bars), (iii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 5.0 μL per million cells (TCR / DNR(5.0), white bars), or (iv) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 10.0 μL per million cells. (v) were transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 2.5 μL per million cells (TCR / DNR(2.5), black bars); (vi) were transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 5.0 μL per million cells (TCR / DNR(5.0), black bars); or (vii) were transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 10.0 μL per million cells (TCR / DNR(10.0), black bars). [Figure 73]FIG. 73 shows the fold amplification of non-transduced cells (NT, both bars) and cells transduced with only a TCR-encoding vector at 2.5 μL per million cells (TCR only, both bars), (ii) cells transduced with a TCR-encoding vector at 2.5 μL per million cells and a dnTGFβRIIvar1-encoding vector at 0.31 μL per million cells (TCR / DNR(0.31), white bars), (iii) cells transduced with a TCR-encoding vector at 2.5 μL per million cells and a dnTGFβRIIvar1-encoding vector at 0.31 μL per million cells (TCR / DNR(0.31), white bars), (iv) cells transduced with a TCR-encoding vector at 2.5 μL per million cells. (iv) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding a dnTGFβRIIvar1 at 1.25 μL per million cells (TCR / DNR(1.25), white bars); (v) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding a dnTGFβRIIvar1 at 2.50 μL per million cells (TCR / DNR(1.25), white bars). (vi) cells transduced with a vector encoding TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 0.31 μL per million cells (TCR / DNR(0.31), black bars); (vii) cells transduced with a vector encoding TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 0.63 μL per million cells (TCR / Figure 1 shows the fold amplification for cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 1.25 μL per million cells (TCR / DNR(0.63), black bars), (viii) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 1.25 μL per million cells (TCR / DNR(1.25), black bars), or (ix) cells transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 2.50 μL per million cells (TCR / DNR(2.50), black bars). [Figure 74-1]Figure 74A shows the percentage of CD3+ cells positive for TCR (black bars) or positive for dnTGFβRIIvar1 (white bars). In the exemplary transductions, cells were not transduced (NT) or (i) transduced with only a vector encoding a TCR at 2.5 μL per million cells (TCR only), (ii) transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 0.31 μL per million cells (TCR / DNR(0.31)), (iii) transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 0.63 μL per million cells. (iv) were transduced with a vector encoding TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 1.25 μL per million cells (TCR / DNR(1.25)), or (v) were transduced with a vector encoding TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 2.50 μL per million cells (TCR / DNR(2.50)). [Figure 74-2]Figure 74B shows the percentage of CD3+ cells positive for TCR (black bars) or positive for dnTGFβRIIvar2 (white bars). In the exemplary transductions, cells were not transduced (NT) or (i) transduced with only a vector encoding a TCR at 2.5 μL per million cells (TCR only), (ii) transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 0.31 μL per million cells (TCR / DNR(0.31)), (iii) transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar2 at 0.63 μL per million cells. (iv) were transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding a dnTGFβRIIvar2 at 1.25 μL per million cells (TCR / DNR(1.25)), or (v) were transduced with a vector encoding a TCR at 2.5 μL per million cells and a vector encoding a dnTGFβRIIvar2 at 2.50 μL per million cells (TCR / DNR(2.50)). [Figure 75]FIG. 75 shows the percentage of CD3+ cells double positive for TCR and dnTGFβRIIvar1 (white bars) or TCR and dnTGFβRIIvar2 (black bars) in exemplary transductions.Cells were either not transduced (NT) or (i) transduced with only a vector encoding the TCR at 2.5 μL per million cells (TCR only, both bars), (ii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 0.313 μL per million cells (TCR / DNR(0.313), white bars), or (iii) transduced with a vector encoding the TCR at 2.5 μL per million cells and a vector encoding dnTGFβRIIvar1 at 0.625 μL per million cells (TCR / DNR(0.313), white bars). (iv) transduced with vector encoding TCR at 2.5 μL per million cells and vector encoding dnTGFβRIIvar1 at 1.25 μL per million cells (TCR / DNR(1.25), white bars); (v) transduced with vector encoding TCR at 2.5 μL per million cells and vector encoding dnTGFβRIIvar1 at 2.50 μL per million cells (TCR / DNR(1.25), white bars). (vi) transduced with vector encoding TCR at 2.5 μL per million cells and vector encoding dnTGFβRIIvar2 at 0.313 μL per million cells (TCR / DNR(0.313), black bars); (vii) transduced with vector encoding TCR at 2.5 μL per million cells and vector encoding dnTGFβRIIvar2 at 0.625 μL per million cells (TCR / DNR( 0.625), black bars), or (viii) transduced with a vector encoding a TCR at 1.25 μL per million cells and a vector encoding dnTGFβRIIvar2 at 10.0 μL per million cells (TCR / DNR(1.25), black bars), or (ix) transduced with a vector encoding a TCR at 1.25 μL per million cells and a vector encoding dnTGFβRIIvar2 at 2.5 μL per million cells (TCR / DNR(2.5), black bars). [Figure 76]FIG. 76 shows the fold amplification of untransduced cells (PS NT) and cells transduced with TCR (2.5 μL per million cells) and dnTGFβRIIvar1 (2.5 μL per million cells) (all other bars). Untransduced cells in "PS NT" were incubated with protamine sulfate (PS). Transduced cells in "PS Mixed" were transduced with TCR and dnTGFβRIIvar1 and PS was added simultaneously. Transduced cells in "PS Sequential" were transduced with TCR and dnTGFβRIIvar1 and PS was added sequentially. Transduced cells in "LB Mixed" were transduced with TCR and dnTGFβRIIvar1 and LentiBOOST® (LB) was added simultaneously. In the exemplary transduction, "LB Sequential" transduced cells were transduced with TCR and dnTGFβRIIvar1 and sequentially added PS. "None Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 and no enhancer was added at the same time. "PS Mixed(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1 and PS was added at the same time and spinoculated. "LB Mixed(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1 and LB was added at the same time and spinoculated. "None(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1 and no enhancer was added at the same time and spinoculated. [Figure 77-1]FIG. 77A shows the percentage of TCR-positive CD3+ cells (black bars) or dnTGFβRIIvar1-positive CD3+ cells (white bars) in the exemplary transductions. Non-transduced cells (NT) are shown as controls. "None Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 without the addition of enhancer at the same time. "PS Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of PS at the same time. "PS Serial" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of PS serially. "LB Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of LB at the same time. "LB Serial" transduced cells were transduced with TCR and dnTGFβRIIvar1 and sequentially added with LB. "None(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1, no enhancer was added at the same time, and spinoculated. "PS(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1, PS was added at the same time, and spinoculated. "LB(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1, LB was added at the same time, and spinoculated. [Figure 77-2]FIG. 77B shows the percentage of CD3+ cells double positive for TCR and dnTGFβRIIvar1 in the exemplary transduction. Non-transduced cells (NT) are shown as a control. "None Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 without the addition of enhancer at the same time. "PS Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of PS at the same time. "PS Serial" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of PS sequentially. "LB Mixed" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of LB at the same time. "LB Serial" transduced cells were transduced with TCR and dnTGFβRIIvar1 with the addition of LB sequentially. "None(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1, no enhancer was added at the same time, and spinoculated. "PS(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1, PS was added at the same time, and spinoculated. "LB(spin)" transduced cells were transduced with TCR and dnTGFβRIIvar1, LB was added at the same time, and spinoculated. [Figure 78] FIG. 78 shows the fold amplification (Y-axis) of non-transduced (NT) cells and cells transduced with TCR (2.5 μL / million cells) and dnTGFβRIIvar1 (2.5 μL / million cells) (2.50) in an exemplary transduction. [Figure 79]Figure 79A shows the percentage of non-transduced CD3+ cells (NT) and TCR and dnTGFβRIIvar1 transduced CD3+ cells (TCR / DNR) that were TCR positive (black bars) or dnTGFβRIIvar1 positive (white bars) in an exemplary transduction. Figure 79B shows the percentage of non-transduced CD3+ cells (NT) and TCR and dnTGFβRIIvar1 transduced CD3+ cells (TCR / DNR) that were TCR and dnTGFβRIIvar1 double positive in an exemplary transduction. [Figure 80-1] 80A-80E show an exemplary cell sorting scheme and plots of the cell fractions obtained for one donor (D120). The Sort1 input (FIG. 80A) can be seen to be composed of a mixture of TCR+dnTGFβRII+, TCR+dnTGFβRII-, TCR-dnTGFβRII+, and TCR-dnTGFβRII- cells. The collected Sort1 output (FIG. 80B) was composed primarily of TCR+dnTGFβRII+ cells. The flow-through cells from Sort1 (Sort1 Waste) (FIG. 80C) were sorted a second time to obtain the Sort2 output cells (FIG. 80D), which contained primarily TCR+dnTGFβRII- cells and were collected. The flow-through cells from Sort2 ("Sort2 Waste" or "waste") (FIG. 80E) were composed primarily of TCR-dnTGFβRII- cells. For each of Figures 80A-80E, the plot on the left (FSC x SSC) shows the parent gate of the plot on the right. On the right, TCR / DNR expression is shown as a percentage of "total lymphocytes." Tet+ cells are TCR+ cells and Tet- cells are TCR- cells. [Figure 80-2] Same as above [Figure 80-3] Same as above [Figure 80-4] Same as above [Figure 80-5] Same as above [Figure 81]FIG. 81 shows fold tumor growth of UACC257-RFP cells normalized to time 0 in an exemplary co-culture assay. Shown are UACC257 cells alone ("UACC257 only", white downward triangles) and UACC257 cells cocultured with TCR-only transduced cells; cocultures were incubated with (i) 8 ng / mL TGF-β1 ("TGF-b 8 ng / mL", white octagons), (ii) 128 ng / mL TGF-β1 ("TGF-b 128 ng / mL", filled circles), (iii) no TGF-β1 or GAL added ("TGF-b 0 ng / mL", open circles), (iv) 8 ng / mL TGF-β1 and 5 μM GAL ("TGF-b 8 ng / mL+GAL", white upward triangles), and (v) 128 ng / mL TGF-β1 and 5 μM GAL ("TGF-b 128 ng / mL+GAL”, open squares), or (vi) treated with 5 μM GAL alone (“TGF-b 0 ng / mL+GAL”, Xs). [Figure 82] Figure 82 shows the mitotic index (average number of divisions per cell) of effector T cells (TCR only transduced) in an exemplary co-culture assay. The mitotic index includes all CD3+ T cells co-cultured with UACC257 cells in the presence (+GAL) or absence (-GAL) of GAL, and in the absence (0 ng / mL) or presence of 2, 4, 8, 16, 32, 64, or 128 ng / mL of TGF-β1. [Figure 83] Figure 83 shows the proliferation of effector cells (transduced with TCR only) co-cultured with UACC257 tumor cells without the addition of TGF-β1 or GAL (TGF-β 0 ng / mL-GAL), with the addition of 8 ng / mL TGF-β1 but no GAL (TGF-β 8 ng / mL-GAL), and with the addition of 8 ng / mL TGF-β1 and 5 μM GAL (TGF-β 8 ng / mL+GAL 5 μM). In the exemplary co-culture assay, cell numbers (Y-axis) range from 0-500 for the "TGF-β 0 ng / mL-GAL" plot, 0-300 for the "TGF-β 8 ng / mL-GAL" plot, and 0-500 for the "TGF-β 8 ng / mL+GAL 5 μM" plot. [Figure 84-1] Figure 84A shows the percentage of all lymphocytes in an exemplary assay that were positive for pSMAD (Y axis). Lymphocytes are from the first donor. Results are shown for non-transduced cells (NT) and for cells transduced with a vector encoding dnTGFβRIIvar1 at 7.5 μL per cell (7.5). Cells were not treated (white bars), treated with 5 ng / mL TGFβ (black bars), or treated with 5 ng / mL TGFβ and 5 μM GAL (black bars). [Figure 84-2] Figure 84B shows the percentage of all lymphocytes in an exemplary assay that were positive for pSMAD (Y axis). Lymphocytes are from the first donor. Results are shown for non-transduced cells (NT) and for cells transduced with a vector encoding dnTGFβRIIvar2 at 7.5 μL per cell (7.5). Cells were not treated (white bars), treated with 5 ng / mL TGFβ (black bars), or treated with 5 ng / mL TGFβ and 5 μM GAL (black bars). [Figure 84-3] Figure 84C shows the percentage of all lymphocytes positive for pSMAD (Y-axis) in an exemplary assay. Lymphocytes are from a second donor. Results are shown for non-transduced cells (NT) and for cells transduced with a vector encoding dnTGFβRIIvar2 at 10 μL per cell (10). Cells were untreated (white bars), treated with 5 ng / mL TGFβ (black bars), or treated with 5 ng / mL TGFβ and 5 μM GAL (black bars). [Figure 85]Figure 85 shows the fold proliferation of UACC257-RFP tumor cells normalized to 0 hours in an exemplary co-culture assay. Tumor cells were added back to the co-culture at about 60 hours and about 134 hours after the start of the co-culture. Proliferation of UACC257-RFP cells cultured alone (UACC257 (No Cyto)) is shown as a control (white square). Proliferation of UACC257-RFP cells co-cultured with cells depleted of TCR+dnTGFβRII+ cells and TCR+dnTGFβRII- cells (Waste (No Cyto)) is shown (black diamond). Proliferation of UACC257-RFP cells co-cultured with TCR+ cells depleted of TCR+dnTGFβRII+ cells (TCR+ (No Cyto)) is shown (white diamond). Proliferation of UACC257-RFP cells (TCR+DNR+(No Cyto)) co-cultured with TCR+dnTGFβRII+ cells is shown (black triangles). The effector:target (E:T) ratio was 4:1. TGF-β1 was not added to the cell cultures. [Figure 86] Figure 86 shows the fold proliferation of UACC257-RFP tumor cells normalized to 0 hours in an exemplary co-culture assay. Tumor cells were added back to the co-culture at about 60 hours and about 134 hours after the start of the co-culture. Proliferation of UACC257-RFP cells cultured alone (UACC257(TGFb)) is shown as a control (white square). Proliferation of UACC257-RFP cells co-cultured with cells depleted of TCR+dnTGFβRII+ cells and TCR+dnTGFβRII- cells (Waste(TGFb)) is shown (black diamond). Proliferation of UACC257-RFP cells co-cultured with TCR+ cells depleted of TCR+dnTGFβRII+ cells (TCR+(TGFb)) is shown (white diamond). Proliferation of UACC257-RFP cells (TCR+DNR+(TGFb)) co-cultured with TCR+dnTGFβRII+ cells is shown (black triangles). The effector:target (E:T) ratio was 4:1. TGF-β1 at a concentration of 10 ng / mL was added to each of the cell cultures at the start of the culture and when the tumor cells were added back. [Figure 87] Figure 87 shows the fold proliferation of UACC257-RFP tumor cells normalized to 0 hours in an exemplary co-culture assay. Tumor cells were added back to the co-culture at about 60 hours and about 134 hours after the start of the co-culture. Proliferation of UACC257-RFP cells cultured alone (UACC257(TGFb Daily)) is shown as a control (white square). Proliferation of UACC257-RFP cells co-cultured with cells depleted of TCR+dnTGFβRII+ cells and TCR+dnTGFβRII- cells (Waste(TGFb Daily)) is shown (black diamond). Proliferation of UACC257-RFP cells co-cultured with TCR+ cells depleted of TCR+dnTGFβRII+ cells (TCR+(TGFb Daily)) is shown (white diamond). Proliferation of UACC257-RFP cells co-cultured with TCR+dnTGFβRII+ cells (TCR+DNR+(TGFb Daily)) is shown (black triangles). The effector:target (E:T) ratio was 4:1. TGF-β1 was added to each of the cell cultures at a concentration of 10 ng / mL every day, including the day of initiation of co-culture. [Figure 88] Figure 88 shows the percentage of dividing cells in an exemplary assay: (i) TCR+dnTGFβRII+ cells not treated with TGF-β1 (TCR+DNR+(Untreated)), (ii) TCR+ cells depleted of TCR+dnTGFβRII+ cells not treated with TGF-β1 (TCR+(Untreated)), (iii) TCR-dnTGFβRII- cells not treated with TGF-β1 (Waste(Untreated)), (iv) TCR-dnTGFβRII- cells depleted of TGF-β1 (Waste(Untreated)), (v) TCR-dnTGFβRII- cells depleted of TGF-β1 (Waste(Untreated)), (vi) TCR-dnTGFβRII- cells depleted of TGF-β1 (Waste(Untreated)), (vi) TCR-dnTGFβRII- cells depleted of TGF-β1 (Waste(Untreated)), (vi) TCR-dnTGFβRII- cells depleted of TGF-β1 (Waste(Untreated)), (vi) TCR-dnTGFβRII- cells depleted of TGF-β1 (Waste(Untreated)), (v ... (v) TCR+dnTGFβRII+ cells depleted and treated with 10 ng / mL TGF-β1 (TCR+(TGF-b)), and (vi) TCR-dnTGFβRII- cells treated with 10 ng / mL TGF-β1 (Waste(TGF-b)). Averages of cells from two donors are shown. [Figure 89]Figure 89 shows the fold expansion of non-transduced (NT) cells and cells transduced with TCR (2.5 μL / million cells), CD8βα.TCR (CDba.TCR), 2.5 μL / million cells, and CD8βα.TCR.dnTGFβRII (CD8ba.TCR.dnTGFbRII) at concentrations of 20, 10, 5, 2.5, or 1.25 μL / million cells in an exemplary assay. [Figure 90] FIG. 90 shows vector copy numbers in cells prepared as described in FIG. 89 in an exemplary assay. [Figure 91] FIG. 91 shows, in an exemplary assay, expression of CD8 and CD4 in cells prepared as described in FIG. [Figure 92] FIG. 92 shows, in an exemplary assay, expression of TCR and TGFbRII in cells prepared as described in FIG. [Figure 93] FIG. 93 shows the fold expansion of non-transduced (NT) cells and cells transduced with TCR (2.5 μL / million cells) and dnTGFβRII.TCR (dnTGFbRII-TCR, 2.5 μL / million cells) in an exemplary assay. [Figure 94] FIG. 94 shows, in an exemplary assay, expression of constructs in cells prepared as described in FIG. 93. [Figure 95-1] Figures 95A-95C show SMAD phosphorylation in TCR+ and TCR+dnTGFβRII+ cells in an exemplary assay in the absence (untreated) or presence (TGFb) of TGFβ. Non-transduced cells (NT) are shown as a control. The percentage of phosphorylated SMAD+ is shown as a percentage of all live CD3+CD8+ T cells. Representative flow plots for one donor are shown. [Figure 95-2] Same as above [Figure 96-1]Figures 96A-96B show the proliferation of TCR+ (TCR) or TCR+dnTGFβRII+ (TCR+DNR) transduced cells in the absence (untreated) or presence (TGF-β) of TGFβ in an exemplary co-culture assay. Tumor cells were added to the co-cultures on D+0, D+3, and D+6 (tumor challenge, #1, #2, and #3, respectively). After 3 days in culture with tumor cells, cells were harvested for flow analysis. Proliferation index values ​​are shown for TCR+ cells. [Figure 96-2] Same as above [Figure 97-1] Figures 97A-97B show fold proliferation of UACC257-RFP tumor cells normalized to 0 hours in an exemplary co-culture assay with TCR+ cells or TCR+dnTGFβRII+ cells derived from three different donors (D645, D776, D897). Tumor cells were added to the co-cultures at 0, 68, 140, 212, and 284 hours, or approximately every 3 days. Proliferation of UACC257-RFP cells cultured alone (UACC257) or cells cultured in co-culture with non-transduced cells (NT) is shown as controls. [Figure 97-2] Same as above [Figure 98-1] Figures 98A-98K show cytokine release from TCR+ cells and TCR+dnTGFβRII+ cells in an exemplary assay in the absence (untreated) or presence of TGFβ (TGFb). [Figure 98-2] Same as above [Figure 98-3] Same as above [Figure 98-4] Same as above [Figure 98-5] Same as above [Figure 98-6] Same as above [Figure 98-7] Same as above [Figure 98-8] Same as above [Figure 98-9] Same as above [Figure 98-10] Same as above [Figure 98-11] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0136] Dominant negative TGFβR In embodiments, one or more TGF-β signaling pathways may be completely or partially disrupted in cells expressing one or more dominant-negative TGF-β receptors (dnTGFβR). In embodiments, one or more dnTGFβRI and / or dnTGFβRII polypeptides are provided. In embodiments, the nucleic acids described herein comprise and / or encode one or more dnTGFβRI and / or dnTGFβRII polypeptides. In embodiments, the vectors described herein comprise and / or encode one or more dnTGFβRI and / or dnTGFβRII polypeptides. In embodiments, the cells described herein comprise and / or express one or more dnTGFβRI and / or dnTGFβRII polypeptides. In embodiments, the compositions described herein comprise one or more dnTGFβRI and / or dnTGFβRII polypeptides or include cells comprising and / or expressing one or more dnTGFβRI and / or dnTGFβRII polypeptides. In some embodiments, the TGFβR is made dominant negative by, for example and without limitation, truncating and / or mutating the TGFβR to remove and / or render inoperative all or part of at least one signaling portion of the TGFβR.

[0137] In some embodiments, TGFβRII is made dominant negative by, for example and without limitation, truncating and / or mutating TGFβRII to remove and / or disable all or part of the intracellular signaling portion of TGFβRII. In some embodiments, dnTGFβRII polypeptides can include an extracellular domain, a transmembrane domain, and / or a cytoplasmic domain. In some embodiments, the cytoplasmic domain can be truncated, mutated, or absent.

[0138] In some embodiments, dnTGFβRII variant 1 (dnTGFβRIIvar1) and / or dnTGFβRII variant 2 (dnTGFβRIIvar2) are provided, which are examples of dnTGFβRII polypeptides. dnTGFβRIIvar1 may comprise or consist of SEQ ID NO: 305, and may be encoded by a nucleic acid comprising or consisting of SEQ ID NO: 306. dnTGFβRIIvar1 may comprise or consist of SEQ ID NO: 307, and may be encoded by a nucleic acid comprising or consisting of SEQ ID NO: 308. TGFβRIIvar1 and TGFβRIIvar2 disclosed herein each lack the cytoplasmic domain required for downstream signaling. Without being bound by theory, in some embodiments, dnTGFβRII may function, for example, as follows: truncated TGFβRII retains the ability to bind TGF-β and form a heteromeric complex with TGFβRI, but the lack of a cytoplasmic domain prevents phosphorylation of TGFβRI and subsequent activation of downstream elements. Furthermore, the inclusion of a single truncated TGFβRII protein in a heteromeric TGF-β receptor complex may be sufficient to abolish signaling, suggesting that the protein functions in a dominant-negative manner.

[0139] In some embodiments, a nucleic acid sequence is provided that encodes a dnTGFβRII polypeptide operably linked to a promoter. In some embodiments, a nucleic acid sequence is provided that encodes a dnTGFβRII polypeptide operably linked to a post-transcriptional regulatory element. In some embodiments, the promoter is an MSCV promoter, and / or the post-transcriptional regulatory element is a WPRE, optionally a mutant WPRE, optionally a WPREmut2. Figure 67A shows a dnTGFβRIIvar1 linked to an MSCV promoter and a WPRE. In some embodiments, the WPRE is a WPRE, such a construct is encoded, for example, by SEQ ID NO: 312. Figure 67B shows a dnTGFβRIIvar2 linked to an MSCV promoter and a WPRE. In some embodiments, the WPRE is a WPRE, such a construct is encoded, for example, by SEQ ID NO: 313. In Figures 67A and 67B, the lines linking the MSCV promoter to the dnTGFβRII and the dnTGFβRII to the WPRE may represent direct bonds with no intervening sequences, or may represent intervening sequences, such as, but not limited to, a linker, a non-translated sequence (in the case of a nucleic acid sequence), a translated sequence, a sequence comprising one or more restriction endonuclease sites (in the case of a nucleic acid sequence), or a combination thereof.

[0140] In some embodiments, an isolated dnTGFβRII polypeptide is provided. In some embodiments, an isolated nucleic acid sequence is provided that includes one or more nucleic acid sequences encoding one or more dnTGFβRII polypeptides. In some embodiments, an isolated vector is provided that includes one or more nucleic acid sequences that include one or more nucleic acid sequences encoding one or more dnTGFβRII polypeptides. In some embodiments, an isolated cell is provided that includes or expresses one or more dnTGFβRII polypeptides. In some embodiments, an isolated cell is provided that includes or expresses one or more nucleic acid sequences that include one or more nucleic acid sequences encoding one or more dnTGFβRII polypeptides. In some embodiments, an isolated cell is provided that includes or expresses one or more vectors that include one or more nucleic acid sequences that include one or more nucleic acid sequences encoding one or more dnTGFβRII polypeptides. In some embodiments, compositions are provided that include such polypeptides, nucleic acids, vectors, and / or cells.

[0141] In certain aspects, the polypeptide and / or nucleic acid sequences described herein can be isolated and / or recombinant sequences.

[0142] In some embodiments, the dnTGFβRII polypeptide has 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 some embodiments, the dnTGFβRII polypeptide has 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 several embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a mutant dnTGFβRII polypeptide, (ii) one or more reduced or absent functions of dnTGFβRII, such as, for example and without limitation, reduced or eliminated signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, are preserved and / or reduced in a mutant dnTGFβRII polypeptide, or (iii) any combination thereof may be found in a mutant dnTGFβRII polypeptide.

[0143] In embodiments, the dnTGFβRII polypeptide comprises (a) SEQ ID NO: 305, which comprises one, two, three, four or five amino acid substitutions, or (b) SEQ ID NO: 307, which comprises one, two, three, four or five amino acid substitutions. In embodiments, (i) a function of dnTGFβRII, such as, but not limited to, the ability of dnTGFβRII to bind TGFβ, is preserved and / or enhanced in the mutant dnTGFβRII polypeptide, (ii) one or more reduced or absent functions of dnTGFβRII, such as, but not limited to, reduced or eliminated signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or reduced in the mutant dnTGFβRII polypeptide, or (iii) any combination thereof may be found in the mutant dnTGFβRII polypeptide.

[0144] In embodiments, the dnTGFβRII polypeptide is encoded by a nucleic acid 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 of SEQ ID NO: 306. In embodiments, the dnTGFβRII polypeptide is encoded by a nucleic acid 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 of SEQ ID NO: 308. In several embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid, (ii) one or more reduced or absent functions of dnTGFβRII, such as, for example and without limitation, reduced or eliminated signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or reduced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid, or (iii) any combination thereof may be found in a dnTGFβRII polypeptide encoded by a mutant nucleic acid.

[0145] In some embodiments, the dnTGFβRII polypeptide is encoded by a nucleic acid comprising (a) SEQ ID NO: 306, which comprises one, two, three, four or five nucleic acid substitutions, or (b) SEQ ID NO: 308, which comprises one, two, three, four or five nucleic acid substitutions. One or more nucleic acid substitutions in a codon may result in a codon that encodes the same amino acid, or may result in a codon that encodes a different amino acid. In some embodiments, one or more nucleic acid substitutions in a codon may result in a codon that encodes a conservative amino acid substitution. One or more nucleic acid substitutions in a codon may result in a codon that encodes the same amino acid. In several embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid, (ii) one or more reduced or absent functions of dnTGFβRII, such as, for example and without limitation, reduced or eliminated signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or reduced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid, or (iii) any combination thereof may be found in a dnTGFβRII polypeptide encoded by a mutant nucleic acid.

[0146] In some embodiments, the nucleic acid encoding the dnTGFβRII polypeptide may include a stop codon (e.g., TAA, TAG or TGA), which, by way of non-limiting example, is located at the 3' end of the nucleotide sequence encoding the dnTGFβRII polypeptide.

[0147] In some embodiments, the dnTGFβRII polypeptide may be encoded by a nucleic acid that also includes and / or encodes one or more MSCV promoters and / or one or more post-transcriptional regulatory elements. 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, but not limited to, 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:366), or a variant thereof, or any combination thereof. In some embodiments, such a construct is encoded by SEQ ID NO:312. In some embodiments, such a construct is encoded by SEQ ID NO:313.

[0148] In embodiments, the dnTGFβRII polypeptide encoded by a nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs may be encoded by a nucleic acid 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 of SEQ ID NO:312. In embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs, and (ii) a reduction or absence of one or more functions of dnTGFβRII, such as, for example and without limitation, a reduction or absence of a signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs. (iii) the function of the MSCV promoter is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; (iv) one or more post-transcriptional functions of a WPRE are preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; or (v) any combination thereof may be found in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs.

[0149] In some embodiments, the dnTGFβRII polypeptide encoded by a nucleic acid that also includes and / or encodes one or more MSCV promoters and one or more WPREs may be encoded by a nucleic acid that includes (a) SEQ ID NO: 312, which includes 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 that encodes the same amino acid, or may result in a codon that encodes a different amino acid. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon that encodes a conservative amino acid substitution. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon that encodes the same amino acid.In embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs, and (ii) a reduction or absence of one or more functions of dnTGFβRII, such as, for example and without limitation, a reduction or absence of a signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs. (iii) the function of the MSCV promoter is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; (iv) the post-transcriptional function of a WPRE is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; or (v) any combination thereof may be found in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs.

[0150] In embodiments, the dnTGFβRII polypeptide encoded by a nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs may be encoded by a nucleic acid 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 of SEQ ID NO:313. In embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs, and (ii) a reduction or absence of one or more functions of dnTGFβRII, such as, for example and without limitation, a reduction or absence of a signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs. (iii) the function of the MSCV promoter is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; (iv) the post-transcriptional function of a WPRE is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; or (v) any combination thereof may be found in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs.

[0151] In some embodiments, the dnTGFβRII polypeptide encoded by a nucleic acid that also includes and / or encodes one or more MSCV promoters and one or more WPREs may be encoded by a nucleic acid that includes (a) SEQ ID NO: 313, which includes 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 that encodes the same amino acid, or may result in a codon that encodes a different amino acid. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon that encodes a conservative amino acid substitution. In some embodiments, the one or more nucleic acid substitutions in a codon may result in a codon that encodes the same amino acid.In embodiments, (i) a function of dnTGFβRII, such as, for example and without limitation, the ability of dnTGFβRII to bind to TGFβ, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs, and (ii) a reduction or absence of one or more functions of dnTGFβRII, such as, for example and without limitation, a reduction or absence of a signaling function of the C-terminal portion of dnTGFβRII, one or more mutations and / or deletions of the C-terminal portion of dnTGFβRII, or any combination thereof, is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs. (iii) the function of the MSCV promoter is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; (iv) the post-transcriptional function of a WPRE is preserved and / or enhanced in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs; or (v) any combination thereof may be found in a dnTGFβRII polypeptide encoded by a mutant nucleic acid that also comprises and / or encodes one or more MSCV promoters and one or more WPREs.

[0152] In some embodiments, the nucleic acid encoding the dnTGFβRII polypeptide may include a stop codon (e.g., TAA, TAG or TGA), which, by way of non-limiting example, is located at the 3' end of the nucleotide sequence encoding the dnTGFβRII polypeptide.

[0153] Expression of dnTGFβRII may improve the persistence, functionality, proliferation, survival, expansion, or a 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 dnTGFβRII. Expression of dnTGFβRII may improve the persistence, functionality, proliferation, survival, expansion, or a 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 dnTGFβRII. Expression of dnTGFβRII 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 dnTGFβRII. Expression of dnTGFβRII 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 a combination thereof, compared to cells that do not express dnTGFβRII. In embodiments, expression of a dnTGFβRII 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.

[0154] Persistence can be assessed by, for example and not by way of limitation, the length of time that 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, and 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. Persistence of cells expressing dnTGFβRII polypeptides 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 dnTGFβRII polypeptides.

[0155] 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 a dnTGFβRII polypeptide to kill tumor cells may be compared, by way of non-limiting example, to the continued ability of similar cells not expressing a dnTGFβRII polypeptide to kill tumor cells, or the continued ability of other control cells to kill tumor cells.

[0156] 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.

[0157] When assessing the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naivety, or other properties of cells expressing dnTGFβRII, 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, may be utilized as control cells. Because dnTGFβRII may act to reduce or eliminate TGFβ signaling, assessment of the persistence, functionality, proliferation, survival, expansion, tumor killing efficacy, naivety, or other properties of cells expressing dnTGFβRII may be performed in the presence of exogenous TGFβ, such as, for example, TGF-β1.

[0158] dnTGFβRII 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 in a combination thereof. If dnTGFβRII acts in trans, cells adjacent to or in the vicinity of cells expressing dnTGFβRII (e.g., in the tumor microenvironment) may exhibit the same or equivalent improvements or combinations as described for cells expressing dnTGFβRII compared to cells not adjacent to or in the vicinity of cells expressing dnTGFβRII. Without being bound by theory, dnTGFβRII may act to reduce the amount of TGF-β in the tumor microenvironment. Also, cells expressing dnTGFβRII may exhibit improved ability to secrete cytokines in response to target antigens in the presence of TGF-β compared to cells not expressing dnTGFβRII.

[0159] Modified CD8 Polypeptides 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.

[0160] 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.

[0161] 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.

[0162] In embodiments, the CD8 polypeptide described herein may comprise: (a) at least about 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 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 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 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 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.

[0163] 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 amino acid substitutions. The CD8 polypeptides 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.

[0164] 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.

[0165] 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 mediate signaling functions in T cells through its association with p56lck and may participate in the T cell activation cascade by phosphorylation.

[0166] 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: 310.

[0167] 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.

[0168] 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.

[0169] T cells The T cells may express dnTGFβRII, modified CD8 polypeptides described herein, or combinations thereof. As another non-limiting example, the T cells may co-express a T cell receptor (TCR) and a dnTGFβRII polypeptide. As another non-limiting example, the T cells may co-express a T cell receptor (TCR) and a modified CD8 polypeptide described herein. As another non-limiting example, the T cells may co-express a T cell receptor (TCR), a dnTGFβRII polypeptide, and a modified CD8 polypeptide described herein. The T cells may also express a chimeric antigen receptor (CAR), a CAR analog, or a CAR derivative. In several 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.

[0170] The T cells, when a population, can be αβ T cells, γδ T cells, natural killer 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 a combination thereof.

[0171] The T cells can be αβ T cells and can express a CD8 polypeptide described herein. The T cells can be αβ T cells and can express a modified CD8 polypeptide 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 a dnTGFβRII 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.

[0172] The T cells may be γδ T cells and may express a CD8 polypeptide as described herein. 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 CD8α polypeptide, or a modified CD8α polypeptide comprising a CD8β stalk region, e.g., m1CD8α in constructs #11 and #12 (FIG. 4), or CD8α* (FIG. 55B). The T cells may be γδ T cells and may express one or any combination of a dnTGFβRII 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.

[0173] T cells can be provided that contain a nucleic acid encoding one or any combination of a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, a CAR, a dnTGFβRII polypeptide, and / or a CD8 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.

[0174] A T cell may be provided that comprises a nucleic acid encoding one or any combination of a TCR comprising an α chain and a β chain, a dnTGFβRII polypeptide, and / or a CD8 polypeptide. A cell may be provided that comprises a nucleic acid encoding one or any combination of a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and / or a CD8 polypeptide. A cell may be provided that comprises a nucleic acid encoding one or any combination of a CAR, a dnTGFβRII polypeptide, and / or a CD8 polypeptide. In some embodiments, the CD8 polypeptide may comprise 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.

[0175] A T cell may be provided that includes a nucleic acid encoding a TCR comprising an α chain and a β chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, a cell may be provided that includes a nucleic acid encoding a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. A cell may be provided that includes a nucleic acid encoding a CAR, a dnTGFβRII polypeptide, 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.

[0176] A T cell may be provided that includes a TCR that includes an α chain and a β chain, and a nucleic acid that encodes a dnTGFβRII polypeptide. A cell may be provided that includes a TCR that includes a γ chain and a δ chain, and a nucleic acid that encodes a dnTGFβRII polypeptide. A cell may be provided that includes a CAR and a nucleic acid that encodes a dnTGFβRII polypeptide. 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.

[0177] A T cell may be provided that comprises a TCR comprising an α chain and a β chain, and a nucleic acid encoding a CD8 polypeptide. In some embodiments, a cell may be provided that comprises a TCR comprising a γ chain and a δ chain, and a nucleic acid encoding a CD8 polypeptide. A cell may be provided that comprises a CAR, and a nucleic acid encoding a CD8 polypeptide. In some embodiments, the CD8 polypeptide may comprise 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. In some embodiments, such a cell may comprise or comprises a nucleic acid encoding at least one dnTGFβRII polypeptide.

[0178] Natural killer (NK) cells 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 by reference in its entirety. In some embodiments, engineered NK cells are provided.

[0179] The NK cells may express dnTGFβRII, a modified CD8 polypeptide described herein, or a combination thereof. As another non-limiting example, the NK cells may co-express a T cell receptor (TCR) and a dnTGFβRII polypeptide. As another non-limiting example, the NK cells may co-express a T cell receptor (TCR) and a modified CD8 polypeptide described herein. As another non-limiting example, the NK cells may co-express a T cell receptor (TCR), a dnTGFβRII polypeptide, and a modified CD8 polypeptide described herein. The NK cells may also express a chimeric antigen receptor (CAR), a CAR analog, or a CAR derivative. In several 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.

[0180] The NK cells may express a CD8 polypeptide as described herein. 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 a dnTGFβRII polypeptide, a CD8 polypeptide, and / or a CAR. In some embodiments, the CD8 polypeptide may comprise a CD8α chain and / or a CD8β chain, which may be independently modified or unmodified.

[0181] NK cells can be provided that contain a nucleic acid encoding one or any combination of a TCR comprising an α chain and a β chain, a TCR comprising a γ chain and a δ chain, a CAR, a dnTGFβRII polypeptide, and / or a CD8 polypeptide. 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.

[0182] NK cells may be provided that include a nucleic acid encoding one or any combination of a TCR comprising an α chain and a β chain, a dnTGFβRII polypeptide, and / or a CD8 polypeptide. Cells may be provided that include a nucleic acid encoding one or any combination of a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and / or a CD8 polypeptide. Cells may be provided that include a nucleic acid encoding one or any combination of a CAR, a dnTGFβRII 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.

[0183] NK cells may be provided that include a nucleic acid encoding a TCR comprising an α chain and a β chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. In some embodiments, cells may be provided that include a nucleic acid encoding a TCR comprising a γ chain and a δ chain, a dnTGFβRII polypeptide, and a CD8 polypeptide. Cells may be provided that include a nucleic acid encoding a CAR, a dnTGFβRII polypeptide, 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.

[0184] A NK cell may be provided that comprises a TCR comprising an α chain and a β chain, and a nucleic acid encoding a dnTGFβRII polypeptide. A cell may be provided that comprises a TCR comprising a γ chain and a δ chain, and a nucleic acid encoding a dnTGFβRII polypeptide. A cell may be provided that comprises a CAR and a nucleic acid encoding a dnTGFβRII polypeptide. In embodiments, the CD8 polypeptide may comprise 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.

[0185] NK cells may be provided that include a TCR comprising an α chain and a β chain, and a nucleic acid encoding a CD8 polypeptide. In some embodiments, cells may be provided that include a TCR comprising a γ chain and a δ chain, and a nucleic acid encoding a CD8 polypeptide. Cells may be provided that include a CAR, and a nucleic acid 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. In some embodiments, such cells may or may include a nucleic acid encoding at least one dnTGFβRII polypeptide.

[0186] T cell receptor T cells may co-express a T cell receptor (TCR), an antigen binding protein, or both, along with a dnTGFβRII polypeptide and / or a CD8 polypeptide described herein, including but not limited to those listed in Table 3 (SEQ ID NOs: 15-92). In embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, which may be independently modified or unmodified.Additionally, the T cells may be provided with a dnTGFβRII polypeptide, a modified CD8 polypeptide as described herein, a TCR, and U.S. Patent Application Publication No. 2017 / 0267738; U.S. Patent Application Publication No. 2017 / 0312350; U.S. Patent Application Publication No. 2018 / 0051080; U.S. Patent Application Publication No. 2018 / 0164315; U.S. Patent Application Publication No. 2018 / 0161396; U.S. Patent Application Publication No. 2018 / 0162922; U.S. Patent Application Publication No. 2018 / 0273602; U.S. Patent Application Publication No. 2019 / 0 016801;US Patent Application Publication No. 2019 / 0002556;US Patent Application Publication No. 2019 / 0135914;US Patent 10,538,573;US Patent 10,626,160;US Patent Application Publication No. 2019 / 0321478;US Patent Application Publication No. 2019 / 0256572;US Patent 10,550,182;US Patent 10,526,407;US Patent Application Publication No. 2019 / 0284276;US Patent Application Publication No. 2019 / 0016802;US Patent Application Publication No. 2019 / 0016803;US Patent US Patent Publication No. 2019 / 0016804; US Patent 10,583,573; US Patent Publication No. 2020 / 0339652; US Patent 10,537,624; US Patent 10,596,242; US Patent Publication No. 2020 / 0188497; US Patent 10,800,845; US Patent Publication No. 2020 / 0385468; US Patent 10,527,623; US Patent 10,725,044; US Patent Publication No. 2020 / 0249233; US Patent 10,702,609; US Patent Publication No. 20 The antigen binding proteins may express one or any combination of the antigen binding proteins described in US Patent Publication 2020 / 0254106; US Patent 10,800,832; US Patent Publication 2020 / 0123221; US ​​Patent 10,590,194; US Patent 10,723,796; US Patent Publication 2020 / 0140540; US Patent 10,618,956; US Patent Publication 2020 / 0207849; US Patent Publication 2020 / 0088726; and US Patent Publication 2020 / 0384028. The contents of each of these publications and the sequence listings set forth therein are incorporated herein by reference in their entirety. The cell may be a T cell or a natural killer cell.The T cells may be CD4+ cells, CD8+ cells, CD4+ / CD8+ cells, αβ T cells, γδ T cells, or natural killer T cells. In embodiments, the TCRs described herein may be single chain TCRs or soluble TCRs.

[0187] Additionally, a TCR that may be co-expressed in a T cell with a dnTGFβRII polypeptide and / or a CD8 polypeptide described herein may be a TCR composed 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 cell may be a T cell or a natural killer cell. The T cell may be an αβ T cell, a γδ T cell, or a natural killer T cell.

[0188] 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]

[0189] Tumor-associated antigens (TAA) Tumor-associated antigen (TAA) peptides can be used with the dnTGFβRII polypeptide and / or CD8 polypeptide constructs, methods, and embodiments described herein.For example, the T cell receptor (TCR) described herein can specifically bind to TAA peptides when bound to human leukocyte antigen (HLA), also known as major histocompatibility complex (MHC) molecules.Human MHC molecules are also designated as human leukocyte antigens (HLA).

[0190] Tumor associated antigen (TAA) peptides that may be used with the dnTGFβRII polypeptides and / or CD8 polypeptides described herein include, but are not limited to, those listed in Table 3, as well as 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; U.S. Patent Application Publication No. 2016 / 0346371;US Patent Application Publication 2016 / 0368965;US Patent Application Publication 2017 / 0022251;US Patent Application Publication 2017 / 0002055;US Patent Application Publication 2017 / 0029486;US Patent Application Publication 2017 / 0037089;US Patent Application Publication 2017 / 0136108;US Patent Application Publication 2017 / 0101473;US Patent Application Publication 2017 / 0096461;US Patent Application Publication 2017 / 0165337;US Patent Application Publication 2017 / 0189505;US Patent Application Publication 2017 / 0173132;US Patent Application Publication 2017 / 0296640;US Patent Application Publication 2017 / 0253633;US Patent Application Publication 2017 / 0260249;US Patent Application Publication 2018 / 0051080;US Patent Application Publication 2018 / 0164315;US Patent Application Publication 2018 / 0291082;US Patent Application Publication 2018 / 0291083;US Patent Application Publication 2019 / 0255110;US Patent 9,717,774; The TAA peptides described in 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.

[0191] 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 or a natural killer cell. The T cells can be αβ T cells, γδ T cells, or natural killer T cells.

[0192] T cell culture Described herein are methods for activating, transducing, and / or expanding T cells, such as 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. The cells can be T cells or natural killer cells. The T cells can be αβ T cells, γδ T cells, or natural killer T cells.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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 combinations thereof.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] In embodiments, the same methodology can be used to isolate, activate, and expand αβ T cells.

[0204] In embodiments, the same methodology can be used to isolate, activate, and expand γδ T cells.

[0205] vector Engineered cells can be produced using a variety of methods, including those recognized in the literature. For example, polynucleotides encoding expression cassettes with tumor recognition moieties or other types of recognition moieties can be stably introduced into T cells by transposon / transposase systems, or by viral gene transfer systems, such as lentivirus and retrovirus systems, or by viral delivery methods, including, for example, transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO4), 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, such as, for example, the methods 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. 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.

[0206] 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 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.

[0207] 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.

[0208] 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.

[0209] Additionally, multiple vectors may be used to transfect cells with the constructs and sequences described herein. One or more vectors may contain a combination of TCR transgenes, dnTGFβRII polypeptide transgenes, and CD8 polypeptides 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 a dnTGFβRII polypeptide, and a third vector may contain a transgene encoding a CD8 polypeptide 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 then be transfected with one or more other transgenes. In embodiments, a CD8 polypeptide can comprise a CD8 α chain and / or a CD8 β chain, which can independently be modified or unmodified.

[0210] One or more vectors may comprise a nucleic acid encoding a dnTGFβRII polypeptide. One or more vectors may comprise a nucleic acid encoding a CD8 polypeptide. One or more vectors may comprise a nucleic acid encoding a CD8α polypeptide. One or more vectors may comprise a nucleic acid encoding a CD8β polypeptide. In embodiments, the CD8 polypeptide may comprise a CD8α chain and / or a CD8β chain, which may be independently modified or unmodified.

[0211] The one or more vectors may comprise a nucleic acid encoding a T cell receptor (TCR) comprising an α chain and a β chain. The one or more vectors may comprise a nucleic acid encoding a T cell receptor (TCR) comprising a γ chain and a δ chain. The one or more vectors may comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

[0212] The two or more vectors can comprise nucleic acids encoding one or any combination of a dnTGFβRII 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 the CD8 β chain can be independently modified or unmodified.

[0213] A vector may contain nucleic acids encoding one or any combination of a dnTGFβRII 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 the CD8 β chain may be independently modified or unmodified.

[0214] 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.

[0215] 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.

[0216] The transgene may further comprise one or more multicistronic elements, which may be located between any, part or each of, as non-limiting examples, the nucleic acid encoding TCRα or a portion thereof, the nucleic acid encoding TCRβ or a portion thereof, the nucleic acid encoding CD8α or a portion thereof, the nucleic acid encoding CD8β or a portion thereof, and / or the nucleic acid encoding a dnTGFβRII polypeptide or a portion thereof. The multicistronic element may be located between any two nucleic acid sequences encoding TCRα, TCRβ, CD8α, CD8β, and / or a dnTGFβRII polypeptide, and these coding sequences may be in any order. The multicistronic element may comprise a sequence encoding a ribosome skip element selected from T2A, P2A, E2A or F2A, or an internal ribosome entry site (IRES).

[0217] 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 (e.g., but not limited to, GSG, SGSG (SEQ ID NO: 266)) before the self-cleaving 2A sequence may allow for efficient synthesis of biologically active proteins such as TCRs.

[0218] 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. In embodiments, the IRES is selected from IRES derived from viruses, IRES derived from cellular mRNAs, particularly IRES derived from 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 IRES derived from cellular mRNAs, translation initiation factors, e.g., eIF4G and DAP5, transcription factors, e.g., c-Myc and NF-κB inhibitors (NRFs), 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.

[0219] 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.

[0220] 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: 366), or a variant thereof, or any combination thereof.

[0221] 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.

[0222] In embodiments, the vector may include one or more Kozak sequences. 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: 365).

[0223] In embodiments, the vector may contain one or more factor Xa sites.

[0224] 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 portions thereof, or combinations thereof.

[0225] In embodiments, the vector may be a viral vector or a non-viral vector.

[0226] 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.

[0227] 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.

[0228] Non-viral vectors can also be used in conjunction with the sequences, constructs, and cells described herein.

[0229] 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 a 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.

[0230] Gene editing In some embodiments, transgenes (e.g., transgenes encoding the alpha and / or beta chains of CD8, transgenes encoding the alpha and / or beta chains of TCR, and / or transgenes encoding dominant negative TGFβRII 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 example, T cells or natural killer cells or combinations thereof. The T cells can be, by way of non-limiting example, 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).

[0231] composition The composition may comprise a dnTGFβRII polypeptide, and / or a CD8 polypeptide as described herein, and / or a TCR as described herein. Additionally, the composition as described herein may comprise a T cell and / or a natural killer cell expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide as described herein. The composition as described herein may comprise a T cell and / or a natural killer cell expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide as described herein, and a T cell and / or a natural killer cell receptor (TCR), optionally a TCR that specifically binds to one of the TAAs described herein complexed with an antigen-presenting protein, such as, for example, MHC, also referred to in humans as HLA for human leukocyte antigen. In some embodiments, the CD8 polypeptide may comprise a CD8 α chain and / or a CD8 β chain, which may be independently modified or unmodified.

[0232] To facilitate administration, the T cells and / or natural killer cells described herein may be made into pharmaceutical compositions or made into implants suitable for in vivo administration with pharma- ceutically acceptable carriers or diluents.Methods for making such compositions or implants are described in the art.See, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980).

[0233] 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- ceutically 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-ceutically acceptable carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, and combinations thereof. Pharmaceutical preparations can be mixed, if desired, with auxiliary agents that do not adversely react with T cells and / or natural killer cells, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers. The T cells and / or natural killer cells can be dnTGFβRII polypeptides and / or CD8 polypeptides described herein, optionally αβ T cells or γδ T cells expressing a TCR described herein. In embodiments, the CD8 polypeptides can include CD8α chains and / or CD8β chains, which can be independently modified or unmodified.

[0234] Compositions of the present disclosure 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 appropriate active agents.

[0235] 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 any combination thereof.

[0236] 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.

[0237] Adjuvants include, but are not limited to, cyclophosphamide, imiquimod, or resiquimod. Other exemplary adjuvants include Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinations thereof.

[0238] 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.

[0239] 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.

[0240] 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]-based 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. In some embodiments, the adjuvant may be Freund's or GM-CSF. Several immune adjuvants specific for dendritic cells and their preparations (e.g., MF59) 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).

[0241] 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.

[0242] Treatment and preparation methods The engineered T cells and / or engineered natural killer (NK) cells may express a dnTGFβRII polypeptide and / or a CD8 polypeptide as described herein. Additionally, the engineered T cells and / or engineered natural killer (NK) cells may express a TCR as described herein. The TCR expressed by the engineered T cells and / or engineered natural killer (NK) cells may recognize a TAA bound to an HLA as described herein. The engineered T cells and / or engineered natural killer (NK) cells of the present disclosure may be used to treat a subject in need of treatment for a disease, such as, for example, a cancer as described herein. The cells may be αβ T cells, γδ T cells and / or natural killer (NK) cells expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide, and optionally a TCR as described herein. In several embodiments, the CD8 polypeptide may include a CD8α chain and / or a CD8β chain, and the CD8α chain and / or the CD8β chain may be independently modified or unmodified.

[0243] A method of treating a disease (e.g., a disorder) in a subject using the T cells and / or natural killer (NK) cells described herein may include administering to the subject a therapeutically effective amount of the engineered T cells and / or engineered natural killer (NK) cells described herein, optionally γδ T cells. The T cells and / or natural killer (NK) 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 (NK) cells of the present disclosure. The population of engineered T cells and / or engineered natural killer (NK) cells may be frozen or cryopreserved prior to administration to the subject. The population of engineered T cells and / or engineered natural killer (NK) 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 (NK) cells may include several distinct engineered T cells and / or engineered natural killer (NK) cells designed to recognize different antigens or different epitopes of the same antigen. The cells may be αβ T cells, γδ T cells and / or natural killer (NK) cells expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide as described herein, 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.

[0244] The T cells and / or natural killer (NK) cells described herein, including αβ T cells and γδ T cells, can be used to treat a variety of conditions. The cells can be αβ T cells, γδ T cells and / or natural killer (NK) cells expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide, and optionally a TCR as described herein. In 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. The T cells and / or natural killer (NK) cells described herein can 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.

[0245] The T cells and / or natural killer (NK) cells described herein may be used to treat infectious diseases. The T cells and / or natural killer (NK) cells described herein may be used to treat infectious diseases that may be caused by viruses. The T cells and / or natural killer (NK) 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 (NK) cells expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide, and optionally a TCR as described herein. In some embodiments, the CD8 polypeptide may include a CD8α chain and / or a CD8β chain, and the CD8α chain and / or the CD8β chain may be independently modified or unmodified.

[0246] Treatment with T cells and / or natural killer (NK) 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 about 1 day, about 1 week, about 6 months, about 12 months, or about 2 years after clinical manifestation of a disease. Treatment may be provided to a subject for multiple days, about 1 week, about 1 month, about 6 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, or more after clinical manifestation of a disease. Treatment may be provided to a subject less than about 1 day, less than about 1 week, less than about 1 month, less than about 6 months, less than about 12 months, or less than about 2 years after clinical manifestation of a disease. Treatment may also include treating humans in clinical trials. The treatment may include administering to the subject a pharmaceutical composition comprising the engineered T cells and / or engineered natural killer (NK) cells described herein. The cells may be αβ T cells, γδ T cells and / or natural killer (NK) cells expressing a dnTGFβRII 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.

[0247] In embodiments, the activity of endogenous lymphocytes in the subject's body may be modulated by administering the engineered T cells and / or engineered natural killer (NK) 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 engineered natural killer (NK) 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 (NK) cells of the present disclosure to a subject. In embodiments, the other immune cell may be a CD8+ T cell. In embodiments, the other immune cell may be a natural killer T cell. In embodiments, the regulatory T cell may be suppressed by administering the engineered γδ T cells and / or engineered natural killer (NK) cells of the present disclosure to a subject. In embodiments, the regulatory T cell may be a FOX3+ Treg cell. In embodiments, the regulatory T cells may be FOX3-Treg cells. Non-limiting examples of cells whose activity may be regulated by the engineered T cells and / or engineered natural killer (NK) 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 T cells may be αβ T cells or γδ T cells expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide, and optionally a TCR as described herein. In embodiments, the CD8 polypeptide may include a CD8α chain and / or a CD8β chain, and the CD8α chain and / or the CD8β chain may be independently modified or unmodified.

[0248] 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 (NK) 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 (NK) cells may be administered to a subject without co-administration with IL-2. In embodiments, the engineered T cells and / or engineered natural killer (NK) cells may be administered to a subject without co-administration with IL-2, for example, during a procedure such as a bone marrow transplant.

[0249] 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 (NK) cells. The chemotherapeutic agent may be cyclophosphamide, fludarabine, or a combination thereof. The chemotherapeutic agent may include a dose of cyclophosphamide at about 400-600 mg / m2 / day. The chemotherapeutic agent may include a dose of fludarabine at about 10-30 mg / m2 / day.

[0250] 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. 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.

[0251] In embodiments, the patient may be HLA-A*02.

[0252] In embodiments, the patient may be HLA-A*06.

[0253] 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 and / or natural killer (NK) cells.

[0254] In some embodiments, the dose of T cells and / or natural killer (NK) cells is about 0.8-1.2×10 9 The dose of T cells and / or natural killer (NK) cells may be about 0.5×10 8 ~About 10×10 9 The dose of T cells and / or natural killer (NK) cells may be about 1.2 to 3 × 10 9 T cells and / or natural killer (NK) cells, approximately 3–6 × 10 9 T cells and / or natural killer (NK) cells, approximately 10 × 10 9 T cells and / or natural killer (NK) cells, approximately 5 × 10 9 T cells and / or natural killer (NK) cells, approximately 0.1 x 10 9 T cells and / or natural killer (NK) cells, approximately 1 × 10 8 T cells and / or natural killer (NK) cells, approximately 5 × 10 8T cells and / or natural killer (NK) cells, approximately 1.2–6 × 10 9 T cells and / or natural killer (NK) cells, approximately 1–6 × 10 9 T cells and / or natural killer (NK) cells, or approximately 1–8 × 10 9 The cells may be T cells and / or natural killer (NK) cells.

[0255] In some embodiments, the T cells and / or natural killer (NK) cells may be administered in three doses. The dose of T cells may be increased with each administration. The T cells and / or natural killer (NK) cells may be administered by intravenous infusion.

[0256] In some embodiments, the dnTGFβRII and / or CD8 sequences described herein, and related products and compositions, can be used for autologous or allogeneic adoptive cell therapy. In another embodiment, the dnTGFβRII sequences, CD8 sequences, T cells and / or natural killer (NK) cells thereof, and compositions can 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. These documents are incorporated herein by reference in their entirety.

[0257] The present disclosure also provides a population of modified T cells and / or natural killer (NK) cells expressing a dnTGFβRII polypeptide and / or displaying an exogenous CD8 polypeptide and a T cell receptor as described herein, where the modified population of T cells is activated and expanded with a combination of IL-2 and IL-15. In some embodiments, the modified population of T cells and / or natural killer (NK) cells is expanded and / or activated with a combination of IL-2, IL-15 and zoledronate. In some embodiments, the modified population of T cells and / or natural killer (NK) cells is expanded with a combination of IL-2, IL-15, and without zoledronate, while being activated with a combination of IL-2, IL-15 and zoledronate. The present disclosure further provides for the use of other interleukins during activation and / or expansion, such as, for example, IL-12, IL-18, IL-21, and any combination thereof.

[0258] 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.

[0259] Compositions comprising engineered T cells and / or natural killer (NK) 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. Engineered T cells and / or engineered natural killer (NK) cells may also be administered to reduce the likelihood of developing, contracting, or worsening the condition. For therapeutic applications, the effective amount of a population of engineered T cells and / or engineered natural killer (NK) cells 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 (NK) cells engineered to express a dnTGFβRII polypeptide and / or a modified or unmodified CD8 polypeptide described herein, and optionally a TCR as described herein.

[0260] Method of administration One or more populations of engineered T cells and / or natural killer (NK) cells described herein may be administered to a subject in any order or simultaneously. If simultaneously, the multiple engineered T cells and / or natural killer (NK) cells may be provided in one unified form, such as, for example, an intravenous injection, or may be provided in multiple forms, such as multiple intravenous infusions, subcutaneous injections, or pills. The engineered T cells and / or engineered natural killer (NK) cells may be packaged together or separately, in one package, or in multiple packages. One or all of the engineered T cells and / or engineered natural killer (NK) cells may be administered in multiple doses. If not simultaneously, the timing between multiple administrations may vary from about one week, about one month, about two months, about three months, about four months, about five months, about six months, or about a year. In some embodiments, the engineered T cells and / or engineered natural killer (NK) cells may be expanded in vivo in the subject's body after administration to the subject. The engineered T cells and / or engineered natural killer (NK) cells can be frozen to provide cells for multiple treatments with the same cell preparation. The engineered T cells and / or engineered natural killer (NK) cells of the present disclosure, as well as pharmaceutical compositions comprising the same, can be packaged as a kit. The kit can include instructions (e.g., written instructions) for the use of the engineered T cells and / or engineered natural killer (NK) cells, as well as compositions comprising the same.

[0261] A method of treating cancer may include administering to a subject a therapeutically effective amount of engineered T cells and / or engineered natural killer (NK) cells, whereby the cancer is treated. In some embodiments, the therapeutically effective amount of engineered γδ T cells and / or engineered natural killer (NK) cells may be administered for at least about 10 seconds, about 30 seconds, about 1 minute, 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, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or about 1 year. In some embodiments, the therapeutically effective amount of engineered T cells and / or engineered natural killer (NK) cells may be administered for at least about 1 week. In embodiments, a therapeutically effective amount of engineered T cells and / or engineered natural killer (NK) cells may be administered for at least about two weeks.

[0262] The engineered T cells and / or engineered natural killer (NK) 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 can vary. For example, the engineered T cells and / or engineered natural killer (NK) 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 (NK) 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 (NK) cells can begin immediately after the onset of symptoms, within about the first 3 hours from the onset of symptoms, within about the first 6 hours from the onset of symptoms, within about the first 24 hours from the onset of symptoms, within about 48 hours from 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 (NK) cells of the present disclosure may be intravenous. One or more doses of engineered T cells and / or engineered natural killer (NK) cells can be administered as soon as possible after the onset of cancer, infectious disease, immune disease, sepsis, or during 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 (NK) cells can 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 (NK) 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 αβ T cell, a γδ T cell and / or a natural killer (NK) cell expressing a dnTGFβRII polypeptide and / or a CD8 polypeptide as described herein, optionally a TCR as described herein. The engineered T cells and / or natural killer (NK) cells, optionally αβ T cells and / or γδ T cells, expressing the dnTGFβRII polypeptide and / or CD8 polypeptide 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 4 Cells / 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 104 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 7 Cells / 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 8Cells / 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, or approximately 1 x 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.

[0263] 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.

[0264] 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, or 305-365. 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 305-365. 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.

[0265] In some embodiments, the disclosure provides sequences with at least 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 embodiment, the disclosure provides sequences 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 embodiment, the disclosure provides sequences 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 embodiment, the sequence substitutions are conservative substitutions.

[0266] 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.

[0267] 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.

[0268] Amino acid substitutions can be conservative or non-conservative, in some embodiments, the substitutions are conservative, where one amino acid is replaced with another amino acid having similar structural and / or chemical properties.

[0269] Conservative substitutions can include those described in "The Atlas of Protein Sequence and Structure. Vol. 5" by Dayhoff, Natl. Biomedical Research. The contents of this document are incorporated herein by reference in their entirety. For example, in some embodiments, amino acids belonging to one of the following groups can be exchanged for one another to constitute conservative exchanges: Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine (S), threonine (T); Group 2: cysteine ​​(C), serine (S), tyrosine (Y), threonine (T); Group 3: valine (V), isoleucine (I), leucine (L), methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K), arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H); Group 6: aspartic acid (D), glutamic acid (E). In embodiments, conservative amino acid substitutions may be selected from the following: T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and / or T→S.

[0270] Conservative amino acid substitutions can include, for example, the replacement of an amino acid with another amino acid of the same class, such as: (1) non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2) uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp, Glu; and (4) basic: Lys, Arg, His. Other conservative amino acid substitutions can also be made, such as: (1) aromatic: Phe, Tyr, His; (2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, e.g., U.S. Pat. No. 10,106,805, the contents of which are incorporated herein by reference in their entirety).

[0271] Conservative substitutions may be made according to Table A. Methods for predicting tolerance to protein modifications can be found, for example, in Guo et al., Proc. Natl. Acad. Sci., USA, 101(25):9205-9210 (2004), the contents of which are incorporated herein by reference in their entirety. [Table 2]

[0272] The sequences described herein may contain 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acid or nucleotide mutations, substitutions, or deletions. Any one of SEQ ID NOs: 1-97, 256-266, 293, 294, and 305-365 may contain 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. In another embodiment, any one of SEQ ID NOs: 1-97, 256-266, 293, 294, and 305-365 may contain up to 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 mutations, substitutions, or deletions. In one embodiment, the mutations or substitutions may be conservative amino acid substitutions.

[0273] Conservative substitutions in the polypeptides described herein may be those shown under the heading of "conservative substitutions" in Table B. If such substitutions result in alterations in biological activity, more substantial changes shown in Table B as "exemplary substitutions" may be introduced and the products screened, if desired. [Table 3]

[0274] A nucleic acid comprising or consisting of a nucleic acid sequence with "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. For example, due to codon degeneracy, a mutant nucleic acid sequence may occur that encodes a protein identical to the protein encoded by the reference sequence, even if there is a mutation or substitution in the reference nucleic acid sequence. A mutant nucleic acid sequence that encodes a protein having a different sequence from the protein encoded by the reference sequence is also contemplated. A mutant nucleic acid sequence that encodes a conservative amino acid mutation is contemplated. In the case of substitutions, a nucleic acid sequence that is at least, or at least approximately, 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.

[0275] Unless otherwise indicated, all terms used herein have the same meaning to one of ordinary skill in the art.

[0276] In this specification and the appended claims, singular forms such as "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0277] It is understood that the above elements, either individually or together, may find useful application in other types of methods different from those described above. Without further analysis, the foregoing fully reveals the gist of the present disclosure, the rest of which can be readily adapted to various applications by applying current knowledge without omitting those features that, in view of the prior art, adequately constitute the essential characteristics of the summary or specific embodiments of the present disclosure as set forth in the appended claims. The above-described embodiments are presented by way of example only, and the scope of the present disclosure is limited only by the following claims.

[0278] All references cited herein are incorporated by reference as if each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate the reference by reason of prior invention. Additional and further information regarding CD8 polypeptides, TCR polypeptides may be found in U.S. Patent Application No. 17 / 563,599, filed December 28, 2021, entitled "CD8 POLYPEPTIDES, COMPOSITIONS, AND METHODS OF USING THEREOF." The document is incorporated by reference in its entirety.

[0279] Unless otherwise stated herein, the ranges of values ​​set forth herein are intended to be affected as a scheme for referencing each separate value falling within each particular range, including but not limited to the endpoints of the ranges, and each separate value of each range set forth herein is incorporated herein as if it were individually recited.

[0280] The specification may include references to "one embodiment," "an embodiment," "embodiments," "an aspect," "an aspect," or "aspects." Each of these words and phrases is not intended to convey a different meaning from the other words and phrases. These words and phrases may refer to the same embodiment or aspect, may refer to different embodiments or aspects, or may refer to multiple embodiments or aspects. The various embodiments and aspects may be combined in any manner consistent with the present disclosure.

[0281] As used herein, "activation" broadly refers to the state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with the induction of cytokine production and detectable effector functions. The term "activated T cells" specifically refers to T cells that are undergoing proliferation.

[0282] As used herein, "antibody" refers to, but is not limited to, an antibody or immunoglobulin of any isotype, including but not limited to, Fab, Fab', Fab'-SH, (Fab') 2 It broadly refers to antibody fragments that retain specific binding to an antigen, including Fv, scFv, bivalent scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins that contain an antigen-specific targeting region of an antibody and a non-antibody protein. Antibodies are organized into five classes: IgG, IgE, IgA, IgD, and IgM.

[0283] As used herein, "antigen" or "antigenic" refers broadly to a peptide or peptide portion capable of being bound by an antibody, which additionally can induce in an animal the production of an antibody capable of binding an epitope of the antigen. An antigen may have one epitope, or it may have two or more epitopes. A specific reaction, as referred to herein, indicates that an antigen reacts in a highly selective manner with its corresponding antibody, but not with a multitude of other antibodies that may be induced by other antigens.

[0284] As used herein, "chimeric antigen receptor", "CAR" or "CARs" broadly refers to genetically engineered receptors that transfer antigen specificity onto cells, such as T cells, NK cells, macrophages and stem cells. A CAR can include at least one antigen-specific targeting region (ASTR), a hinge or stalk domain, a transmembrane domain (TM), one or more co-stimulatory domains (CSD), and an intracellular activating domain (IAD). In certain embodiments, the CSD is optional. In some embodiments, the CAR is a bispecific CAR, specific for two different antigens or epitopes. After the ASTR specifically binds to the target antigen, the IAD activates intracellular signaling. For example, the IAD can redirect the specificity and reactivity of T cells to a target of choice in a non-MHC-restricted manner, utilizing the antigen-binding properties of antibodies. Non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thus avoiding a major mechanism of tumor escape. Moreover, when expressed in T cells, it is beneficial that they do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0285] As used herein, "cytotoxic T lymphocytes (CTLs)" refers broadly to T lymphocytes that express CD8 on their surface (e.g., CD8+ T cells). Such cells are classified as antigen-experienced "memory" T cells (T M The present invention may be directed to a method for detecting a cell.

[0286] As used herein, "effective amount," "therapeutically effective amount," or "effective amount" refers broadly to the amount of an agent, or the combined amount of two agents, that, when administered to a mammal or other subject for the purpose of treating a disease, is sufficient to affect treatment of that disease. A "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0287] As used herein, "genetically modified" refers broadly to any method of introducing an exogenous nucleic acid into a cell, whether or not the exogenous nucleic acid is integrated into the genome of the cell. As used herein, "genetically modified cell" refers broadly to a cell that contains an exogenous nucleic acid, whether or not the exogenous nucleic acid is integrated into the genome of the cell.

[0288] As used herein, "immune cells" refers broadly to white blood cells derived from hematopoietic stem cells (HSCs) produced in the bone marrow, including, but not limited to, lymphocytes (T cells, B cells, natural killer (NK) (CD3-CD56+) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" includes all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs) and gamma-delta T cells, and NK T cells (CD3+ and CD56+). Those skilled in the art will understand that T cells and / or NK cells, as used throughout this disclosure, may include only T cells, only NK cells, or both T cells and NK cells. In certain exemplary embodiments and aspects provided herein, T cells are activated and transduced. Additionally, T cells are provided in certain exemplary composition embodiments and aspects provided herein. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, NK-T cells, γδ T cells, and neutrophils, which are cells capable of mediating a cytotoxic response.

[0289] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer broadly to mammals, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, dogs, cats, and ungulates (e.g., horses, cows, sheep, pigs, goats).

[0290] As used herein, "peripheral blood mononuclear cells" or "PBMCs" refer broadly to any peripheral blood cell with a round nucleus. PBMCs include, for example, lymphocytes such as T cells, B cells, and NK cells, as well as monocytes.

[0291] As used interchangeably herein, "polynucleotide" and "nucleic acid" refer broadly to any length of nucleotide polymeric form, either ribonucleotide or deoxyribonucleotide.Thus, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or polymers that contain purine and pyrimidine bases, or polymers that contain other natural bases or chemically or biochemically modified non-natural bases or derivatized nucleotides.

[0292] As used herein, "T cells" or "T lymphocytes" refer broadly to thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. Examples of populations of T cells suitable for use in certain embodiments include, but are not limited to, helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, natural killer T cells, T cells expressing αβTCR (αβ T cells), T cells expressing γδTCR (γδ T cells), or any other T cell subset. Other examples of populations of T cells suitable for use in certain embodiments include, but are not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR, which can be further isolated, if desired, by positive or negative selection methods.

[0293] In the present disclosure, the term "homology" refers to the degree of identity between two amino acid sequences, such as, for example, peptide or polypeptide sequences. The aforementioned "homology" is determined by comparing two sequences aligned under optimal conditions to the entire sequence being compared. Such sequence homology can be calculated, for example, by generating an alignment using the ClustalW algorithm. Publicly available sequence analysis software, more specifically, Vector NTI, GENETYX or other tools, are provided by public databases.

[0294] The terms "sequence homology" or "sequence identity" are used interchangeably herein. For the purposes of this disclosure, to determine the percentage of sequence homology or sequence identity of two amino acid sequences or two nucleotide sequences, the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps may be introduced into either of the two sequences being compared. Such alignments may be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over a shorter length, for example, about 5, about 10, about 20, about 50, about 100 or more nucleotides or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the entire reported alignment region.

[0295] The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be achieved using a mathematical algorithm. Those skilled in the art are aware of the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison. In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Addison Wesley). The percentage of sequence identity between two amino acid sequences or the percentage of sequence identity between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for alignment of two sequences. (Needleman, SB and Wunsch, CD (1970) J. Mal. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by algorithms. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of this disclosure, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden, and Bleasby, Trends in Genetics 16, (6) 276-277, emboss.bioinformatics.nl / ). For amino acid sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5.One of skill in the art will understand that all these different parameters will produce slightly different results, but the overall percent identity of the two sequences will not change significantly when using different algorithms.

[0296] After alignment by the above program NEEDLE, the percentage of sequence identity between the query sequence and the sequence of the present disclosure is calculated as follows: the number of corresponding positions in the alignment that show the same amino acid or the same nucleotide in both sequences is divided by the total length of the alignment after subtracting the total number of gaps in the alignment. Identity can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the program output as "longest-identity". The nucleotide and amino acid sequences of the present disclosure can further be used as "query sequences" to perform searches against sequence databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST program of Altschul et al. (1990) J.Mal.Biol.215:403-10 and the XBLAST program (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program with score=100 and word length=12 to obtain nucleotide sequences homologous to the polynucleotides of the present disclosure. BLAST protein searches can be performed using the XBLAST program with score=50 and word length=3 to obtain amino acid sequences homologous to the polypeptides of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0297] As used herein, "T cell receptor (TCR)" refers broadly to a protein receptor on T cells composed of a heterodimer of an alpha (α) and a beta (β) chain, although in some cells the TCR consists of a gamma and a delta (γ / δ) chain. TCRs can be engineered on any cell that contains a TCR, including helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, or gamma delta T cells.

[0298] TCRs are generally found on the surface of T lymphocytes (or T cells) that are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. 95% of T cells have a heterodimer consisting of an alpha and a beta chain, while 5% of T cells have a TCR consisting of a gamma and a delta chain. The association of antigen and MHC with the TCR results in the activation of the T lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors and specialized accessory molecules. In immunology, the CD3 antigen (CD stands for cluster of differentiation) is a protein complex that in mammals is composed of four separate chains (CD3-γ, CD3δ, and two CD3ε) that associate with a molecule known as the T cell receptor (TCR) and the zeta chain to generate an activation signal in T lymphocytes. Together, the TCR, the zeta chain, and the CD3 molecule constitute the TCR complex. The CD3-γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single extracellular immunoglobulin domain. The transmembrane regions of the CD3 chains are negatively charged, allowing these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular tail of the CD3 molecule contains a conserved motif known as the immunoreceptor tyrosine-based activation motif, or ITAM for short, which is essential for the signaling capacity of the TCR.

[0299] As used herein, "treatment," "treating," and the like refer broadly to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or may be therapeutic, in that a disease and / or adverse effects caused by the disease are partially or completely cured. As used herein, "treatment" covers any treatment of a mammalian, e.g., human, disease, including (a) preventing the disease from developing in a subject prone to developing the disease, but not diagnosed as having the disease, (b) inhibiting the disease, e.g., arresting its development, and (c) alleviating the disease, e.g., causing regression of the disease.

[0300] The ability of dendritic cells (DCs) to activate and expand antigen-specific CD8+ T cells may depend on the maturation stage of DCs, and DCs may need to receive a "licensing" signal related to IL-12 production to initiate a cytolytic immune response. In particular, signaling via CD40 ligand (CD40L)-CD40 interactions on CD4+ T cells and DCs may be considered important for DC licensing and induction of cytotoxic CD8+ T cells, respectively. DC licensing may result in upregulation of costimulatory molecules, increased survival, and good DC cross-presentation. This process can be mediated through CD40 / CD40L interactions [SR Bennet et al., "Help for cytotoxic T-cell responses is mediated by CD40 signalling," Nature 393(6684):478-480(1998);SP Schoenberger et al., "T-cell help for cytotoxic T-cell help is mediated by CD40-CD40L interactions," Nature 393(6684):480-483(1998)]. However, CD40 / CD40L-independent mechanisms also exist (CD70, LTβR). Furthermore, a direct interaction between CD40L expressed on DCs and CD40 expressed on CD8+ T cells has also been proposed, providing a possible explanation for the generation of helper-independent CTL responses [S.Johnson et al., "Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T-cell priming by upregulating CD40L on dendritic cells," Immunity 30(2):218-227(2009)]. EXAMPLES

[0301] Example 1 Exemplary Nucleic Acid and Amino Acid Sequences [Table 4]

[0302] The inventors found that various CD8 elements in the vectors resulted in unexpected increases in expression and activity. For example, construct #10 was observed to have lower viral titers than constructs #9b, #11 and #12 (Figure 5A), but construct #10 expressing the CD8αβ heterodimer and TCR was used at the lowest viral volume concentration, e.g., 1.25 μl / 10 6 T cells transduced with IL-16 were more CD8+CD4+TCR+ cells (56.7%, Figure 9B) than cells transduced with construct #9b expressing CD8α and TCR (42.3%, Figure 9A), construct #11 expressing the transmembrane and intracellular domains of CD8α and the CD8αCD8β stalk together with TCR (51.6%, Figure 9C), and construct #12 expressing the transmembrane and intracellular domains of Neural Cell Adhesion Molecule 1 (NCAM1) and the CD8αCD8β stalk together with TCR (14.9%, Figure 9D).

[0303] The inventors also surprisingly found that expression of dnTGFβRII and exogenous TCR in T cells increases the ability of the T cells to maintain killing capacity following multiple stimulation with tumor cells, particularly in the presence of exogenous TGF-β.

[0304] The vector can include any one or more of the nucleic acid sequences of SEQ ID NOs: 72, 73, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 306, 308, 309-312 or 313.

[0305] T cells and / or natural killer cells can be transduced to express any one or more of the nucleic acids of SEQ ID NOs: 72, 73, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 306, 308, 309-312 or 313.

[0306] Some of the elements of the constructs in Table 2 are listed in Table 3. [Table 5] JPEG2025515604000007.jpg193138JPEG2025515604000008.jpg180138JPEG20255156040000 09.jpg182139JPEG2025515604000010.jpg176137JPEG2025515604000011.jpg180138JPEG202 5515604000012.jpg173138JPEG2025515604000013.jpg178138JPEG2025515604000014.jpg1 93138JPEG2025515604000015.jpg193138JPEG2025515604000016.jpg196138JPEG2025515604 000017.jpg193137JPEG2025515604000018.jpg194139JPEG2025515604000019.jpg196139JP EG2025515604000020.jpg196139JPEG2025515604000021.jpg196139JPEG2025515604000022. jpg196139JPEG2025515604000023.jpg196139JPEG2025515604000024.jpg196139JPEG202551 5604000025.jpg196139JPEG2025515604000026.jpg191137JPEG2025515604000027.jpg74138

[0307] The constructs of Table 2 may be an assembly of individual components described in Table 3. The inventors have found that the combinations, orders, and inclusions of the transcriptional enhancers of Table 3 described in Table 2 resulted in unexpected improvements in transfection efficiency, expression levels, and induction of cytotoxic T cell activity, such as unexpected improvements in IL-12 secretion, IFN-γ secretion, TNF-α secretion, Granzyme A secretion, MIP-1a secretion, IP-10 secretion, Granzyme B secretion, and any combination thereof.

[0308] Tumor-associated antigens (TAA) In an MHC class I-dependent immune response, a peptide not only needs to be able to bind to a specific MHC class I molecule expressed by a tumor cell, but also needs to be subsequently recognized by T cells bearing a specific T cell receptor (TCR).

[0309] In order for a protein to be recognized by T lymphocytes as a tumor-specific or tumor-associated antigen and used therapeutically, certain prerequisites must be met. The antigen must be expressed primarily by tumor cells and not expressed or expressed in relatively low amounts in normal healthy tissues. In some embodiments, the peptide may be over-presented by tumor cells compared to normal healthy tissues. It is further desirable that the respective antigen is not present only in certain tumors, but is present in high concentration (e.g., number of copies of the respective peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transformation of normal cells into tumor cells by their functions, such as cell cycle control or apoptosis inhibition. Furthermore, granule targets of proteins directly responsible for transformation may also be upregulated and therefore indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets for vaccination approaches. Singh-Jasuja et al.Cancer Immunol.Immunother.53(2004):187-195. Epitopes are present in the amino acid sequence of an antigen, making the peptide an "immunogenic peptide," and are derived from tumor-associated antigens that elicit T cell responses both in vitro and in vivo.

[0310] Any peptide that can bind to MHC molecule can function as T cell epitope. For induction of T cell response, TAA must be presented to T cell with corresponding TCR, and host must not have immune tolerance to this particular epitope. Exemplary tumor-associated antigens (TAA) that can be used with the CD8 polypeptide described herein are disclosed herein. [Table 6] JPEG2025515604000029.jpg63127

[0311] Example 2 CD8α molecule and dnTGFβRII polypeptide CD8 Polypeptide A CD8α homodimer (CD8αα) can be composed of two α subunits held together by two disulfide bonds at the stalk region. Figure 1 shows a CD8α polypeptide, e.g., SEQ ID NO: 258 (CD8α1), which contains five domains: (1) one signal peptide (-21 to -1), e.g., SEQ ID NO: 6, (2) one Ig-like domain-1 (1 to 115), e.g., SEQ ID NO: 1, (3) one stalk region (116 to 160), e.g., SEQ ID NO: 260, (4) one transmembrane (TM) domain (161 to 188), e.g., SEQ ID NO: 3, and (5) one cytoplasmic tail (Cyto) (189 to 214) containing an lck-binding motif, e.g., SEQ ID NO: 4. Another example of a CD8α subunit, e.g., CD8α2 (sequence number 259), differs from CD8α1 at position 112, where CD8α2 contains a cysteine ​​(C) while CD8α1 contains a tyrosine (Y).

[0312] Modified CD8 Polypeptides Unlike CD8α polypeptides, e.g., CD8α1 (SEQ ID NO:258) and CD8α2 (SEQ ID NO:259), modified CD8α polypeptides, e.g., m1CD8α (SEQ ID NO:7) and m2CD8α (SEQ ID NO:262), may contain additional regions, e.g., sequence stretches from CD8β polypeptides. In several embodiments, SEQ ID NO:2 or variants thereof are used with CD8α polypeptides. In other embodiments, portions of the CD8α polypeptide, e.g., SEQ ID NO:260, are removed or not included in the modified CD8 polypeptides described herein. Figure 2 shows a sequence alignment between CD8α1 (SEQ ID NO:258) and m1CD8α (SEQ ID NO:7). Figure 3 shows a sequence alignment between CD8α2 (SEQ ID NO:259) and m2CD8α (SEQ ID NO:262), in which cysteine ​​substitutions are indicated with arrows. The stalk region is shown in a box.

[0313] Cells expressing modified CD8 showed improved functionality in terms of cytotoxicity and cytokine responses compared to TCR-transduced T cells expressing the original CD8.

[0314] dnTGFβRII polypeptide The dnTGFβRII polypeptide may comprise or consist of the appropriate amino acid sequence specified herein. The dnTGFβRII polypeptide may be encoded by one or more nucleic acids comprising or consisting of the appropriate nucleic acid sequence specified herein. For example, in some embodiments, dnTGFβRII variant 1 (dnTGFβRIIvar1) and / or dnTGFβRII variant 2 (dnTGFβRIIvar2) are provided, which are examples of dnTGFβRII polypeptides. The dnTGFβRIIvar1 may comprise or consist of SEQ ID NO:305 and may be encoded by a nucleic acid comprising or consisting of SEQ ID NO:306. The dnTGFβRIIvar1 may comprise or consist of SEQ ID NO:307 and may be encoded by a nucleic acid comprising or consisting of SEQ ID NO:308. In embodiments, the dnTGFβRII polypeptide encoded by a nucleic acid that also comprises and / or encodes the MSCV promoter and WPRE may be encoded by a nucleic acid comprising or consisting of SEQ ID NO:312 or SEQ ID NO:313.

[0315] Example 3 Lentiviral Vectors The lentiviral vectors used herein contain several elements that enhance vector function, including a central polypurine tract for improved replication and nuclear import, a promoter (SEQ ID NO: 263) derived from the murine stem cell virus (MSCV) to reduce vector silencing in some cell types, and a woodchuck hepatitis virus posttranscriptional response element (WPRE) (SEQ ID NO: 264) for improved transcription termination, and the backbone is a deleted 3'-LTR self-inactivating (SIN) vector design to improve safety, sustained gene expression and anti-silencing properties. Yang et al. Gene Therapy (2008) 15, 1411-1423.

[0316] In some embodiments, the vectors, constructs, or sequences described herein contain a mutant form of the WPRE. In some embodiments, the sequences or vectors described herein contain a mutation in WPRE version 1, e.g., WPREmut1 (SEQ ID NO: 256), or WPRE version 2, e.g., WPREmut2 (SEQ ID NO: 257). Construct #9 and construct #9b represent two LV production batches and are the same constructs containing SEQ ID NO: 257 as WPREmut2, but the difference between construct #9 and construct #9b is in the titer consistent with Table 4. In some embodiments, the WPRE mutant contains at most 1 mutation, at most 2 mutations, at most 3 mutations, at least 4 mutations, or at most 5 mutations. In some embodiments, the vectors, constructs, or sequences described herein do not contain a WPRE. In some aspects, the WPRE sequence described in US2021 / 0285011 (the contents of which are incorporated by reference in their entirety) can be used with the vectors, sequences, or constructs described herein.

[0317] In some embodiments, the vector, construct or sequence described herein does not contain X protein promoter.The WPRE mutant described herein does not express X protein.It is concluded that WPRE promotes the accumulation of mRNA and promotes the transport of mRNA from nucleosome to cytoplasm to promote the translation of transgene mRNA.

[0318] Various lentiviral vector designs were generated to obtain optimal co-expression levels of TCRαβ, mCD8α (e.g., m1CD8α (which can be encoded by SEQ ID NO:7 (SEQ ID NO:311)) and m2CD8α (SEQ ID NO:262)) and CD8β (e.g., any one of CD8β1-7 (SEQ ID NOs:8-14)), and / or dnTGFβRII (e.g., any one or both of SEQ ID NOs:305 or 307) in transduced CD4+ T cells, CD8+ T cells and / or γδ T cells. T cells may be transduced with two separate lentiviral vectors (2-in-1), for example one expressing TCRα and TCRβ and the other expressing mCD8α and CD8β to co-express the TCRαβ and CD8αβ heterodimer, or one expressing TCRα and TCRβ and the other expressing mCD8α to co-express the TCRαβ and mCD8α homodimer. Alternatively, T cells may be transduced with one lentiviral vector (4-in-1) that co-expresses TCRα, TCRβ, mCD8α and CD8β to co-express the TCRαβ and CD8αβ heterodimer. In the 4-in-1 vector, the nucleotides encoding the TCR α chain, TCR β chain, mCD8 α chain, and CD8 β chain may be shuffled in various orders, for example, from 5' to 3', in the following order: TCR α-TCR β-mCD8 α-CD8 β, TCR α-TCR β-CD8 β-mCD8 α, TCR β-TCR α-mCD8 α-CD8 β, TCR β-TCR α-CD8 β-mCD8 α, mCD8 α-CD8 β-TCR α-TCR β, mCD8 α-CD8 β-TCR β-TCR α, CD8 β-mCD8 α-TCR α-TCR β, and CD8 β-mCD8 α-TCR β-TCR α. The various 4-in-1 vectors so generated may be used to transduce CD4+ T cells, CD8+ T cells, and / or γδ T cells, and then the level of co-expression of TCRαβ / mCD8α / CD8β by the transduced cells may be measured using techniques known in the art, such as, for example, flow cytometry.Similarly, T cells may be transduced with a single lentiviral vector (3-in-1) co-expressing TCRα, TCRβ, and mCD8α (e.g., m1CD8α and m2CD8α) to co-express TCRαβ and mCD8α homodimers. In the 3-in-1 vector, the nucleotides encoding the TCRα, TCRβ, and mCD8α chains may be shuffled in various orders, such as TCRα-TCRβ-mCD8α, TCRβ-TCRα-mCD8α, mCD8α-TCRα-TCRβ, and mCD8α-TCRβ-TCRα. The various 3-in-1 vectors so generated may be used to transduce CD4+ T cells, CD8+ T cells, and / or γδ T cells, followed by measuring the level of co-expression of TCRαβ / mCD8α by the transduced cells using techniques known in the art. Similarly, one or more dnTGFβRII polypeptides may be encoded by a separate vector, or may be encoded by a vector that also encodes one or more CD8 and / or one or more TCRs. Vectors may be generated that co-express any combination of TCRα, TCRβ, mCD8α, CD8β, and / or dnTGFβRII, in any order.

[0319] To generate lentiviral vectors co-expressing TCRαβ and mCD8α and / or CD8β, nucleotides encoding the Furin linker (GSG or SGSG (SEQ ID NO: 266))-2A peptide may be placed between the TCRα and TCRβ chains, between the mCD8α and CD8β chains, and between the TCR and CD8 chains, and / or between CD8 or TCR and dnTGFβRII to allow highly efficient gene expression. 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).

[0320] Lentiviral vectors may contain post-transcriptional regulatory elements (PREs), such as WPRE (SEQ ID NO: 264), WPREmut1 (SEQ ID NO: 256), or WPREmut2 (SEQ ID NO: 257), which may function to enhance expression of one or more transgenes by increasing both nuclear and cytoplasmic mRNA levels. One or more regulatory elements, including mouse RNA transport element (RTE), simian retrovirus type 1 (SRV-1) constitutive transport element (CTE), and human heat shock protein 70 5' untranslated region (Hsp70 5'UTR), may also be used and / or in combination with WPRE to increase transgene expression. WPREmut1 and WPREmut2 do not express the X protein, but still act to enhance translation of transgene mRNA.

[0321] Lentiviral vectors may be pseudotyped with RD114TR (e.g., SEQ ID NO: 97), a chimeric glycoprotein that includes the extracellular and transmembrane domains of feline endogenous virus (RD114) fused to the cytoplasmic tail (TR) of murine leukemia virus. Other viral envelope proteins may be used, such as, for example, VSV-G env, MLV 4070A env, RD114 env, chimeric envelope protein RD114pro, baculovirus GP64 env, or GALV env, or derivatives thereof. RD114TR variants that include at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% relative to SEQ ID NO: 97 are also provided.

[0322] For example, Figure 4 shows exemplary vectors including two 4-in-1 vectors, e.g., constructs #10 and #2, co-expressing TCR (TCR alpha and TCR beta chains), CD8 alpha and CD8 beta; three 3-in-1 vectors, e.g., constructs #1 and #9, expressing TCR and CD8 alpha, two 3-in-1 vectors, e.g., constructs #11 and #12, expressing TCR and m1CD8 alpha (SEQ ID NO: 7), and construct #8, expressing only TCR. To improve transcription termination, wild-type WPRE (WPRE) (SEQ ID NO: 264) is included in constructs #1, #2, and #8, and WPREmut (SEQ ID NO: 257) is included in constructs #9, #10, #11, and #12.

[0323] As another example, FIG. 68 shows an exemplary vector provided in multiple embodiments. For example, constructs C-L shown in FIG. 68 are provided in multiple embodiments. The TCR in FIG. 68 can be, for example, a TCRβ fused directly or indirectly to a TCRα with or without a linker and / or other elements therebetween, or a TCRα fused directly or indirectly to a TCRβ with or without a linker and / or other elements therebetween. The dnTGFβRII polypeptide in FIG. 68 can include or consist of, as non-limiting examples, dnTGFβRIIvar1 (which is SEQ ID NO: 305 and can be encoded by SEQ ID NO: 306), and / or dnTGFβRIIvar2 (which is SEQ ID NO: 307 and can be encoded by SEQ ID NO: 308). The CD8α, CD8β, and TCR polypeptides in FIG. 68 can be, independently, as described herein, and / or can be, independently, modified or unmodified. In embodiments, CD8α may comprise or consist of CD8α1 (SEQ ID NO:258, which may be encoded by SEQ ID NO:310). In embodiments, CD8α may comprise or consist of m1CD8α (SEQ ID NO:7, which may be encoded by SEQ ID NO:311). In embodiments, CD8β may comprise or consist of CD8β1 (SEQ ID NO:8, which may be encoded by SEQ ID NO:309).

[0324] Further exemplary constructs (constructs #13-#19 and #21-#26) are described in Table 2 above. In particular, constructs #13, #14, and #16 are 4-in-1 constructs that co-express TCR, CD8α, and CD8β3 with various signal peptide combinations (SEQ ID NO:6 [WT CD8α signal peptide], SEQ ID NO:293 [WT CD8β signal peptide], and SEQ ID NO:294 [S19 signal peptide]) and various element orders. Constructs #15 and #17 are 4-in-1 constructs that co-express TCR, CD8α, and CD8β5. Construct #15 contains WT CD8α signal peptide (SEQ ID NO:6) and WT CD8β signal peptide (SEQ ID NO:293), while construct #17 contains S19 signal peptide (SEQ ID NO:294) at the N-terminus of both CD8α and CD8β5. Construct #21 is a 4-in-1 construct co-expressing TCR, CD8α, and CD8β2, including the WT CD8α signal peptide (SEQ ID NO:6) and the WT CD8β signal peptide (SEQ ID NO:293). Construct #18 is a variant of construct #10, in which the WT signal peptides of CD8α and CD8β1 (SEQ ID NO:6 and 293, respectively) have been replaced with the S19 signal peptide (SEQ ID NO:294). Construct #19 is a variant of construct #11, in which the WT CD8α signal peptide (SEQ ID NO:6) has been replaced with the S19 signal peptide (SEQ ID NO:294). Construct #22 is a variant of construct #11, in which the transmembrane and intracellular domains of CD4 have been fused to the C-terminus of the CD8β stalk sequence in place of the transmembrane and intracellular domains of CD8α. Construct #25 is a variant of construct #22 in which the CD8β stalk sequence (SEQ ID NO:2) has been replaced with the CD8α stalk sequence (SEQ ID NO:260).

[0325] Example 4 Vector screening (constructs #1, #2, #8, #9, #10, #11 and #12) Viral titer Figure 5A shows the viral titers of constructs #1, #2, #8, #9, #10, #11 and #12. Table 5 shows the viral titers of constructs #9, #10, #11 and #12, as well as the lentiviral P24 ELISA data. [Table 7]

[0326] For construct 12, NCAMfu refers to the NCAMFusion protein expressing a modified CD8a extracellular domain and neural cell adhesion molecule 1 (CD56) intracellular domain.

[0327] For Table 5, the WPREmut2 portion refers to SEQ ID NO:257.

[0328] T cell manufacturing activation FIG. 6 shows PBMCs (approximately 9×10 8 Figure 7A shows that PBMCs (cells) were thawed and allowed to rest. The cells were activated in anti-CD3 and anti-CD28 antibody-coated bags (AC290) in the presence of serum. Activation markers, such as CD25, CD69, and human low-density lipoprotein receptor (H-LDL-R), were present on CD8+ and CD4+ cells and were subsequently measured. Figure 7A shows that the percentages of CD3+CD8+CD25+ cells, CD3+CD8+CD69+ cells, and CD3+CD8+H-LDL-R+ cells were increased after activation (Post-A) compared to before activation (Pre-A). Similarly, Figure 7B shows that the percentages of CD3+CD4+CD25+ cells, CD3+CD4+CD69+ cells, and CD3+CD4+H-LDL-R+ cells were increased after activation (Post-A) compared to before activation (Pre-A). These results support the activation of PBMCs.

[0329] Transduction FIG. 6 shows that on day +1, activated PBMCs were cultured in serum-free medium at approximately 5×10 6 Cells / well were transduced in G-Rex® 6-well plates. The amount of virus used for transduction is shown in Table 6. [Table 8]

[0330] amplification Figure 6 shows that on day +2, transduced PBMCs were expanded in the presence of serum. On day +6, cells were harvested and cryopreserved for subsequent analysis, e.g., FACS-Dextramer and vector copy number (VCN). Figures 8A and 8B show the day +6 fold expansion of transduced T cell products obtained from donor #1 and donor #2, respectively. Cell viability was greater than 90% on day +6. Characterization of T cell products

[0331] Cell counts, FACS-dextramers, and vector copy numbers (VCN) were determined. Tetramer panels can include live / dead cells, CD3, CD8α, CD8β, CD4, and peptide / MHC tetramers, such as PRAME-004 (SLLQHLIGL) (SEQ ID NO: 147) / MHC tetramer. FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+tetramer (Tet)+ and CD8+Tet+.

[0332] Figures 9A, 9B, 9C and 9D show representative flow plots of cells obtained from donor #1, showing the % CD8, CD4 and PRAME-004 / MHC tetramer (Tet) percentages of cells transduced with constructs #9b, #10, #11 or #12, respectively.

[0333] Figure 10 shows the 1×10 6The percentages of CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8 (TCR), #9, #10, #11 or #12 at 1.25 μl, 2.5 μl or 5 μl per cell are shown. These results show that the percentages of CD8+CD4+ cells obtained by transduction with vectors expressing CD8α and TCR together with wild-type WPRE (construct #1) and WPREmut2 (construct #9) were higher than those transduced with constructs #10, #11 or #12. Construct #8 (TCR only) served as a negative control. Figure 11 shows the percentages of CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with 1×10 6 The % Tet percentage of CD8+CD4+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8(TCR), #9, #10, #11 and #12 at 1.25 μl, 2.5 μl or 5 μl per cell is shown. From these results, the % Tet percentage of CD8+CD4+ cells appeared to be comparable between cells transduced with constructs #9, #10 and #11 and higher than cells transduced with construct #12. FACS analysis was gated on viable singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+.

[0334] Figure 12 shows the 1×10 6 The Tet MFI of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8(TCR), #9, #10, #11 or #12 at 1.25 μl, 2.5 μl or 5 μl per cell is shown. These results show that the tetramer MFI in CD4+CD8+Tet+ differs between donors. FIG. 13 shows the Tet MFI of CD8+CD4+Tet+ cells from 1×10 6CD8α MFI of CD8+CD4+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per cell. These results show that CD8α MFI in cells transduced with vectors expressing CD8α and TCR together with wild-type WPRE (construct #1) and WPREmut2 (construct #9) was higher than those transduced with other constructs. 5 μl / 10 6 The transduction volume was 1.25 μl / 10 6 and 2.5μl / 10 6 FACS analysis was gated on viable singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+, followed by Tet MFI / CD8α MFI.

[0335] Figure 14 shows the 1×10 6 Shown are CD8 frequencies (% of CD8+CD4- of CD3+) in cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per cell. These results showed no difference in CD8 frequency between constructs. Non-transduced (NT) served as a negative control. Figure 15 shows the CD8 frequencies (% of CD8+CD4- of CD3+) in cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per cell. 6 The percentage of CD8+Tet+ cells (of CD3+) from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8(TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per cell is shown. These results show that the frequency of CD8+Tet+ (of CD3+) in cells transduced with constructs #9, #11, and #12 is higher than in cells transduced with construct #10. FACS analysis was gated on viable singlets, followed by CD3+, followed by CD8+CD4−, followed by CD8+Tet+.

[0336] Figure 16 shows the 1×10 6 Figure 17 shows the Tet MFI of CD8+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8(TCR), #9, #10, #11 or #12 at 1.25 μl, 2.5 μl or 5 μl per cell. These results show that the tetramer MFI of CD8+tet+ cells differs between donors. Figure 17 shows the Tet MFI of CD8+tet+ cells from 1×10 6 CD8α MFI of CD8+Tet+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8(TCR), #9, #10, #11 or #12 at 1.25 μl, 2.5 μl or 5 μl per cell is shown. These results show that the CD8α MFI of CD8+Tet+ is comparable between cells transduced with different constructs. FACS analysis was gated on viable singlets, followed by CD3+, followed by CD4+CD8+, followed by CD4+CD8+Tet+, followed by Tet MFI / CD8α MFI.

[0337] Figure 18 shows the 1×10 6 The percentage of Tet+ CD3+ cells from donor #1 (upper panel) and donor #2 (lower panel) transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per 1x10 cells is shown. These results show that the frequency of CD3+Tet+ in cells transduced with constructs #9 or #11 is higher than in cells transduced with constructs #10 or #12. 6 A higher percentage of Tet+CD3+ cells appeared in cells transduced with construct #10 (WPREmut2) than in cells transduced with construct #2 (wild type WPRE) at 5 μl per cell. FACS analysis was gated on viable singlets, followed by CD3+, followed by CD3+, followed by Tet+.

[0338] Figure 19 (top panel) shows the 1×10 6Figure 19 shows the vector copy number (VCN) of cells from donor #1 transduced with constructs #1, #2, #8 (TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per cell. These results indicate that the VCN was higher in cells transduced with constructs #11 or #12 than in cells transduced with constructs #9 or #10 (presumably due to higher titers). Figure 19 (lower panel) shows that the VCN was higher in cells transduced with constructs #11 or #12 than in cells transduced with constructs #9 or #10. 6 The CD3+Tet+ / VCN of cells from donor #1 transduced with constructs #1, #2, #8(TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per cell is shown. These results show that the CD3+Tet+ / VCN in cells transduced with construct #9 is higher than that in cells transduced with constructs #10, #11, or #12.

[0339] In summary, these results show that (1) by transducing cells with vectors expressing CD8α and TCR together with wild-type WPRE (construct #1) or WPREmut2 (construct #9), a higher percentage of CD8+CD4+ cells was obtained than cells transduced with constructs #10, #11, or #12, (2) the percentage of CD8+CD4+Tet+ cells was comparable between cells transduced with the various constructs, and (3) there was a dose-dependent increase in the percentage of tetramers, e.g., at 1x10 6 5 μl per cell, 1x10 6(4) the % of CD8+ cells was comparable between cells transduced with the various constructs; (5) the frequency of CD8+Tet+ was higher in cells transduced with constructs #9, #11 or #12 than in cells transduced with construct #10; (6) the frequency of CD3+Tet+ was higher in cells transduced with constructs #9 or #11 than in cells transduced with constructs #10 or #12; (7) the VCN was higher in cells transduced with constructs #11 or #12 than in cells transduced with constructs #9 or #10; and (8) the CD3+tet+ / VCN was higher in cells transduced with constructs #9 than in cells transduced with constructs #10, #11 or #12.

[0340] T cell products transduced with viral vectors expressing transgenic TCRs and modified CD8 coreceptors showed superior cytotoxicity and increased cytokine production against target-positive cell lines.

[0341] Example 5 Tumor death assay Figures 20A-C 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 and A375 at 50 copies per cell). [Table 9]

[0342] Construct #9 loses tumor control over time against low target antigen expressing A375 cell line

[0343] Example 6 IFNγ secretion assay IFNγ secretion was measured in UACC257 and A375 cell lines. IFNγ secretion in response to UACC257 cell line was comparable between constructs. However, in A375 cell line, construct #10 showed higher IFNγ secretion than other constructs. IFNγ was quantified in the supernatant from Incucyte plates. Figures 21A-B

[0344] FIG. 22 shows an exemplary experimental design for evaluating dendritic cell (DC) maturation and cytokine secretion by PBMC-derived T cell products in response to exposure to target-positive tumor cell lines UACC257 and A375.

[0345] IFNγ secretion in response to A375 is increased in the presence of immature DCs (iDCs). In triple cocultures with iDCs, IFNγ secretion is higher in construct #10 compared to the other constructs. However, when comparing constructs #9 and #11, which express wild-type and modified CD8 coreceptor sequences, respectively, T cells transduced with #11 strongly induced a cytokine response measured as IFNγ quantified in the culture supernatant of three-dimensional cocultures using donor D600115, E:T:iDC::1:1 / 10:1 / 4. Figure 23A-B

[0346] 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 the other constructs. IFNγ was quantified in supernatants from DC co-cultures D150081, E:T:iDC::1:1 / 10:1 / 4. Figures 24A-B

[0347] IFNγ secretion in response to UACC257 is increased in the presence of iDCs. In triple coculture 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 coreceptor sequences, respectively, T cells transduced with construct #11 strongly induced cytokine responses measured as IFNγ quantified in culture supernatants of three-dimensional cocultures using donor D600115, E:T:iDC::1:1 / 10:1 / 4 (Figure 25A-B). These results demonstrate that T cell products coexpressing transgenic TCR and CD8 coreceptor (αβ heterodimer or modified CD8α homodimer) are capable of licensing DCs in the microenvironment via antigen cross-presentation, and thus may mount stronger antitumor responses and modulate the tumor microenvironment.

[0348] Example 7 Vector screening (constructs #13 to #21) Viral titer Figure 5B shows the viral titers of constructs #10, #10n (new batch), #11, #11n (new batch), #13 to #21, and TCR only as a control.

[0349] T cell manufacturing activation Figure 26 shows that PBMCs obtained from two HLA-A02+ donors (Donor #1 and Donor #2) were thawed and rested on day +0. The cells were activated in anti-CD3 and anti-CD28 antibody-coated bags (AC290) in the absence of serum. Activation markers, such as CD...

Claims

1. (i) Sequence ID 307, 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 307, (ii) Sequence ID 305, 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 305, A nucleic acid that encodes a polypeptide containing both (iii)(i) and (ii).

2. (i) Sequence ID 306, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID 306, (ii) Sequence ID 308, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID 308, The nucleic acid according to claim 1, comprising both (iii)(i) and (ii).

3. The nucleic acid according to claim 1, further comprising a nucleic acid sequence encoding at least one TCR polypeptide, at least one CD8 polypeptide, or at least one TCR polypeptide and at least one CD8 polypeptide.

4. (i) Sequence ID 312, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence ID 312, (ii) Sequence ID 313, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to Sequence ID 313, The nucleic acid according to claim 1, comprising both (iii)(i) and (ii).

5. (a) (i) a T cell receptor (TCR) comprising the α chain and β chain of TCR, and a CD8 polypeptide comprising the α chain and β chain of CD8, or (ii) a TCR comprising the α chain and β chain of TCR, and a CD8 polypeptide comprising the α chain of CD8 but not the β chain of CD8, and (b) A nucleic acid sequence encoding at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide, The α chain and β chain of the TCR are 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 The TCRα chain and the TCRβ chain are selected from sequence numbers 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, 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 α chain of CD8 is sequence numbers 7, 258, 259, 262, or a variant thereof. The nucleic acid according to claim 3, wherein the β-chain of CD8 is sequence number 8, 9, 10, 11, 12, 13, or 14.

6. The nucleic acid according to claim 3, wherein the nucleic acid sequence encoding the at least one TCR polypeptide and the at least one CD8 polypeptide is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NOs: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301.

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

8. The vector according to claim 7, further comprising a post-transcriptional regulatory element (PRE) selected from woodchuck PRE (WPRE), woodchuck PRE (WPRE) variant 1, woodchuck PRE (WPRE) variant 2, or hepatitis B virus (HBV) PRE (HPR) (SEQ ID NO: 366).

9. The vector according to claim 7, wherein the vector is a viral vector or a non-viral vector.

10. The vector according to claim 7, 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.

11. The aforementioned vector includes N1, N2, N3, N4, N5, L1, L2, L3, and L4 in any order, N1 comprises and may or may not comprise a nucleic acid sequence encoding the CD8β chain as defined in claim 5. N2 comprises a nucleic acid sequence encoding the CD8α chain as defined in claim 5, N3 comprises a nucleic acid sequence encoding the TCRβ chain as defined in claim 5, N4 comprises a nucleic acid sequence encoding the TCRα chain as defined in claim 5, and N5 comprises a nucleic acid sequence encoding at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide as defined in claim 5, and The vector according to claim 7, wherein each of L1 to L4 comprises a nucleic acid sequence encoding at least one linker, each of L1 to L4 is independently identical or different, and each of L1 to L4 is independently present or absent.

12. 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 11, including the vector described in claim 11.

13. (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) 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 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, The vector according to claim 11, wherein the 2A peptide is P2A (SEQ ID NO: 93), T2A (SEQ ID NO: 94), E2A (SEQ ID NO: 95), or F2A (SEQ ID NO: 96).

14. The vector according to claim 7, wherein the vector further comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

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

16. 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.

17. The method according to claim 16, further comprising isolating CD4+CD8+ T cells from the transduced T cells and / or natural killer cells, and amplifying the isolated CD4+CD8+ transduced T cells.

18. The method according to claim 16, wherein the blood sample comprises peripheral blood mononuclear cells (PMBCs).

19. 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.

20. The activation and / or amplification is carried out in the presence of a combination of IL-2 and IL-15, or The method according to claim 16, wherein the activation and / or amplification is carried out using zoledronate in the presence of a combination of IL-2 and IL-15.

21. A method for increasing the persistence, lifespan, functionality, naivety, ability to kill antigen-presenting cells, IFN-γ secretion, or 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 or natural killer (NK) cells, A method wherein the persistence, lifespan, functionality, naivety, ability to kill antigen-presenting cells, IFN-γ secretion, or a combination thereof of the transduced T cells and / or natural killer (NK) cells are increased compared to control cells.

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

23. The method according to claim 21, 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.

24. The method according to claim 21, wherein the persistence, lifespan, functionality, naivety, ability to kill antigen-presenting cells, IFN-γ secretion, or combination thereof of the transduced T cells and / or natural killer (NK) cells and the control cells is 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 more challenges using antigen-presenting cells.

25. The transduced T cells and / or natural killer (NK) cells, and the control cells, are cultured in the presence of exogenous TGF-β, or The method according to claim 21, wherein the transduced T cells and / or natural killer (NK) cells, and the control cells, are cultured in the presence of exogenous TGF-β and TGF-β1.

26. The exogenous TGF-β is added to the cell culture daily. The exogenous TGF-β and TGF-β1 are added to the cell culture daily. The exogenous TGF-β is added to the cell culture daily, and is added to the cell culture at the same time as or multiple times when tumor cells are added to the cell culture, or The exogenous TGF-β and TGF-β1 are added to the cell culture daily, and are added to the cell culture simultaneously with or multiple times when tumor cells are added to the cell culture. The method according to claim 25.

27. The method according to claim 21, wherein the antigen-presenting cells present an antigen on their cell surface, and the transduced T cells and / or natural killer (NK) cells, as well as the control cells, have the ability to kill the antigen-presenting cells.

28. The method according to claim 27, wherein the antigen comprises a peptide in a complex with an MHC molecule on the cell surface.

29. A T cell or natural killer (NK) cell transduced with the nucleic acid described in Claim 1.

30. (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) a TCR comprising the α chain and β chain of the TCR, and a CD8 polypeptide comprising the α chain of CD8 but not the β chain of CD8, and (b) at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide, The α chain and β chain of the TCR are 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 The TCRα and TCRβ chains are selected from 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, and in particular the TCRα and TCRβ 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 α chain of CD8 is sequence numbers 7, 258, 259, 262, or a variant thereof. If present, the β chain of CD8 is sequence number 8, 9, 10, 11, 12, 13, or 14, or (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) a TCR comprising the α chain and β chain of the TCR, and a CD8 polypeptide comprising the α chain of CD8 but not the β chain of CD8, and (b) at least one dominant-negative TGFβ receptor II (dnTGFβRII) polypeptide, The α chain and β chain of the TCR are 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 The TCRα and TCRβ chains are selected from 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, and in particular the TCRα and TCRβ 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 α chain of CD8 is sequence numbers 7, 258, 259, 262, or a variant thereof. If present, the β chain of CD8 is encoded by a nucleic acid sequence that is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14, and also includes the MSCV promoter and WPRE sequence, and comprises a dnTGFβRII polypeptide selected from (i) SEQ ID NO: 312, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 312, or (ii) SEQ ID NO: 313, or a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

313. The T cell and / or natural killer (NK) cell according to claim 29.

31. The T cell or natural killer (NK) cell according to claim 30, wherein the cell is one or more selected from the group consisting of αβ T cells, γδ T cells, natural killer T cells and natural killer (NK) cells.

32. The T cell or natural killer (NK) cell according to claim 31, wherein the αβ T cell is a CD4+ T cell or a CD8+ T cell.

33. The T cell or natural killer (NK) cell according to claim 31, wherein the γδ T cell is a Vγ9Vδ2+ T cell.

34. The nucleic acid according to claim 1, wherein the nucleic acid is isolated, recombinant, or both isolated and recombinant.

35. A composition comprising T cells and / or natural killer (NK) cells as described in claim 29.

36. The composition according to claim 35, wherein the composition is a pharmaceutical composition.

37. The composition according to claim 35, wherein the composition further comprises an adjuvant, an excipient, a carrier, a diluent, a buffer, a stabilizer, or a combination thereof.

38. 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 37, 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.

39. A therapeutic pharmaceutical comprising T cells and / or natural killer (NK) cells according to any one of claims 29 to 34, or a composition according to any one of claims 35 to 38.

40. A pharmaceutical agent for cancer treatment comprising T cells and / or natural killer (NK) cells according to any one of claims 29 to 34, or a composition according to any one of claims 35 to 38.

41. A therapeutic agent comprising T cells and / or natural killer (NK) cells according to any one of claims 29 to 34, or a composition according to any one of claims 35 to 38.

42. A cancer treatment agent comprising T cells and / or natural killer (NK) cells according to any one of claims 29 to 34, or a composition according to any one of claims 35 to 38.

43. Use of T cells and / or natural killer (NK) cells according to any one of claims 29 to 34, or the composition according to any one of claims 35 to 38, in the manufacture of a pharmaceutical product for the treatment of cancer.

44. The use according to claim 43, 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.

45. A cancer treatment agent and / or cancer immune response inducer comprising the composition according to any one of claims 35 to 38, 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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