The present disclosure relates to cells capable of co-expressing T cell receptors (“TCR”) together with membrane-bound IL-15 polypeptides and/or CD8 polypeptides and the use thereof in adoptive cellular therapy. The present disclosure further provides for modified IL-15, IL-15Rα, IL-15/IL-15Rα fusion polypeptide, and IL-15Rα/IL-15 fusion polypeptide sequences, vectors, and associated methods of making and using the same. The present disclosure further provides for modified CD8 sequences, vectors, and associated methods of making and using the same.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

A Sequence Listing is submitted herewith as an XML file named “3000011-035001_Sequence_Listing_ST26” created on 30 Oct. 2024 and having a size of 758,927 bytes as permitted under 37 C.F.R. § 1.821 (c). The material in the aforementioned file is hereby incorporated by reference in its entirety.

BACKGROUND

Field

The present disclosure relates to cells capable of co-expressing one or any combination of T cell receptors (“TCR”), CD8 polypeptides, and/or membrane-bound interleukin 15 (IL-15) and the use thereof in adoptive cellular therapy (“ACT”). The present disclosure further provides for modified CD8 sequences, IL-15 sequences, IL-15 receptor a (IL-15-Rα) sequences, IL-15/IL-15Rα fusion polypeptides, vectors, compositions, transformed cells, and associated methods thereof. Specifically, the present disclosure relates to sequences encoding specific TCRs and IL-15/IL-15Rα fusion polypeptides and optionally CD8. Further, the present disclosure relates to vectors and cells, specifically T cells such as αβ T cells, comprising and capable of co-expressing such sequences.

CD8 and CD4 are transmembrane glycoproteins characteristic of distinct populations of T lymphocytes whose antigen responses are restricted by class I and class II MHC molecules, respectively. They play major roles both 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 an interface distinct from the region presenting the antigenic peptide, but the structural basis for their similar functions appears to be very different. Their sequence similarity is low and, whereas CD4 is expressed on the cell surface as a monomer, CD8 is expressed as an αα homodimer (e.g., FIG. 55C) or an αβ heterodimer (e.g., FIG. 55A). In humans, this CD8αα homodimer may functionally substitute for the CD8αβ heterodimer. CD8 contacts an acidic loop in the α3 domain of Class I MHC, thereby increasing the avidity of the T cell for its target. CD8 is also involved in the phosphorylation events leading to CTL activation through the association of its a chain cytoplasmic tail with the tyrosine kinase p56lck.

Pleiotropic cytokine interleukin-15 (“IL-15” or “IL15”) is a member of the 4 α-helix bundle cytokine family. (Waldmann T A and Tagaya Y, Ann. Rev. Immunol. 17:19-49, 1999, the content of which is incorporated herein by reference). A 14-15 kDa glycoprotein, wild type IL-15 shares partial structural homology with IL-2. (Id.). Wild type IL-15 can be expressed in two isoforms, one having a 48 amino acid signal peptide and the other having a 21 amino acid signal peptide. (Id.). The mature form of wild type IL-15 consists of 114 amino acids. (Id.). Wild type IL-15 expression is regulated at the transcriptional, translational, and intracellular trafficking levels. (Id.). Wild type IL-15 utilizes a private receptor, IL-15Rα (or “IL15Rα”), which, in lymphocytes, binds IL-15 with high affinity and trimerizes with IL-2Rβ (also referred to as IL-2/IL-15Rβ) and IL-2Rγ (also referred to as γc). (Id.; Okada S et al., Immunol. and Cell Biol. 93: 461-471, 2015, the content of which is incorporated herein by reference). Wild type IL-15Rα comprises a signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. (Waldmann and Tagaya). The extracellular domain of wild type IL-15Rα comprises a sushi domain (also referred to as a GP-1 motif). (Id.).

Adoptive cell therapy (ACT) is a promising approach to 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 content of which is incorporated by reference in its entirety. However, cells used in ACT often fail to persist in the tumor microenvironment and quickly lose their ability to kill tumor cells. Accordingly, there is a need for T cells and natural killer cells that exhibit longer persistence in the tumor microenvironment and/or sustained capability to kill tumor cells. It is also desirable to develop methods of manufacturing T cells and natural killer cells with enhanced, specific cytotoxic activity for immunotherapy.

BRIEF SUMMARY

In a first aspect, the present disclosure relates to nucleic acids comprising a nucleotide sequence according to any one of SEQ ID NOs: 454, 451, 448, 449, 450, 452 or 453 or a sequence 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 any one of SEQ ID NOs: 454, 451, 448, 449, 450, 452 or 453. In some embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence according to any one of SEQ ID NOs: 454, 451, 448 or a sequence 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 any one of SEQ ID NOs: 454, 451, 448. In specific embodiments, the nucleic acid of the present disclosure comprises a nucleotide sequence according to any one of SEQ ID NO: 454 or a sequence 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: 454. The nucleic acid(s) provided herein encode one or more (fusion) polypeptide(s). Specifically, such polypeptides and fusion polypeptides may include a TCR having an alpha chain variable domain as comprised in SEQ ID NO: 15, and a TCR beta chain variable domain as comprised in SEQ ID NO: 16. Such polypeptides may also include an IL-15Rα fusion polypeptide having a CD28 or CD25 transmembrane domain. Further, the present disclosure provides vectors comprising the nucleic acids according to said first aspect, T cells and/or natural killer cell transduced with such nucleic acids or vectors and/or expressing the polypeptides encoded by said nucleic acids or vectors. In other words, the T cells and/or natural killer cells of the invention may express a TCR having an alpha chain variable domain as comprised in SEQ ID NO: 15, and a beta chain variable domain as comprised in SEQ ID NO: 16, and further an IL-15/IL-15Rα fusion polypeptide having a CD28 or CD25 transmembrane domain as comprised in SEQ ID NO: 329, 327, 325, 323 or 321. Specifically, the TCR alpha chain variable domain may have an amino acid sequence comprising the sequence of SEQ ID NO: 455, the TCR beta chain variable domain may have an amino acid sequence comprising SEQ ID NO: 456, and the IL-15/IL-15 Receptor alpha fusion polypeptide may have an amino acid sequence as comprised in SEQ ID NO: 329. In some embodiments, the T cells and/or natural killer cells may additionally express CD8 encoded by the nucleic acid(s) and/or vector(s). Finally, compositions comprising said nucleic acids, vectors and/or said transduced T cells and/or natural killer cells are also provided. Said compositions are envisaged for medical purposes and in particular in the treatment of cancer.

In some embodiments, a membrane-bound IL-15 polypeptide (membrane-bound IL-15 or mbIL-15) may be provided. In some embodiments, nucleic acids described herein may comprise and/or encode a membrane-bound IL-15 polypeptide. In some embodiments, vectors described herein may comprise and/or encode a membrane-bound IL-15 polypeptide. In some embodiments, cells described herein may comprise and/or express a membrane-bound IL-15 polypeptide. In some embodiments, compositions described herein may comprise a membrane-bound IL-15 polypeptide or may comprise cells comprising and/or expressing a membrane-bound IL-15 polypeptide. In some embodiments, IL-15 may be rendered membrane-bound by expressing an IL-15 polypeptide and an IL-15Rα polypeptide in an IL-15/IL-15Rα fusion polypeptide (IL-15/IL-15Rα). IL-15/IL-15Rα fusion polypeptides and other membrane-bound forms of IL-15 may be referred to as membrane-bound IL-15 (mbIL-15).

In some embodiments, isolated membrane-bound IL-15 polypeptides may be provided. Isolated nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides may be provided. In some embodiments, isolated vectors comprising one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides may be provided. In some embodiments, cells comprising and/or expressing one or more membrane-bound IL-15 polypeptides may be provided. In some embodiments, cells comprising or expressing one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides may be provided. In some embodiments, cells comprising or expressing one or more vectors comprising one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides may be provided. In some embodiments, compositions comprising such polypeptides, nucleic acids, vectors, and/or cells may be provided.

In some embodiments, an IL-15 polypeptide may be located N-terminal to an IL-15Rα polypeptide in a membrane-bound IL-15 polypeptide. (FIG. 67A). In some embodiments, an IL-15 polypeptide may be located C-terminal to an IL-15Rα polypeptide in a membrane-bound IL-15 polypeptide. (FIG. 67B). The IL-15 polypeptide in FIGS. 67A and 67B may be immature wild type (“wt”), immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptide in FIGS. 67A and 67B may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments, the IL-15 polypeptide in 67A and 67B is mature and may or may not be mutated, and the IL-15Rα polypeptide in 67A and 67B is mature and may or may not be mutated. In some embodiments, the IL-15 polypeptide in 67A and 67B is mature and may or may not be mutated, and the IL-15Rα polypeptide in in 67A and 67B is mature and mutated. Although a linker is depicted in FIGS. 67A and 67B, a mbIL-15 polypeptide may or may not comprise a linker.

In some embodiments, an IL-15 polypeptide and an IL-15Rα polypeptide may be linked by one or more linker. In some embodiments, a membrane-bound IL-15 may comprise and/or be encoded by a structure as shown in FIG. 67A or FIG. 67B. In FIGS. 67A and 67B, the lines connecting the IL-15 to the one or more linker (L) and the one or more linker (L) to the IL-15Rα may represent direct linkages, with no intervening sequences, or may represent intervening sequences, such as, but not limited to, a linker, an untranslated 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.

In some embodiments, an IL-15/IL-15Rα polypeptide may comprise one or more signal peptide. In some embodiments, a membrane-bound IL-15 comprising one or more signal peptide and, optionally, one or more linker may comprise and/or be encoded by a structure as shown in FIG. 68A or FIG. 68B. The IL-15 polypeptide in FIGS. 68A and 68B may be immature wild type, immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptide in FIGS. 68A and 68B may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments, the IL-15 polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated. In some embodiments, the IL-15 polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 68A and FIG. 68B is mature and mutated. Although a linker is depicted in FIGS. 68A and 68B, a mbIL-15 polypeptide comprising a signal peptide may or may not comprise a linker. In FIGS. 68A and 68B, the lines connecting (a) the one or more signal peptide (SP) to the IL-15, the IL-15 to the one or more linker (L), and the one or more linker to the IL-15Rα (as in FIG. 68A) or (b) the one or more signal peptide (SP) to the IL-15α, the IL-15α to the one or more linker (L), and the one or more linker to the IL-15 (as in FIG. 68B) may represent direct linkages, with no intervening sequences, or may represent intervening sequences, such as, but not limited to, a linker, an untranslated 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.

In some embodiments, CD8 polypeptides described herein may comprise a CD8α immunoglobulin (Ig)-like domain, a CD8β region, a CD8α transmembrane domain, and a CD8α cytoplasmic domain. In some embodiments, a CD8β region may be a CD8β stalk region or domain.

In some embodiments, CD8 polypeptides described herein may comprise (a) an immunoglobulin (Ig)-like domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, (b) a CD8β region comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity sequence identity to the amino acid sequence of SEQ ID NO: 2, (c) a transmembrane domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, and (d) a cytoplasmic domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

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

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

In some embodiments, CD8 polypeptides described herein may comprise one or more signal peptide with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 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 directly or indirectly fused to the N-terminus or to the C-terminus of CD8 polypeptides described herein.

In some embodiments, CD8 polypeptides described herein may be CD8α or modified CD8α polypeptides.

In some embodiments, CD8 polypeptides described herein may be CD8αβ or modified CD8α polypeptides.

In some embodiments, a CD8β polypeptide may comprise the amino acid sequence of any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.

In some embodiments, a TCR α chain variable domain and a TCR β chain variable domain may be selected from SEQ ID NO: 455 and 456.

In some embodiments, an isolated nucleic acid may comprise a nucleic acid sequence encoding a T-cell receptor comprising an α chain and a β chain and a CD8 polypeptide comprising an α chain and a β chain. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified. An isolated nucleic acid may comprise a nucleic acid at least about 80% identical to the nucleic acid sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301. An isolated nucleic acid may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, or 301.

In an aspect, polypeptide sequences and/or nucleic acid sequences described herein may be isolated and/or recombinant sequences.

In an aspect, cells described herein may be isolated and/or recombinant cells.

In some embodiments, an isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 267.

In some embodiments, an isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 279.

In some embodiments, isolated polypeptide(s) may be encoded by nucleic acids described herein or, due, for example, to codon degeneration, by nucleic acids encoding the same polypeptide.

In some embodiments, an isolated polypeptide may comprise the amino acid sequence of SEQ ID NO: 268.

In some embodiments, an isolated polypeptide may comprise the amino acid sequence of SEQ ID NO: 280.

In some embodiments, a nucleic acid encoding a fusion polypeptide of Formula I:

wherein P6 and P7 are each independently a first and second polypeptides and PL is a linker, wherein PL comprises SEQ ID NO: 387 or 389 or a sequence 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: 387 or 389 may be provided.

In some embodiments, a nucleic acid comprising formula II:

wherein N6 and N7 each independently encode a first and second polypeptides and NL encodes a linker, wherein NL comprises SEQ ID NO: 388 or 390 or a sequence 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: 388 or 390 may be provided.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 309, 311, 313, or 315 or a polypeptide at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 309, 311, 313, or 315 may be provided.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 311, 313, or 315 or a polypeptide at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 311, 313, or 315 may be provided.

In some embodiments, a nucleic acid comprising SEQ ID NO: 310, 312, 314, or 316 or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 310, 312, 314, or 316 may be provided. In some embodiments, a nucleic acid comprising SEQ ID NO: 312, 314, or 316 or a sequence 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, 314, or 316 may be provided.

In some embodiments, the nucleic acid comprises the sequence SEQ ID NO: 314, or a sequence 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: 314.

In some embodiments, a nucleic acid encoding (i) a polypeptide comprising SEQ ID NO: 307 fused directly or indirectly to an N terminus of a polypeptide comprising any of SEQ ID NO: 309, 311, 313, or 315 or (ii) a polypeptide 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 fused directly or indirectly to a polypeptide 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 any of SEQ ID NO: 309, 311, 313, or 315 may be provided.

In some embodiments, a nucleic acid encoding (i) a polypeptide comprising SEQ ID NO: 307 fused directly or indirectly to an N terminus of a polypeptide comprising any of SEQ ID NO: 311, 313, or 315 or (ii) a polypeptide 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 fused directly or indirectly to a polypeptide 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 any of SEQ ID NO: 311, 313, or 315 may be provided.

In some embodiments, a nucleic acid encoding (i) a polypeptide comprising SEQ ID NO: 307 fused directly or indirectly to an N terminus of a polypeptide comprising any of SEQ ID NO: 313 or (ii) a polypeptide 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 fused directly or indirectly to a polypeptide 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 any of SEQ ID NO: 313 may be provided.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: 307 or to an N terminus of a sequence 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 signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising (i) SEQ ID NO: 308 fused directly or indirectly to a 5′ end of any of SEQ ID NO: 310, 312, 314, or 316 or (ii) a sequence 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 fused directly or indirectly to the 5′ end of any of a sequence 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 any of SEQ ID NO: 310, 312, 314, or 316 may be provided.

In some embodiments, a nucleic acid comprising (i) SEQ ID NO: 308 fused directly or indirectly to a 5′ end of any of SEQ ID NO: 312, 314, or 316 or (ii) a sequence 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 fused directly or indirectly to the 5′ end of any of a sequence 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 any of SEQ ID NO: 312, 314, or 316 may be provided.

In some embodiments, a nucleic acid comprising (i) SEQ ID NO: 308 fused directly or indirectly to a 5′ end of any of SEQ ID NO: 314 or (ii) a sequence 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 fused directly or indirectly to the 5′ end of any of a sequence 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 any of SEQ ID NO: 314 may be provided.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of SEQ ID NO: 308 or to 5′ end of a sequence 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. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 368 or a sequence 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 thereto.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335 or a polypeptide 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: 317, 321, 325, 327, 329, 331, 333, or 335 may be provided.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or a polypeptide 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: 317, 321, 325, 327, 329, 331, or 333 may be provided.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 329 or a polypeptide 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: 329 may be provided.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or to an N terminus of a sequence 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: 317, 321, 325, 327, 329, 331, or 333. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO.: 329 or to an N terminus of a sequence 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: 329. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising SEQ ID NO: 318, 322, 326, 328, 330, 332, 334, or 336 or a sequence 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: 318, 322, 326, 328, 330, 332, 334, or 336 may be provided.

In some embodiments, a nucleic acid comprising SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334 or a sequence 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: 318, 322, 326, 328, 330, 332, or 334 may be provided.

In some embodiments, a nucleic acid comprising SEQ ID NO: 330 or a sequence 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: 330 may be provided.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334 or to 5′ end of a sequence 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: 318, 322, 326, 328, 330, 332, or 334. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may be comprise SEQ ID NO: 368 or a sequence 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 thereto.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of SEQ ID NO: 330, or to 5′ end of a sequence 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: 330. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may be comprise SEQ ID NO: 368 or a sequence 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 thereto.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 337, 341, 345, 347, 349, 351, 353, or 355 or a polypeptide at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 341, 345, 347, 349, 351, 353, or 355 may be provided.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 or a polypeptide at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 may be provided.

In some embodiments, a nucleic acid encoding a polypeptide comprising SEQ ID NO: 349 or a polypeptide 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: 349 may be provided.

In some embodiments, a nucleic acid comprising SEQ ID NO: 338, 342, 346, 348, 350, 352, 354, or 356 or a sequence 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: 338, 342, 346, 348, 350, 352, 354, or 356 may be provided.

In some embodiments, a nucleic acid comprising SEQ ID NO: 338, 342, 346, 348, 350, 352, or 354 or a sequence 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: 338, 342, 346, 348, 350, 352, or 354 may be provided.

In some embodiments, a nucleic acid comprising SEQ ID NO: 350 or a sequence 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: 350 may be provided.

In some embodiments, a nucleic acid described herein may further comprise a nucleic acid encoding (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) both 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) both an α chain and a β chain.

In some embodiments, a polypeptide, polypeptides, or fusion polypeptide encoded by a nucleic acid described herein may be provided.

In some embodiments, the polypeptide or fusion polypeptide comprises an amino acid sequence at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 329.

In some embodiments, a fusion polypeptide comprising a polypeptide 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 fused directly or indirectly to an N terminus of any of a polypeptide 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 any of SEQ ID NO: 309, 311, 313, or 315 may be provided.

In some embodiments, a fusion polypeptide comprising a polypeptide 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 fused directly or indirectly to an N terminus of any of a polypeptide 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 any of SEQ ID NO: 311, 313, or 315 may be provided.

In some embodiments, a fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of a polypeptide 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 signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from (i) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 309 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (iii) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; or (iv) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to the N terminus of SEQ ID NO: 307 of any of (i), (ii), (iii), or (iv) or to the N terminus of a sequence 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 of any of (i), (ii), (iii), or (iv).

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from (i) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; or (iii) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to the N terminus of SEQ ID NO: 307 of any of (i), (ii), or (iii) or to the N terminus of a sequence 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 of any of (i), (ii), or (iii).

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335, or a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335, or directly or indirectly fused to an N terminus of a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333, or a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, or 333 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333, or directly or indirectly fused to an N terminus of a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, or 333.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding a fusion polypeptide comprising (i) SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an N terminus of an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the nucleic acid of (b) encodes a fusion polypeptide selected from SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355 may be provided.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding a fusion polypeptide comprising (i) SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an N terminus of an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the nucleic acid of (b) encodes a fusion polypeptide selected from SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, or 353, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, or 353 may be provided.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335 or a sequence 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: 317, 321, 325, 327, 329, 331, 333, or 335 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335, or to an N terminus of a sequence 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: 317, 321, 325, 327, 329, 331, 333, or 335.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or a sequence 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: 317, 321, 325, 327, 329, 331, or 333 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333, or to an N terminus of a sequence 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: 317, 321, 325, 327, 329, 331, or 333.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding a fusion polypeptide comprising (i) SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an N terminus of an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the nucleic acid of (b) encodes a fusion polypeptide selected from SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 345, 347, 349, 351, 353, or 355 may be provided.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding a fusion polypeptide comprising (i) SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an N terminus of an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the nucleic acid of (b) encodes a fusion polypeptide selected from SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 may be provided.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 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; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; and wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 317 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 319 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 321 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 323 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 325 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 327 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 329 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 331 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 333 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 335 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of the IL-15/IL-15Rα fusion polypeptide. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a nucleic acid comprising: (a) a nucleic acid 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 without a β chain, and (b) a nucleic acid encoding a fusion polypeptide comprising (i) SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an N terminus of an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 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; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; and wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14 may be provided.

In some embodiments, the nucleic acid of (b) may encode SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353 or a sequence 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 any of the aforementioned SEQ ID NOs. 337, 339, 341, 343, 345, 347, 349, 351, 353.

In some embodiments, a nucleic acid comprising: (a) 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 and (b) a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may be provided.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may be selected from (i) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 310 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween; (ii) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 312 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween; (iii) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 314 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween; or (iv) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 316 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide, wherein the nucleic acid encoding the signal peptide is directly or indirectly fused to the 5′ end of SEQ ID NO: 308 of any of (i), (ii), (iii), or (iv) or to the 5′ end of sequence 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 of any of (i), (ii), (iii), or (iv).

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may be selected from (i) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 312 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween; (ii) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 314 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween; or (iii) SEQ ID NO: 308 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, directly or indirectly fused to a 5′ end of SEQ ID NO: 316 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, with or without a nucleic acid encoding a linker therebetween.

In some embodiments, a nucleic acid may further comprise a nucleic acid encoding a signal peptide, wherein the nucleic acid encoding the signal peptide is directly or indirectly fused to the 5′ end of SEQ ID NO: 308 of any of (i), (ii), or (iii) or to the 5′ end of sequence 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 of any of (i), (ii), or (iii).

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the nucleic acid encoding the signal peptide may comprise SEQ ID NO: 368 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may further comprise a nucleic acid encoding a signal peptide, wherein the nucleic acid encoding the signal peptide is directly or indirectly fused to a 5′ end of SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, 334, or 336 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, 334, or 336.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may further comprise a nucleic acid encoding a signal peptide, wherein the nucleic acid encoding the signal peptide is directly or indirectly fused to a 5′ end of SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, or 334 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, or 334.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the nucleic acid encoding the signal peptide may comprise SEQ ID NO: 368 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide (i) may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) may be selected from SEQ ID NO: 338, 340, 342, 344, 346, 348, 350, 352, 354, or 356 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 338, 340, 342, 344, 346, 348, 350, 352, 354, or 356.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide (i) may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) may be selected from SEQ ID NO: 338, 340, 342, 344, 346, 348, 350, 352, or 354 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 338, 340, 342, 344, 346, 348, 350, 352, or 354.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may further comprise an nucleic acid encoding a signal peptide, wherein the nucleic acid encoding the signal peptide is directly or indirectly fused to a 5′ end of SEQ ID NO: 318, 322, 326, 328, 330, 332, 334, or 336 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 318, 322, 326, 328, 330, 332, 334, or 336.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may further comprise an nucleic acid encoding a signal peptide, wherein the nucleic acid encoding the signal peptide is directly or indirectly fused to a 5′ end of SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the nucleic acid encoding the signal peptide may comprise SEQ ID NO: 368 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide (i) may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) may be selected from SEQ ID NO: 338, 342, 346, 348, 350, 352, 354, or 356 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 338, 342, 346, 348, 350, 352, 354, or 356.

In some embodiments, the nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide (i) may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) may be selected from SEQ ID NO: 338, 342, 346, 348, 350, 352, or 354 or a sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid of SEQ ID NO: 338, 342, 346, 348, 350, 352, or 354.

In some embodiments, a vector comprising a nucleic acid encoding at least one CD8α chain, at least one TCRα chain, at least one TCRβ chain, at least one IL-15/IL-15Rα fusion polypeptide, and optionally at least one CD8β chain may be provided.

In some embodiments, a vector comprising N1, N2, N3, N4, N5, L1, L2, L3, and L4, in any order, wherein N1 comprises a nucleic acid encoding a CD8β chain and is present or absent, N2 comprises a nucleic acid encoding a CD8α chain, N3 comprises a nucleic acid encoding a TCRβ chain, N4 comprises a nucleic acid encoding a TCRα chain, and N5 comprises a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide; and wherein L1-L4 each comprises a nucleic acid encoding at least one linker, wherein each of L1-L4 is independently the same or different, and wherein each of L1-L4 is independently present or absent may be provided.

In some embodiments, a vector may comprise Formula III or Formula IV:

In some embodiments, N2 comprises a nucleic acid encoding a SEQ ID NO: 7, 258, 259, 262, or a variant thereof.

In some embodiments, N5 may comprise a nucleic acid encoding (i) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 309 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 309, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 311, with or without a linker therebetween; (iii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence 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, with or without a linker therebetween; or (iv) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 315, with or without a linker therebetween.

In some embodiments, N5 may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of the nucleic acid encoding SEQ ID NO: 307 of any of (i), (ii), (iii), or (iv) or to the 5′ end of the nucleic acid encoding the sequence 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 of any of (i), (ii), (iii), or (iv).

In some embodiments, N5 may comprise a nucleic acid encoding (i) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 311, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence 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, with or without a linker therebetween; or (iii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 315, with or without a linker therebetween.

In some embodiments, N5 may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of the nucleic acid encoding SEQ ID NO: 307 of any of (i), (ii), or (iii) or to the 5′ end of the nucleic acid encoding the sequence 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 of any of (i), (ii), or (iii).

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, N5 may comprise a nucleic acid encoding SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335 or a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335.

In some embodiments, N5 may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of the nucleic acid encoding SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335 or to the 5′ end of the nucleic acid encoding the sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335.

In some embodiments, N5 may comprise a nucleic acid encoding SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333 or a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, or 333.

In some embodiments, N5 may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of the nucleic acid encoding SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333 or to the 5′ end of the nucleic acid encoding the sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, or 333.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, (i) N5 may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) N5 may encode SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355.

In some embodiments, (i) N5 may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) N5 may encode SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, or 353, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, or 353.

In some embodiments, N5 may comprise a nucleic acid encoding SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335 or a sequence 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: 317, 321, 325, 327, 329, 331, 333, or 335.

In some embodiments, N5 may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of the nucleic acid encoding SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335 or to the 5′ end of the nucleic acid encoding the sequence 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: 317, 321, 325, 327, 329, 331, 333, or 335.

In some embodiments, N5 may comprise a nucleic acid encoding SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or a sequence 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: 317, 321, 325, 327, 329, 331, or 333.

In some embodiments, N5 may further comprise a nucleic acid encoding a signal peptide directly or indirectly fused to the 5′ end of the nucleic acid encoding SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or to the 5′ end of the nucleic acid encoding the sequence 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: 317, 321, 325, 327, 329, 331, or 333.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, (i) N5 may further comprise a nucleic acid encoding a signal peptide derived from an IgE polypeptide and (ii) N5 may encode SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353.

In some embodiments, (i) the vector may further encode a 2A peptide or an internal ribosome entry site (IRES) positioned 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) the vector may further encode a 2A peptide or an internal ribosome entry site (IRES) positioned 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 some embodiments, (i) the vector may further encode a furin positioned 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) the vector may further encode a furin positioned 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 some embodiments, the IRES may be selected from the group consisting of IRES from picornavirus, IRES from flavivirus, IRES from pestivirus, IRES from retrovirus, IRES from lentivirus, IRES from insect RNA virus, and IRES from cellular mRNA.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from (i) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 309 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 309, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 311, with or without a linker therebetween; (iii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence 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, with or without a linker therebetween; or (iv) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 315, with or without a linker therebetween may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to the N terminus of SEQ ID NO: 307 of any of (i), (ii), (iii), or (iv) or to the N terminus of a sequence 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 of any of (i), (ii), (iii), or (iv).

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from (i) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 311, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence 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, with or without a linker therebetween; or (iii) SEQ ID NO: 307 or a sequence 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, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 315, with or without a linker therebetween may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to the N terminus of SEQ ID NO: 307 of any of (i), (ii), or (iii) or to the N terminus of a sequence 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 of any of (i), (ii), or (iii).

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335, or a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335, or directly or indirectly fused to an N terminus of a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333, or a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, or 333 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333, or directly or indirectly fused to an N terminus of a sequence 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: 317, 319, 321, 323, 325, 327, 329, 331, or 333.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) a fusion polypeptide comprising (i) a signal peptide comprising SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the fusion polypeptide of (b) is selected from SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, 353, or 355 may be provided.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) a fusion polypeptide comprising (i) a signal peptide comprising SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the fusion polypeptide of (b) is selected from SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, or 353, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 339, 341, 343, 345, 347, 349, 351, or 353 may be provided.

In some embodiments, a T cell and/or natural killer cell transduced to express (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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335 or a sequence 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: 317, 321, 325, 327, 329, 331, 333, or 335 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, or 335, or directly or indirectly fused to an N terminus of a sequence 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: 317, 321, 325, 327, 329, 331, 333, or 335.

In some embodiments, a T cell and/or natural killer cell transduced to express (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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof, wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the IL-15/IL-15Rα fusion polypeptide is selected from SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or a sequence 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: 317, 321, 325, 327, 329, 331, or 333 may be provided.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of SEQ ID NO: SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333, or directly or indirectly fused to an N terminus of a sequence 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: 317, 321, 325, 327, 329, 331, or 333.

In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) a fusion polypeptide comprising (i) a signal peptide comprising SEQ ID NO: 367 or a sequence 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: 367 directly or indirectly fused to (ii) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 303, 304 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, and 91 and 92; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; and wherein the fusion polypeptide of (b) is selected from SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353, or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 may be provided.

In some embodiments, a T cell and/or natural killer 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 without a β chain, and (b) an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 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; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; and wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 317 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 319 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 321 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 323 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 325 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 327 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 329 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 331 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 333 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may comprise SEQ ID NO: 335 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the IL-15/IL-15Rα fusion polypeptide may further comprise a signal peptide directly or indirectly fused to an N terminus of the IL-15/IL-15Rα fusion polypeptide. In some embodiments, the signal peptide may be derived from an IgE polypeptide. In some embodiments, the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, T cell and/or natural killer 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 without a β chain, and (b) a fusion polypeptide comprising (i) SEQ ID NO: 367 or a sequence 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: 367 fused to (ii) an N terminus of an IL-15/IL-15Rα fusion polypeptide, wherein the TCR α chain and the TCR β chain are selected from SEQ ID NO: 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; wherein the CD8 α chain is SEQ ID NO: 7, 258, 259, 262, or a variant thereof; and wherein, if present, the CD8 β chain is SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 337 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 339 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 341 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO:

343 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 345 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 347 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO:

349 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 351 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 353 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the fusion polypeptide of (b) may comprise SEQ ID NO: 355 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the T cell may be an αβ T cell, a γδ T cell, a natural killer T cell, or any combination thereof. 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.

In some embodiments, a composition comprising a T cell and/or natural killer cell 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, excipient, carrier, diluent, buffer, stabilizer, or a combination thereof.

In some embodiments, a method of preparing T cells and/or natural killer cells for immunotherapy may be provided, the method comprising: isolating or enriching 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 or enriching 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 (PMBC). In some embodiments, the activating may comprise contacting the T cells and/or natural killer cells with an anti-CD3 and an anti-CD28 antibody. In some embodiments, the T cell may be a CD4+ T cell. In some embodiments, the T cell may be a CD8+ T cell. In some embodiments, the T cell may be a γδ T cell or an αβ T cell. In some embodiments, the activation, the expanding, or both may be in the presence of a combination of IL-2 and IL-15 and optionally with zoledronate.

In some embodiments, a method of increasing persistence, functionality, naivety, longevity, capacity to kill antigen-presenting cells, or a combination thereof, of T cells and/or natural killer cells may be provided, the method comprising: isolating or enriching 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, a vector described herein, or a combination thereof, to obtain transduced T cells and/or natural killer cells, and obtaining the transduced T cells and/or natural killer cells, wherein the persistence, longevity, naivety, capacity to kill antigen-presenting cells, or a combination thereof of the transduced T cells and/or natural killer cells is increased as compared with that of control cells. In some embodiments, the method may further comprise expanding the transduced T cells and/or natural killer cells.

In some embodiments, the control cells may comprise non-transduced T cells and/or natural killer cells, T cells and/or natural killer cells transduced with TCR only, or a combination thereof. In some embodiments, the control may cells comprise non-transduced T cells and/or natural killer cells, T cells and/or natural killer cells transduced with TCR only, T cells and/or natural killer cells transduced with TCR and CD8 only, or a combination thereof. In some embodiments, the persistence, longevity, functionality, naivety, capacity to kill antigen-presenting cells, or a combination thereof of the transduced T cells and/or natural killer 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, the persistence, longevity, functionality, naivety, capacity to kill antigen-presenting cells, or a combination thereof of the transduced T cells and/or natural killer cells and control cells may be determined after five or more challenges with antigen-presenting cells or more challenges with antigen-presenting cells.

In some embodiments, a method of increasing interferon γ (IFNγ) secretion by T cells and/or natural killer cells may be provided, the method comprising: isolating or enriching 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, a vector described herein, or a combination thereof, to obtain transduced T cells and/or natural killer cells, and obtaining the transduced T cells and/or natural killer cells, wherein the IFNγ secretion of the transduced T cells and/or natural killer cells is increased as compared with that of control cells. In some embodiments, the method may further comprise expanding the transduced T cells and/or natural killer cells. In some embodiments, the control cells may comprise non-transduced T cells and/or natural killer cells, T cells and/or natural killer cells transduced with TCR only, or a combination thereof.

In some embodiments, the control cells may comprise non-transduced T cells and/or natural killer cells, T cells and/or natural killer cells transduced with TCR only, T cells and/or natural killer cells transduced with TCR and CD8 only, or a combination thereof. In some embodiments, the IFNγ secretion by the transduced T cells and/or natural killer 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 IFNγ secretion by the transduced T cells and/or natural killer cells and control cells may be determined after five or more challenges with antigen-presenting cells or more challenges with antigen-presenting cells.

In some embodiments, the antigen presenting cells may present an antigen on a cell surface, and the transduced T cells and/or natural killer cells and control cells may be capable of killing the antigen presenting cells. In some embodiments, the antigen may comprise a peptide. In some embodiments, the peptide may be in a complex with an MHC molecule on the cell surface.

In some embodiments, a polypeptide, polypeptides, or fusion polypeptide encoded by a nucleic acid described herein may be provided.

In some embodiments, a nucleic acid described herein may be isolated, recombinant, or both isolated and recombinant.

In some embodiments, a vector described herein may be isolated, recombinant, or both isolated and recombinant.

In some embodiments, a T cell and/or natural killer cell described herein may be isolated, recombinant, engineered, or any combination thereof.

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

In some embodiments, a vector comprising a nucleic acid described herein may be provided. In some embodiments, a vector described herein may further comprise a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) positioned between a nucleic acid encoding a CD8 α chain and a nucleic acid encoding a CD8 β chain. In some embodiments, the vector may further comprise a nucleic acid encoding a 2A peptide or an IRES positioned between a nucleic acid encoding a TCR α chain and a nucleic acid encoding a TCR β chain. In some embodiments, the vector may further comprise a nucleic acid encoding a 2A peptide or an IRES positioned between a nucleic acid encoding a TCR chain or a CD8 chain and a nucleic acid encoding a membrane-bound IL-15, such as an IL-15/IL-15Rα fusion polypeptide. 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 IRES from picornavirus, IRES from flavivirus, IRES from pestivirus, IRES from retrovirus, IRES from lentivirus, IRES from insect RNA virus, and IRES from cellular mRNA. In some embodiments, the vector may further comprise a post-transcriptional regulatory element (PRE) sequence selected from a Woodchuck PRE (WPRE) (SEQ ID NO: 264), Woodchuck PRE (WPRE) mutant 1 (SEQ ID NO: 256), Woodchuck PRE (WPRE) mutant 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 a Woodchuck PRE (WPRE) mutant 1 comprising the nucleic acid sequence of SEQ ID NO: 256. In some embodiments, the post-transcriptional regulatory element (PRE) sequence may be a Woodchuck PRE (WPRE) mutant 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 comprising myeloproliferative sarcoma virus enhancer (MNDU3), Ubiqitin C promoter, EF-1 alpha promoter, or Murine Stem Cell Virus (MSCV) promoter. In some embodiments, the promoter may be a Murine Stem Cell Virus (MSCV) promoter. In some embodiments, 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 adenoviruses, poxviruses, alphaviruses, arenaviruses, flaviviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and any combination thereof. In some embodiments, the viral vector may be pseudotyped with an envelope protein of a virus selected from the native feline endogenous virus (RD114), a version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), a version of 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 retroviral 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).

In some embodiments, a T cell and/or natural killer cell expressing a polypeptide as described herein and/or comprising a vector described herein and/or produced by a method described herein may be provided. In some embodiments, the T cell may be an αβ T cell, a γδ T cell, a natural killer T cell, or any combination thereof. 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.

In some embodiments, a method of treating a patient who has cancer may be provided, the method comprising administering to the patient a composition 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, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer. In some embodiments, a method of eliciting an immune response in a patient who has cancer may be provided, the method comprising administering to the patient a composition 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, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer. In some embodiments, the T cell and/or natural killer cell may kill cancer cells that present a peptide in a complex with an MHC molecule on a cell surface.

In some embodiments, nucleic acid sequences disclosed herein may be mutated such that the amino acids encoded remain the same, but the nucleic acid codons are changed to maintain improved expression in a target cell and/or by a target vector. In some embodiments, nucleic acids disclosed herein may be codon optimized. In some embodiments, nucleic acid sequences set forth herein are codon optimized. In some embodiments, nucleic acid sequences set forth herein may be codon optimized, and nucleic acid sequences encoding polypeptides set forth herein may be codon optimized. In some embodiments, mutation of nucleic acid sequences set forth herein may encompass codon optimization.

In some embodiments, expression of membrane-bound IL-15 may improve immune cell, such as but not limited to, T cell and/or natural killer cell, persistence, functionality, growth, viability, expansion, or any combination thereof, as compared to cells not expressing membrane-bound IL-15. In some embodiments, expression of membrane-bound IL-15 may improve immune cell, such as but not limited to, T cell and/or natural killer cell, persistence, functionality, growth, viability, expansion, or any combination thereof, in a tumor microenvironment, as compared to cells not expressing membrane-bound IL-15. In some embodiments, expression of membrane-bound IL-15 may increase efficacy of immune cells, such as, but not limited to, T cells and/or natural killer cells, in killing tumor cells, as compared to cells not expressing membrane-bound IL-15. In some embodiments, expression of membrane-bound IL-15 may increase ability of immune cells, such as, but not limited to, T cells and/or natural killer cells, to survive in a tumor microenvironment, to persist in killing tumor cells, or any combination thereof, as compared to cells not expressing membrane-bound IL-15. In some embodiments, expression of membrane-bound IL-15 may increase ability of immune cells, such as, but not limited to, T cells and/or natural killer cells, to maintain a naive phenotype.

Persistence may be assessed, as a non-limiting example, by the length of time cells are detectable in an individual (e.g., patient) after infusion. As non-limiting examples, persistence may be measured at days, weeks, months, or years after infusion, as non-limiting examples, at about 1 week, about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, and/or about 30 months after infusion. Persistence may be assessed, as non-limiting examples, by PCR of peripheral blood sample(s), by flow cytometry of peripheral blood samples(s), and/or by analysis of tumor biopsy sample(s). Persistence of cells expressing membrane-bound IL-15 may be compared, as non-limiting examples, to typical persistence of infused ACT cells or persistence of similar cells not expressing membrane-bound IL-15.

Continued ability to kill tumor cells may be measured, as non-limiting examples, via (i) serial killing assays using an IncuCyte (wherein ability to kill/impair tumor growth as measured by fold growth during repeated tumor stimulations over a duration of time is assessed), and/or (ii) via cytokine/effector molecule production (IFNγ via ELISAs and other pro-inflammatory cytokines via Luminex (cytokines measured may include, as non-limiting examples, IFNγ, TNFα, Granzyme B, perforin, IL-2, IL-6, MIP-1β, MIP-1α, GM-CSF, RANTES, IL-18, IL-4, IL-10, and IP10)). Continued ability of cells expressing membrane-bound IL-15 to kill tumor cells may be compared, as non-limiting examples, to continued ability of similar cells not expressing membrane-bound IL-15 to kill tumor cells or continued ability of other control cells to kill tumor cells.

Naivety of phenotype may be assessed, as a non-limiting example, via Tmem panel assay via flow cytometry. Typically, flow cytometer gating is off of CD8+TCR+ cells. Typically, a more naïve phenotype may be indicated by higher frequencies of the T memory subsets Tnaïve/scm (CD45RA+CCR7+), and Tcm (CD45RA−CCR7+) and an increase or retention of the CD39−CD69− and CD27+CD28+ populations. Low CD57 expression may also be desirable.

When assessing the persistence, functionality, growth, viability, expansion, tumor killing efficacy, naivety, or other characteristics of cells expressing dnTGFRβRII, cells such as non-transduced cells, cells transduced with TCR only, cells transduced with CD8 and TCR, or a combination thereof, may serve as control cells, as non-limiting examples.

In some embodiments, membrane-bound IL-15 may act in a cis manner (e.g., affecting cells in which it is expressed), in a trans manner (e.g., affecting cells in which it is not expressed), or any combination thereof. In some embodiments in which membrane-bound IL-15 acts in trans, cells adjacent to or near (e.g., within the tumor microenvironment) cells expressing membrane-bound IL-15 may exhibit any or combination of improvements the same or similar to those described for cells expressing membrane-bound IL-15, as compared to cells not adjacent to or near cells expressing membrane-bound IL-15.

In some embodiments, the disclosure provides for nucleic acid(s) encoding polypeptide(s) described herein. In some embodiments, the disclosure provides for vectors comprising nucleic acids encoding polypeptide(s) described herein. In some embodiments, one or more vector may comprise a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide. In some embodiments, one or more vector may comprise a nucleic acid encoding a CD8 polypeptide. In some embodiments, one or more vector may comprise a nucleic acid encoding a CD8α polypeptide. In some embodiments, one or more vector may comprise a nucleic acid encoding a CD8β polypeptide.

In some embodiments, one or more vector may comprise one or more nucleic acid encoding a T cell receptor (TCR) comprising an α chain and a β chain. In some embodiments, one or more vector may comprise one or more nucleic acid encoding a T cell receptor (TCR) comprising an γ chain and a δ chain. In some embodiments, one or more vector may comprise one or more nucleic acid encoding a chimeric antigen receptor (CAR).

In some embodiments, a vector or vectors comprising one or more nucleic acid(s) 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, an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8 chain may independently be modified or unmodified.

In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding one or any combination of a TCR comprising an α chain and a β chain, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding one or any combination of a TCR comprising a γ chain and a δ chain, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding one or any combination of a CAR, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a TCR comprising an α chain and a β chain, an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a TCR comprising a γ chain and a δ chain, an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a CAR, an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a TCR comprising an α chain and a β chain and an IL-15/IL-15Rα fusion polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a TCR comprising a γ chain and a δ chain and an IL-15/IL-15Rα fusion polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a CAR and an IL-15/IL-15Rα fusion polypeptide may be provided.

In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a TCR comprising an α chain and a β chain and a CD8 polypeptide may be provided. In some embodiments, a vector or vectors comprising one or more nucleic acid(s) encoding a TCR comprising a γ chain and a δ chain and a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising one or more nucleic acid(s) encoding a CAR and a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, more than one vector may be co-transduced into one or more cells, co-expressed in one or more cells, or any combination thereof. In some embodiments, a cell or cells may comprise an αβ T cell, a γδ T cell, a natural killer (NK) cell, a natural killer T cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof. Specifically, the cell or cells may be a γδ1 T cell or a γδ2 T cell.

In some embodiments, more than one vector may comprise a nucleic acid or nucleic acids encoding one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a single vector may comprise a nucleic acid or nucleic acids encoding one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

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

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

In some embodiments, a vector may comprise nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) positioned between a nucleic acid encoding a modified CD8α polypeptide and a nucleic acid encoding a CD8β polypeptide.

In some embodiments, a vector may comprise nucleic acid encoding a 2A peptide positioned between a nucleic acid encoding a TCR α chain and a nucleic acid encoding a TCR β chain.

In some embodiments, a vector may comprise nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) positioned between a nucleic acid encoding a modified CD8α polypeptide, a nucleic acid encoding a CD8β polypeptide, a nucleic acid encoding a TCR α chain, or a nucleic acid encoding a TCR β chain and a nucleic acid encoding a membrane-bound IL-15.

In some embodiments, a single vector may comprise a nucleic acid or nucleic acids encoding one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, and/or a CAR, and a vector may comprise a nucleic acid encoding a 2A peptide or an internal ribosome entry site (IRES) positioned between any or each of nucleic acids encoding polypeptides or fusion polypeptides. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a 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) (such as, but not limited to wild type WPRE, such as but not limited to SEQ ID NO: 264, or a mutated 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: 437), variant(s) thereof, or any combination thereof.

In some embodiments, a vector may further comprise one or more promoter. In some embodiments, promoter(s) may be selected from cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR comprising myeloproliferative sarcoma virus enhancer (MNDU3), Ubiqitin C promoter, EF-1 alpha promoter, Murine Stem Cell Virus (MSCV) promoter, the promoter from CD69, nuclear factor of activated T-cells (NFAT) promoter, IL-2 promoter, minimal IL-2 promoter, or a combination thereof.

In some embodiments, a vector may be a viral vector or a non-viral vector.

In some embodiments, a vector may be pseudotyped with an envelope protein of a virus selected from the native feline endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), a chimeric version of 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 retroviral envelope glycoprotein (BaEV), lymphocytic choriomeningitis virus (LCMV), or a combination thereof.

In some embodiments, a vector may comprise one or more Kozak sequence. In some embodiments, a Kozak sequence may initiate, increase, or facilitate translation, or a combination thereof. In some embodiments, the Kozak sequence may be GCCACC. In some embodiments, the Kozak sequence may be ACCATGG. In some embodiments, the Kozak sequence may be GCCNCCATGG. where N is a purine (A or G) (SEQ ID NO:382).

In some embodiments, a vector may comprise one or more Factor Xa sites.

In some embodiments, a vector may comprise one or more enhancer. In some embodiments, an enhancer may comprise Conserved Non-Coding Sequence (CNS) 0, CNS 1, CNS2, CNS 3, CNS 4, or portions or any combination thereof.

In some embodiments, the disclosure provides for one or more cells transduced with and/or expressing one or more vectors comprising nucleic acids encoding polypeptide(s).

In some embodiments, a cell may comprise an αβ T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof.

In some embodiments, a T cell may be a CD4+ T cell. In some embodiments, a T cell may be a CD8+ T cell. In some embodiments, a T cell may be a CD4+/CD8+ T cell. In some embodiments, a T cell may be a αβ T cell. In some embodiments, a T cell may be a γδ T cell.

In some embodiments, a T cell may be an αβ T cell and may express a CD8 polypeptide described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified. In some embodiments, a T cell may be an αβ T cell and may express a modified CD8 polypeptide described herein, for example, a modified CD8α polypeptide or a modified CD8α polypeptide with a CD8β stalk region, e.g., m1CD8α in Constructs #11 and #12 (FIG. 4) and CD8α* (FIG. 55B). In some embodiments, a T cell may be an αβ T cell and may express one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a modified CD8 polypeptide, and/or a CAR.

In some embodiments, a T cell may be a γδ T cell and may express a CD8 polypeptide described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified. In some embodiments, a T cell may be a γδ T cell and may express a modified CD8 polypeptide described herein, for example, a modified CD8α polypeptide or a modified CD8α polypeptide with a CD8β stalk region, e.g., m1CD8α in Constructs #11 and #12 (FIG. 4) and CD8α* (FIG. 55B). In some embodiments, a T cell may be a γδ T cell and may express one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) 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, an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, one or any combination of a TCR comprising an α chain and a β chain, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, one or any combination of a TCR comprising a γ chain and a δ chain, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, one or any combination of a CAR, an IL-15/IL-15Rα fusion polypeptide, and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a TCR comprising an α chain and a β chain, an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain, an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a CAR, an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a TCR comprising an α chain and a β chain and an IL-15/IL-15Rα fusion polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain and an IL-15/IL-15Rα fusion polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a CAR and an IL-15/IL-15Rα fusion polypeptide may be provided.

In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a TCR comprising an α chain and a β chain and a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain and a CD8 polypeptide may be provided. In some embodiments, a cell or cells comprising, or comprising one or more nucleic acid(s) encoding, a CAR and a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

In some embodiments, one or more nucleic acid(s) may be comprised in and/or expressed from a vector or vectors.

In some embodiments, a cell or cells may comprise an αβ T cell, a γδ T cell, a natural killer cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof.

In some embodiments, populations of cells as described herein may be provided. As a non-limiting example, the disclosure provides for a population of modified cells comprising, or comprising one or more nucleic acid(s) encoding one or any combination of an exogenous CD8 co-receptor comprising a polypeptide described herein, for example, amino acid sequences at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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: 5, 7, 258, 259, 8, 9, 10, 11, 12, 13, or 14; a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), as described herein; and/or a T cell receptor. In some embodiments, populations of cells may comprise αβ T cells, γδ T cells, natural killer cells, a natural killer T cell, CD4+ T cells, CD8+ T cells, CD4+/CD8+ cells, or any combination thereof.

In an aspect, polypeptide sequences and/or nucleic acid sequences described herein may be isolated and/or recombinant sequences.

In an aspect, cells described herein may be isolated and/or recombinant cells.

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

In some embodiments, a method of treating a patient who has cancer may comprise administering to the patient a composition comprising the population of expanded cells, wherein the cells kill cancer cells that present a peptide in a complex with an MHC molecule on the surface, wherein the peptide is selected from SEQ ID NO: 98-255, 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, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, prostate cancer, or a combination thereof. In some embodiments, a cell may comprise an αβ T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof.

In some embodiments, a method of eliciting an immune response in a patient who has cancer may comprise administering to the patient a composition comprising the population of expanded cells, wherein the cells kill cancer cells that present a peptide in a complex with an MHC molecule on the surface, wherein the peptide is selected from SEQ ID NO: 98-255, 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, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, prostate cancer, or a combination thereof. In some embodiments, a cell may comprise an αβ T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof. Specifically, the cell may comprise a γδ1 T cell and/or a γδ2 T cell.

DETAILED DESCRIPTION

In some embodiments a membrane-bound IL-15 polypeptide (membrane-bound IL-15 or mbIL-15) is provided. In some embodiments nucleic acids described herein comprise and/or encode a membrane-bound IL-15 polypeptide. In some embodiments vectors described herein comprise and/or encode a membrane-bound IL-15 polypeptide. In some embodiments cells described herein comprise and/or express a membrane-bound IL-15 polypeptide. In some embodiments compositions described herein comprise a membrane-bound IL-15 polypeptide or comprise cells comprising and/or expressing a membrane-bound IL-15 polypeptide. In some embodiments IL-15 is rendered membrane-bound by expressing an IL-15 polypeptide and an IL-15Rα polypeptide in an IL-15/IL-15Rα fusion polypeptide (IL-15/IL-15Rα).

Membrane-bound IL-15 polypeptides are provided. Isolated nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides are provided. Vectors comprising one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides are provided. Cells comprising and/or expressing one or more membrane-bound IL-15 polypeptides are provided. Cells comprising or expressing one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides are provided. Cells comprising or expressing one or more vectors comprising one or more nucleic acid sequences comprising one or more nucleic acid sequences encoding one or more membrane-bound IL-15 polypeptides are provided. In some embodiments, cells described herein may comprise a membrane-bound IL-15 polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments a cell may comprise an αβ T cell, an 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. In some embodiments such polypeptides, nucleic acids, vectors, and/or cells may be isolated, recombinant, and/or engineered. Compositions comprising such polypeptides, nucleic acids, vectors, and/or cells are provided.

In an aspect, polypeptide sequences and/or nucleic acid sequences described herein may be isolated and/or recombinant sequences.

In an aspect, cells described herein may be isolated and/or recombinant cells.

Membrane-bound IL-15 may comprise, for example, an IL-15/IL-15Rα fusion polypeptide and/or an IL-15Rα/IL-15 fusion polypeptide. One or more linkers may be disposed between IL-15 and IL-15Rα or between IL-15Rα and IL-15. In some embodiments an IL-15 polypeptide is located N-terminal to an IL-15Rα polypeptide in a membrane-bound IL-15 polypeptide. (FIG. 67A). In some embodiments, an IL-15 polypeptide is located C-terminal to an IL-15Rα polypeptide in a membrane-bound IL-15 polypeptide. (FIG. 67B). The IL-15 polypeptide in FIGS. 67A and 67B may be immature wild type, immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptide in FIGS. 67A and 67B may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments the IL-15 polypeptide in FIG. 67A and FIG. 67B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 67A and FIG. 67B is mature and may or may not be mutated. In some embodiments the IL-15 polypeptide in FIG. 67A and FIG. 67B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 67A and FIG. 67B is mature and mutated. Although a linker is depicted in FIGS. 67A and 67B, a mbIL-15 may or may not comprise a linker.

In some embodiments an IL-15 polypeptide and an IL-15Rα polypeptide is linked by one or more linker. An IL-15/IL-15Rα fusion polypeptide and/or an IL-15Rα/IL-15 fusion polypeptide may also comprise one or more linker. In some embodiments a membrane-bound IL-15 comprises and/or is encoded by a structure as shown in FIG. 67A or FIG. 67B. In FIGS. 67A and 67B, the lines connecting the IL-15 to the one or more linker (L) and the one or more linker (L) to the IL-15Rα may represent direct linkages, with no intervening sequences, or may represent intervening sequences, such as, but not limited to, a linker, an untranslated 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.

In some embodiments IL-15/IL-15Rα fusion polypeptide and/or an IL-15Rα/IL-15 fusion polypeptide may comprise one or more signal peptide. In some embodiments a membrane-bound IL-15 comprising one or more signal peptide and, optionally, one or more linkers may comprise and/or be encoded by a structure as shown in FIG. 68A or FIG. 68B. An exemplary IL-15/IL-15Rα fusion polypeptide comprising, optionally, at least one linker and at least one signal peptide is depicted in FIG. 68A. An exemplary 15Rα/IL-15 fusion polypeptide comprising, optionally, at least one linker and at least one signal peptide is depicted in FIG. 68B. The IL-15 polypeptide in FIGS. 68A and 68B may be immature wild type, immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptide in FIGS. 68A and 68B may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments the IL-15 polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated. In some embodiments the IL-15 polypeptide in FIGS. 68A and 68B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIGS. 68A and 68B is mature and mutated. In FIGS. 68A and 68B, the lines connecting (a) the one or more signal peptide (SP) to the IL-15, the IL-15 to the one or more linker (L), and the one or more linker to the IL-15Rα (as in FIG. 68A) or (b) the one or more signal peptide (SP) to the IL-15a, the IL-15a to the one or more linker (L), and the one or more linker to the IL-15 (as in FIG. 68B) may represent direct linkages, with no intervening sequences, or may represent intervening sequences, such as, but not limited to, a linker, an untranslated 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.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprises an entire IL-15 polypeptide, an entire IL-15Rα polypeptide, or both. In some embodiments an entire, or full, wild type IL-15 polypeptide may comprise SEQ ID NO: 305. In some embodiments an entire, or full, wild type IL-15Rα polypeptide may comprise SEQ ID NO: 306.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprises a mature IL-15 polypeptide (e.g., SEQ ID NO: 307), a mature IL-15Rα polypeptide (e.g., SEQ ID NO: 309), which may be mutated (e.g., SEQ ID NO: 311, 313, 315), or both. In some embodiments a mature wild type IL-15 polypeptide may comprise or consist of SEQ ID NO: 307 or may comprise or consist of amino acids 49-162 of SEQ ID NO: 305. In some embodiments a mature wild type IL-15Rα polypeptide may comprise or consist of SEQ ID NO: 309 or may comprise or consist of amino acids 31-267 of SEQ ID NO: 306. In some embodiments a mature wild type IL-15 polypeptide is encoded by a nucleic acid comprising or consisting of the nucleic acid set forth in SEQ ID NO: 308. In some embodiments a mature wild type IL-15Rα polypeptide is encoded by a nucleic acid comprising or consisting of the nucleic acid set forth in SEQ ID NO: 310. However, In some embodiments an IL-15/IL-15Rα fusion polypeptide does not comprise a mature wild type IL-15Rα as in SEQ ID NO: 309 or sequences having about 95% or more sequence identity thereto. In some embodiments an IL-15/IL-15Rα fusion polypeptide does not comprise a mature wild type IL-15Rα encoded by SEQ ID NO: 310 or sequences having about 80%, about 85%, about 90%, or about 95% or more sequence identity thereto.

In some embodiments an IL-15 polypeptide is mutated and/or truncated, an IL-15Rα polypeptide is mutated and/or truncated, or both are mutated and/or truncated.

In some embodiments an IL-15 polypeptide may comprise or may lack a native signal peptide (which may have a sequence comprising SEQ ID NO: 369), may comprise or may lack a native propeptide (which may have a sequence comprising SEQ ID NO:371), or any combination thereof.

In some embodiments an IL-15Rα polypeptide may comprise or may lack a native signal sequence (which may have a sequence comprising SEQ ID NO: 370).

In some embodiments the disclosure provides for nucleic acids encoding polypeptide(s) described herein.

In an aspect, polypeptide sequences and/or nucleic acid sequences described herein may be isolated and/or recombinant sequences.

In an aspect, cells described herein may be isolated and/or recombinant cells.

In some embodiments an IL-15 polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 305. In some embodiments, function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, are preserved and/or enhanced in a mutated IL-15 polypeptide.

In some embodiments an IL-15 polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 307. In some embodiments, function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, are preserved and/or enhanced in a mutated IL-15 polypeptide.

In some embodiments an IL-15 polypeptide comprises (a) SEQ ID NO: 305 comprising one, two, three, four, or five amino acid substitutions or (b) SEQ ID NO: 307 comprising one, two, three, four, or five amino acid substitutions. In some embodiments, amino acid substitutions are conservative or non-conservative. In some embodiments amino acid substitution(s) are conservative amino acid substitution(s). In some embodiments, function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, are preserved and/or enhanced in a mutated IL-15 polypeptide.

In some embodiments an IL-15 polypeptide is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 308. In some embodiments, function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, are preserved and/or enhanced in an IL-15 polypeptide encoded by a mutated nucleic acid sequence.

In some embodiments an IL-15 polypeptide is encoded by a nucleic acid comprising (a) SEQ ID NO: 308 comprising one, two, three, four, or five nucleic acid substitutions. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid. In some embodiments, function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, are preserved and/or enhanced in an IL-15 polypeptide encoded by a mutated nucleic acid sequence.

In some embodiments, a nucleic acid encoding an IL-15 polypeptide may comprise a stop codon (such as TAA, TAG, or TGA), positioned at, as a non-limiting example, at the 3′ end of a nucleotide encoding an IL-15 polypeptide.

In some embodiments an IL-15Rα polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 306. In some embodiments an IL-15Rα polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 309. However, In some embodiments an IL-15Rα polypeptide does not have a sequence comprising or consisting of SEQ ID NO: 309 or a sequence having about 95% or more sequence identity thereto. In some embodiments an IL-15Rα polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 311. In some embodiments an IL-15Rα polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 313. In some embodiments an IL-15Rα polypeptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 315. In some embodiments, function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15a are preserved and/or enhanced in a mutated IL-15Rα polypeptide.

In some embodiments an IL-15Rα polypeptide may comprise (a) SEQ ID NO: 306 comprising one, two, three, four, or five amino acid substitutions; (b) SEQ ID NO: 309 comprising one, two, three, four, or five amino acid substitutions; (c) SEQ ID NO: 311 comprising one, two, three, four, or five amino acid substitutions; (d) SEQ ID NO: 313 comprising one, two, three, four, or five amino acid substitutions; or (e) SEQ ID NO: 315 comprising one, two, three, four, or five amino acid substitutions. However, In some embodiments an IL-15Rα polypeptide does not have a sequence comprising or consisting of SEQ ID NO: 309 or a sequence having about 95% or more sequence identity thereto. In some embodiments, amino acid substitutions are conservative or non-conservative. In some embodiments amino acid substitution(s) are conservative amino acid substitution(s). In some embodiments, function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15a are preserved and/or enhanced in a mutated IL-15Rα polypeptide.

In some embodiments an IL-15Rα polypeptide is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 310. However, In some embodiments an IL-15Rα polypeptide is not encoded by a nucleic acid comprising SEQ ID NO: 310 or a having about 85%, about 90%, about 95% or more sequence identity thereto. In some embodiments an IL-15Rα polypeptide is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 312. In some embodiments an IL-15Rα polypeptide is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 314. In some embodiments an IL-15Rα polypeptide is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 316. In some embodiments, function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15a are preserved and/or enhanced in an IL-15a polypeptide encoded by a mutated nucleic acid sequence.

In some embodiments an IL-15Rα polypeptide is encoded by a nucleic acid comprising (a) SEQ ID NO: 310 comprising one, two, three, four, or five nucleic acid substitutions; (b) SEQ ID NO: 312 comprising one, two, three, four, or five nucleic acid substitutions; (c) SEQ ID NO: 314 comprising one, two, three, four, or five nucleic acid substitutions, and (d) SEQ ID NO: 316 comprising one, two, three, four, or five nucleic acid substitutions. However, In some embodiments an IL-15Rα polypeptide is not encoded by a nucleic acid comprising SEQ ID NO: 310 or a having about 85%, about 90%, about 95% or more sequence identity thereto. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid. In some embodiments, function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15a are preserved and/or enhanced in an IL-15a polypeptide encoded by a mutated nucleic acid sequence.

In some embodiments, a nucleic acid encoding an IL-15Rα polypeptide may comprise a stop codon (such as TAA, TAG, or TGA), positioned at, as a non-limiting example, at the 3′ end of a nucleotide encoding an IL-15Rα polypeptide.

In some embodiments an IL-15 polypeptide and an IL-15Rα polypeptide is linked by one or more linker. In some embodiments, a linker is a peptide linker. In some embodiments, a peptide linker is rigid or flexible. In some embodiments, a linker is cleavable. In some embodiments, a linker may promote stability or proper folding of a fusion polypeptide, may increase expression of a fusion polypeptide, may improve biological activity of a fusion polypeptide, may facilitate targeting of a fusion polypeptide, may alter the PK of a fusion polypeptide, or any combination thereof.

In some embodiments one or more linker of an IL-15/IL-15Rα fusion polypeptide independently comprises or consists of any of GSG, LE, SEQ ID NO: 266, 383, 385, 387, 389, 391, or 393, or 395-432 or a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any of SEQ ID NO: 266, 383, 385, 387, 389, 391, or 393, or 395-432. However, In some embodiments, one or more linker of an IL-15/IL-15Rα fusion polypeptide is not SEQ ID NO: 391 and/or SEQ ID NO: 395. In some embodiments one or more linker of an IL-15/IL-15Rα fusion polypeptide independently comprises or consists of any of GSG, LE, SEQ ID NO: 266, 383, 385, 387, 389, 393, or 396-432 or a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any of SEQ ID NO: 266, 383, 385, 387, 389, 393, or 396-432. In some embodiments one or more linker of an IL-15/IL-15Rα fusion polypeptide independently comprises or consists of any of SEQ ID NO: 383, 385, 387, or 389 or a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any of SEQ ID NO: 383, 385, 387, or 389.

In some embodiments one or more linker of an IL-15/IL-15Rα fusion polypeptide is independently encoded by one or more nucleic acid comprising or consisting of any of SEQ ID NO: 384, 386, 388, 390, or 392, by a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of SEQ ID NO: 384, 386, 388, 390, or 392, by one or more nucleic acid encoding any linker comprising or consisting of GSG, LE, or one or more linker set forth in SEQ ID NO: 266 or 393-432, or by one or more nucleic acid encoding any linker having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of SEQ ID NO: 266 or 393-432. However, In some embodiments, one or more linker of an IL-15/IL-15Rα fusion polypeptide is not encoded by SEQ ID NO: 392 and is not encoded by a nucleic acid encoding SEQ ID NO: 391 or 395.

In some embodiments one or more linker of an IL-15/IL-15Rα fusion polypeptide is independently encoded by one or more nucleic acid comprising or consisting of any of SEQ ID NO: 384, 386, 388, or 390, by a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of SEQ ID NO: 384, 386, 388, or 390, by one or more nucleic acid encoding any linker comprising or consisting of GSG or one or more linker set forth in SEQ ID NO: 266, 383, 385, 387, 389, 393, or 396-432, or by one or more nucleic acid encoding any linker having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of SEQ ID NO: 266, 383, 385, 387, 389, 393, or 396-432.

In some embodiments one or more linker of an IL-15/IL-15Rα fusion polypeptide is independently encoded by any of SEQ ID NO: 384, 386, 388, or 390 or by a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of SEQ ID NO: 384, 386, 388, or 390.

In some embodiments a linker has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 383. In some embodiments a linker has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 385. In some embodiments a linker has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 387. In some embodiments a linker has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 389. In some embodiments, one or more function(s) of a linker, such as, but not limited to, one or more of flexibility, rigidity, cleavability, ability to promote stability or proper folding of a fusion polypeptide, ability to increase expression of a fusion polypeptide, ability improve biological activity of a fusion polypeptide, ability facilitate targeting of a fusion polypeptide, ability to alter the PK of a fusion polypeptide, or a combination thereof, of the linker, are preserved and/or enhanced in a mutated linker.

In some embodiments a linker comprises (a) SEQ ID NO: 383 comprising one, two, three, four, or five amino acid substitutions; (b) SEQ ID NO: 385 comprising one, two, three, four, or five amino acid substitutions; (c) SEQ ID NO: 387 comprising one, two, three, four, or five amino acid substitutions; (d) SEQ ID NO: 389 comprising one, two, three, four, or five amino acid substitutions; or (e) SEQ ID NO: 391 comprising one, two, three, four, or five amino acid substitutions. In some embodiments, amino acid substitutions may be conservative or non-conservative. In some embodiments amino acid substitution(s) may be conservative amino acid substitution(s). In some embodiments, one or more function(s) of a linker, such as, but not limited to, one or more of flexibility, rigidity, cleavability, ability to promote stability or proper folding of a fusion polypeptide, ability to increase expression of a fusion polypeptide, ability improve biological activity of a fusion polypeptide, ability facilitate targeting of a fusion polypeptide, ability to alter the PK of a fusion polypeptide, or a combination thereof, of the linker, are preserved and/or enhanced in a mutated linker.

In some embodiments a linker is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 384. In some embodiments a linker is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 386. In some embodiments a linker is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 388. In some embodiments a linker is encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 390. In some embodiments, one or more function(s) of a linker, such as, but not limited to, one or more of flexibility, rigidity, cleavability, ability to promote stability or proper folding of a fusion polypeptide, ability to increase expression of a fusion polypeptide, ability improve biological activity of a fusion polypeptide, ability facilitate targeting of a fusion polypeptide, ability to alter the PK of a fusion polypeptide, or a combination thereof, of the linker, are preserved and/or enhanced in a mutated linker.

In some embodiments a linker is encoded by a nucleic acid comprising (a) SEQ ID NO: 384 comprising one, two, three, four, or five nucleic acid substitutions; (b) SEQ ID NO: 386 comprising one, two, three, four, or five nucleic acid substitutions; (c) SEQ ID NO: 388 comprising one, two, three, four, or five nucleic acid substitutions; (d) SEQ ID NO: 390 comprising one, two, three, four, or five nucleic acid substitutions; or (e) SEQ ID NO: 392 comprising one, two, three, four, or five nucleic acid substitutions. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid. In some embodiments, one or more function(s) of a linker, such as, but not limited to, one or more of flexibility, rigidity, cleavability, ability to promote stability or proper folding of a fusion polypeptide, ability to increase expression of a fusion polypeptide, ability improve biological activity of a fusion polypeptide, ability facilitate targeting of a fusion polypeptide, ability to alter the PK of a fusion polypeptide, or a combination thereof, of the linker, are preserved and/or enhanced in a mutated linker.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker comprises SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, or 335. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is encoded by a nucleic acid comprising SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, 334, or 336. However, In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker does not comprise or consist of SEQ ID NO: 335 or sequences having about 95% or more sequence identity thereto. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is not encoded by a nucleic acid comprising or consisting of SEQ ID NO: 336 or by sequences having about 80%, about 85%, about 90%, or about 95% or more sequence identity thereto. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker comprises SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, or 333. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is encoded by a nucleic acid comprising SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, or 334. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker comprises SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is encoded by a nucleic acid comprising SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker may comprise (a) SEQ ID NO: 317 comprising one, two, three, four, or five amino acid substitutions; (b) SEQ ID NO: 319 comprising one, two, three, four, or five amino acid substitutions; (c) SEQ ID NO: 321 comprising one, two, three, four, or five amino acid substitutions; (d) SEQ ID NO: 323 comprising one, two, three, four, or five amino acid substitutions; (e) SEQ ID NO: 325 comprising one, two, three, four, or five amino acid substitutions; (f) SEQ ID NO: 327 comprising one, two, three, four, or five amino acid substitutions; (g) SEQ ID NO: 329 comprising one, two, three, four, or five amino acid substitutions; (h) SEQ ID NO: 331 comprising one, two, three, four, or five amino acid substitutions; (i) SEQ ID NO: 333 comprising one, two, three, four, or five amino acid substitutions; or (j) SEQ ID NO: 335 comprising one, two, three, four, or five amino acid substitutions. However, In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker does not comprise or consist of SEQ ID NO: 335 or sequences having about 95% or more sequence identity thereto. In some embodiments, amino acid substitutions may be conservative or non-conservative. In some embodiments amino acid substitution(s) may be conservative amino acid substitution(s). In some embodiments, (i) function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, (ii) function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound, and signaling function(s) of IL-15Rα or (iii) both (i) and (ii), are preserved and/or enhanced in a mutated IL-15/IL-15Rα fusion polypeptide.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker may be encoded by a nucleic acid comprising (a) SEQ ID NO: 318 comprising one, two, three, four, or five nucleic acid substitutions; (b) SEQ ID NO: 320 comprising one, two, three, four, or five nucleic acid substitutions; (c) SEQ ID NO: 322 comprising one, two, three, four, or five nucleic acid substitutions; (d) SEQ ID NO: 324 comprising one, two, three, four, or five nucleic acid substitutions; (e) SEQ ID NO: 326 comprising one, two, three, four, or five nucleic acid substitutions; (f) SEQ ID NO: 328 comprising one, two, three, four, or five nucleic acid substitutions; (g) SEQ ID NO: 330 comprising one, two, three, four, or five nucleic acid substitutions; (h) SEQ ID NO: 332 comprising one, two, three, four, or five nucleic acid substitutions; or (i) SEQ ID NO: 334 comprising one, two, three, four, or five nucleic acid substitutions. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid. In some embodiments, (i) function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, (ii) function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15Rα or (iii) both (i) and (ii), are preserved and/or enhanced in an IL-15/IL-15Rα fusion polypeptide encoded by a mutated nucleic acid sequence.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising one or more linker comprises or consists of, e.g., SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333 or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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: 317, 321, 325, 327, 329, 331, or 333.

In some embodiments an IL-15/IL-15Rα fusion polypeptide is encoded by a nucleic acid comprising or consisting of, e.g., SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334 or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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: 318, 322, 326, 328, 330, 332, or 334.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker (L) comprises or consists of any construct A-J as set forth in FIG. 69A. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker (L) comprises or consists of any construct A-I as set forth in FIG. 69A. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker comprises or consists of any construct A, C, or E-I as set forth in FIG. 69A. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is encoded by one or more nucleic acid comprising or consisting of any construct A′-J′ as set forth in FIG. 69B. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is encoded by one or more nucleic acid comprising or consisting of any construct A′-I′ as set forth in FIG. 69B. In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker is encoded by one or more nucleic acid comprising or consisting of any construct A′, C′, or E′-I′ as set forth in FIG. 69B. In some embodiments, sequences comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to any of the sequences set forth in FIGS. 69A and 69B are also provided. In FIGS. 69A and 69B, the lines connecting the IL-15 to the linker and the linker to the IL-15Rα may represent direct linkages, with no intervening sequences, or may represent intervening sequences, such as, but not limited to, a linker, an untranslated sequence (in the case of FIG. 69B), a translated sequence, a sequence comprising one or more restriction endonuclease sites (in the case of FIG. 69B), or a combination thereof.

In some embodiments, a nucleic acid encoding an IL-15/IL-15Rα fusion polypeptide may comprise a stop codon (such as TAA, TAG, or TGA), positioned at, as non-limiting examples, at the 3′ end of a nucleotide encoding an IL-15Rα polypeptide, such as where the encoded fusion polypeptide is in an orientation shown in FIG. 67A or FIG. 668A or at the 3′ end of the IL-15 polypeptide, such as where the encoded fusion polypeptide is in an orientation shown in FIG. 67B or FIG. 668B.

In some embodiments IL-15/IL-15Rα fusion polypeptide and/or an IL-15Rα/IL-15 fusion polypeptide may comprise one or more signal peptide. In some embodiments a fusion polypeptide may comprise the entirety or a portion(s) of the short or the long signal peptide of IL-15 or the entirety or a portion(s) of the signal peptide of IL-15Rα. In some embodiments the entire signal peptide or part of the signal peptide of IL-15, IL-15Rα, or both, may be mutated or deleted. In some embodiments a fusion polypeptide may comprise one or more heterologous signal peptide, i.e., the entirety or a portion of the signal peptide from a molecule other than IL-15 and IL-15Rα. In some embodiments, a heterologous signal peptide may be derived from IL-2, CD33, IgVκ, or IgE. In some embodiments, a signal peptide may be a signal peptide derived from IgE. In some embodiments a signal peptide derived from IgE may comprise or consist of SEQ ID NO: 367. In some embodiments a signal peptide derived from IgE may be encoded by a nucleic acid comprising or consisting of the sequence set forth in SEQ ID NO: 368.

In some embodiments a signal peptide may be cleaved or otherwise removed from an IL-15/IL-15Rα fusion polypeptide.

In some embodiments, a signal peptide may increase or facilitate transcription, translation, translocation, or a combination thereof, of a fusion polypeptide, as compared to a native IL-15Rα signal peptide, a native IL-15 signal peptide, or both. In some embodiments, the signal peptide may be directly or indirectly fused to the N-terminus or to the C-terminus of an IL-15/IL-15Rα fusion polypeptide.

In some embodiments a signal peptide has a sequence comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 367. In some embodiments, function(s) of a signal peptide, such as, but not limited to, one or more signaling function(s) of the signal peptide, are preserved and/or enhanced in a mutated signal peptide.

In some embodiments a signal peptide may comprise SEQ ID NO: 367 comprising one, two, three, four, or five amino acid substitutions. In some embodiments, function(s) of a signal peptide, such as, but not limited to, one or more signaling function(s) of the signal peptide, are preserved and/or enhanced in a mutated signal peptide.

In some embodiments a signal peptide may be encoded by a nucleic acid comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid of SEQ ID NO: 368. In some embodiments, function(s) of a signal peptide, such as, but not limited to, one or more signaling function(s) of the signal peptide, are preserved and/or enhanced in a signal peptide that is encoded by a mutated nucleic acid sequence.

In some embodiments a signal peptide may be encoded by a nucleic acid comprising SEQ ID NO: 368 comprising one, two, three, four, or five nucleic acid substitutions. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid. In some embodiments, function(s) of a signal peptide, such as, but not limited to, one or more signaling function(s) of the signal peptide, are preserved and/or enhanced in a signal peptide that is encoded by a mutated nucleic acid sequence.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE may comprise (a) SEQ ID NO: 337 comprising one, two, three, four, or five amino acid substitutions; (b) SEQ ID NO: 339 comprising one, two, three, four, or five amino acid substitutions; (c) SEQ ID NO: 341 comprising one, two, three, four, or five amino acid substitutions; (d) SEQ ID NO: 343 comprising one, two, three, four, or five amino acid substitutions; (e) SEQ ID NO: 345 comprising one, two, three, four, or five amino acid substitutions; (f) SEQ ID NO: 347 comprising one, two, three, four, or five amino acid substitutions; (g) SEQ ID NO: 349 comprising one, two, three, four, or five amino acid substitutions; (h) SEQ ID NO: 351 comprising one, two, three, four, or five amino acid substitutions; (i) SEQ ID NO: 353 comprising one, two, three, four, or five amino acid substitutions; or (j) SEQ ID NO: 355 comprising one, two, three, four, or five amino acid substitutions. However, In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE does not comprise or consist of SEQ ID NO: 355 or sequences having about 95% or more sequence identity thereto. In some embodiments, amino acid substitutions may be conservative or non-conservative. In some embodiments amino acid substitution(s) may be conservative amino acid substitution(s). In some embodiments, (i) function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, (ii) function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15Rα, (iii) function(s) of a signal peptide derived from IgE, such as, but not limited to, one or more signaling function(s) of the signal peptide, or (iv) all of (i), (ii), and (iii), are preserved and/or enhanced in a mutated IL-15/IL-15Rα fusion polypeptide comprising a signal peptide derived from IgE.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE may be encoded by a nucleic acid comprising (a) SEQ ID NO: 338 comprising one, two, three, four, or five nucleic acid substitutions; (b) SEQ ID NO: 340 comprising one, two, three, four, or five nucleic acid substitutions; (c) SEQ ID NO: 342 comprising one, two, three, four, or five nucleic acid substitutions; (d) SEQ ID NO: 344 comprising one, two, three, four, or five nucleic acid substitutions; (e) SEQ ID NO: 346 comprising one, two, three, four, or five nucleic acid substitutions; (f) SEQ ID NO: 348 comprising one, two, three, four, or five nucleic acid substitutions; (g) SEQ ID NO: 350 comprising one, two, three, four, or five nucleic acid substitutions; (h) SEQ ID NO: 352 comprising one, two, three, four, or five nucleic acid substitutions; (i) SEQ ID NO: 354 comprising one, two, three, four, or five nucleic acid substitutions; or (j) SEQ ID NO: 356 comprising one, two, three, four, or five nucleic acid substitutions. However, In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE is not encoded by SEQ ID NO: 356 or sequences having about 80%, about 85%, about 90%, or about 95% or more sequence identity thereto. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, (i) function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, (ii) function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and one or more signaling function(s) of IL-15Rα, (iii) function(s) of a signal peptide derived from IgE, such as, but not limited to, one or more signaling function(s) of the signal peptide, or (iv) all of (i), (ii), and (iii), are preserved and/or enhanced in an IL-15/IL-15Rα fusion polypeptide comprising a signal peptide derived from IgE, that is encoded by a mutated nucleic acid sequence.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a signal peptide derived from IgE, and comprising a linker, comprises or consists of, e.g., SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353 or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 337, 341, 345, 347, 349, 351, or 353.

However, In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE is not encoded by a nucleic acid comprising or consisting of (i) SEQ ID NO: 356 or by sequences having about 80%, about 85%, about 90%, or about 95% or more sequence identity thereto or (ii) SEQ ID NO: 368 directly or indirectly fused to the 5′ end of SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 310 or by sequences having about 80%, about 85%, about 90%, or about 95% or more sequence identity thereto. In some embodiments one or more linkers is encoded by one or more nucleic acid comprising or consisting of a nucleic acid encoding a linker set forth herein.

In some embodiments an IL-15/IL-15Rα fusion polypeptide comprising a signal peptide derived from IgE, and comprising one or more linker is encoded by a nucleic acid comprising or consisting of, e.g., SEQ ID NO: 338, 342, 346, 348, 350, 352, or 354 or a sequence at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 338, 342, 346, 348, 350, 352, or 354.

In some embodiments a 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) (such as, but not limited to wild type WPRE, such as but not limited to SEQ ID NO: 264, or a mutated 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: 437), variant(s) thereof, or any combination thereof.

In some embodiments (i) an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE (ii) followed by WPREmut2 or wild type WPRE (wt where indicated) may be encoded by a nucleic acid comprising (a) SEQ ID NO: 357 comprising one, two, three, four, or five nucleic acid substitutions; (b) SEQ ID NO: 358 comprising one, two, three, four, or five nucleic acid substitutions; (c) SEQ ID NO: 359 comprising one, two, three, four, or five nucleic acid substitutions; (d) SEQ ID NO: 360 comprising one, two, three, four, or five nucleic acid substitutions; (e) SEQ ID NO: 361 comprising one, two, three, four, or five nucleic acid substitutions; (f) SEQ ID NO: 362 comprising one, two, three, four, or five nucleic acid substitutions; (g) SEQ ID NO: 363 comprising one, two, three, four, or five nucleic acid substitutions; (h) SEQ ID NO: 364 comprising one, two, three, four, or five nucleic acid substitutions; (i) SEQ ID NO: 365 comprising one, two, three, four, or five nucleic acid substitutions; or (j) SEQ ID NO: 366 (wt WPRE) comprising one, two, three, four, or five nucleic acid substitutions. However, In some embodiments (i) an IL-15/IL-15Rα fusion polypeptide comprising a linker and a signal peptide derived from IgE (ii) followed by a wild type or mutant WPRE is not encoded by SEQ ID NO: 366 or sequences having about 80%, about 85%, about 90%, or about 95% or more sequence identity thereto. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid or may result in a codon encoding a different amino acid. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding a conservative amino acid substitution. In some embodiments, one or more nucleic acid substitution in a codon may result in a codon encoding the same amino acid. In some embodiments, (i) function(s) of IL-15, such as, but not limited to, one or more signaling function(s) of IL-15, (ii) function(s) of IL-15Rα, such as, but not limited to, the ability of IL-15Rα be membrane-bound and signaling function(s) of IL-15Rα, (iii) function(s) of a signal peptide derived from IgE, such as, but not limited to, one or more signaling function(s) of the signal peptide, (iv) post-transcriptional regulatory function(s) of mutant or wild type WPRE, or (v) all of (i), (ii), (iii), and (iv) are preserved and/or enhanced in an IL-15/IL-15Rα fusion polypeptide comprising a signal peptide derived from IgE that is encoded by a mutated nucleic acid sequence.

In some embodiments nucleic acid sequences encoding a mbIL-15 polypeptide operatively coupled to a promoter are provided. In some embodiments nucleic acid sequences encoding a mbIL-15 polypeptide operatively coupled to a post-transcriptional regulatory element are provided. In some embodiments the promoter is an MSCV promoter and/or the post-transcriptional regulatory element is a WPRE, optionally a mutated WPRE, optionally WPREmut2. In some embodiments the promoter is MSCV promoter. In some embodiments the WPRE is WPREmut2.

In some embodiments one or more cells comprising one or more nucleic acids (such as in one or more vectors) encoding SEQ ID NO: 305, 306, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, or combinations thereof are provided. In some embodiments one or more cells comprising one or more nucleic acids (such as in one or more vectors) encoding SEQ ID NO: 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 337, 339, 341, 343, 345, 347, 349, 351, 353, or combinations thereof are provided. In some embodiments one or more cells comprising one or more nucleic acids (such as in one or more vectors) encoding SEQ ID NO: 311, 313, 315, 317, 321, 325, 327, 329, 331, 333, 337, 341, 345, 347, 349, 351, 353, or combinations thereof are provided. Such cells may also comprise one or more nucleic acids (such as in one or more vectors) encoding one or more TCRα, one or more TCRβ, one or more CD8α, one or more CD8β, or combinations thereof. Each of TCRα, TCRβ, CD8α, and CD8β may independently be modified or unmodified.

In some embodiments one or more cells comprising one or more nucleic acids (such as in one or more vectors) comprising SEQ ID NO: 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356-366, or combinations thereof are provided. In some embodiments one or more cells comprising one or more nucleic acids (such as in one or more vectors) SEQ ID NO: 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 338, 340, 342, 344, 346, 348, 350, 352, 354, 357-365 or combinations thereof are provided. In some embodiments one or more cells comprising one or more nucleic acids (such as in one or more vectors) SEQ ID NO: 312, 314, 316, 318, 322, 326, 328, 330, 332, 334, 338, 342, 346, 348, 350, 352, 354, 357, 359, 361-365 or combinations thereof are provided. Such cells may also comprise one or more nucleic acids (such as in one or more vectors) encoding one or more TCRα, one or more TCRβ, one or more CD8α, one or more CD8β, or combinations thereof. Each of TCRα, TCRβ, CD8α, and CD8β may independently be modified or unmodified.

In some embodiments nucleic acids do not encode, vectors do not encode, and/or cells do not comprise and/or are not transduced to express SEQ ID NO: 335 or 355 or any sequence having about 95% or more sequence identity to SEQ ID NO: 335 or 355. In some embodiments nucleic acids, vectors, and/or cells do not comprise SEQ ID NO: 336, 356, or 366 or any sequence having about 80%, about 85%, about 90%, or about 95% or more sequence identity to SEQ ID NO: 336, 356, or 366.

In some embodiments cells described herein may comprise a membrane-bound IL-15 and a CD8 polypeptide as described herein. In some embodiments cells described herein may comprise an IL-15/IL-15Rα fusion polypeptide and a CD8 polypeptide as described herein. In some embodiments, cells described herein may comprise an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, a cell receptor (TCR) comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, a chimeric antigen receptor (CAR), or any combination thereof. In some embodiments a cell may comprise an αβ T cell, an γδ T cell, a natural killer cell, a natural killer T cell, a CD4+ cell, a CD8+ cell, a CD4+/CD8+ cell, or combination thereof.

In some embodiments expression of membrane-bound IL-15 may improve immune cell, such as but not limited to, T cell and/or natural killer cell, persistence, functionality, growth, viability, expansion, or any combination thereof, as compared to cells not expressing membrane-bound IL-15. In some embodiments expression of membrane-bound IL-15 may improve immune cell, such as but not limited to, T cell and/or natural killer cell, persistence, functionality, growth, viability, expansion, or any combination thereof, in a tumor microenvironment, as compared to cells not expressing membrane-bound IL-15. In some embodiments expression of membrane-bound IL-15 may increase efficacy of immune cells, such as, but not limited to, T cells and/or natural killer cells, in killing tumor cells, as compared to cells not expressing membrane-bound IL-15. In some embodiments expression of membrane-bound IL-15 may increase ability of immune cells, such as, but not limited to, T cells and/or natural killer cells, to survive in a tumor microenvironment, to persist in killing tumor cells, or any combination thereof, as compared to cells not expressing membrane-bound IL-15. In some embodiments expression of membrane-bound IL-15 may increase ability of immune cells, such as, but not limited to, T cells and/or natural killer cells, to maintain a naive phenotype.

Persistence may be assessed, as a non-limiting example, by the length of time cells are detectable in an individual (e.g., patient) after infusion. As non-limiting examples, persistence may be measured at days, weeks, months, or years after infusion, as non-limiting examples, at about 1 week, about 2 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 6 months, about 9 months, about 12 months, about 18 months, about 24 months, and/or about 30 months after infusion. Persistence may be assessed, as non-limiting examples, by PCR of peripheral blood sample(s), by flow cytometry of peripheral blood samples(s), and/or by analysis of tumor biopsy sample(s). Persistence of cells expressing membrane-bound IL-15 may be compared, as non-limiting examples, to typical persistence of infused ACT cells or persistence of similar cells not expressing membrane-bound IL-15.

Continued ability to kill tumor cells may be measured, as non-limiting examples, via (i) serial killing assays using an IncuCyte (wherein ability to kill/impair tumor growth as measured by fold growth during repeated tumor stimulations over a duration of time is assessed), and/or (ii) via cytokine/effector molecule production (IFNγ via ELISAs and other pro-inflammatory cytokines via Luminex (cytokines measured may include, as non-limiting examples, IFNγ, TNFα, Granzyme B, perforin, IL-2, IL-6, MIP-1β, MIP-1α, GM-CSF, RANTES, IL-18, IL-4, IL-10, and IP10)). Continued ability of cells expressing membrane-bound IL-15 to kill tumor cells may be compared, as non-limiting examples, to continued ability of similar cells not expressing membrane-bound IL-15 to kill tumor cells or continued ability of other control cells to kill tumor cells.

Naivety of phenotype may be assessed, as a non-limiting example, via Tmem panel assay via flow cytometry. Typically, flow cytometer gating is off of CD8+TCR+ cells. Typically, a more naïve phenotype may be indicated by higher frequencies of the T memory subsets Tnaïve/scm (CD45RA+CCR7+), and Tcm (CD45RA−CCR7+) and an increase or retention of the CD39−CD69− and CD27+CD28+ populations. Low CD57 expression may also be desirable.

When assessing the persistence, functionality, growth, viability, expansion, tumor killing efficacy, naivety, or other characteristics of cells expressing dnTGFRβRII, cells such as non-transduced cells, cells transduced with TCR only, cells transduced with CD8 and TCR, or a combination thereof, may serve as control cells, as non-limiting examples.

In some embodiments membrane-bound IL-15 may act in a cis manner (e.g., affecting cells in which it is expressed), in a trans manner (e.g., affecting cells in which it is not expressed), or any combination thereof. In some embodiments in which membrane-bound IL-15 acts in trans, cells adjacent to or near (e.g., within the tumor microenvironment) cells expressing membrane-bound IL-15 may exhibit any or combination of improvements the same or similar to those described for cells expressing membrane-bound IL-15, as compared to cells not adjacent to or near cells expressing membrane-bound IL-15.

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

CD8 polypeptides described herein may comprise the general structure of a N-terminal signal peptide (optional), CD8α immunoglobulin (Ig)-like domain, a stalk domain or region, CD8α transmembrane domain, and a CD8α cytoplasmic domain.

In some embodiments CD8 polypeptides described herein may comprise (a) an immunoglobulin (Ig)-like domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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) a region comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 2; (c) a transmembrane domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, and (d) a cytoplasmic domain comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. The CD8 polypeptides described herein may be co-expressed with a T-cell receptor or CAR-T in a T-cell and used in methods of adoptive cell therapy (ACT). The T-cell may be an αβ T-cell or a γδ T-cell.

In some embodiments, CD8 polypeptides 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 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: 2; (c) at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 3, and (d) a at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. The CD8 polypeptides described herein may be co-expressed with a T-cell receptor or CAR-T in a T-cell and used in methods of adoptive cell therapy (ACT). The T-cell may be an αβ T-cell or a γδ T-cell.

In some embodiments, CD8 polypeptides described herein may comprise (a) SEQ ID NO: 1 comprising one, two, three, four, or five amino acid substitutions; (b) SEQ ID NO: 2 comprising one, two, three, four, or five amino acid substitutions; (c) SEQ ID NO: 3 comprising one, two, three, four, or five amino acid substitutions, and (d) SEQ ID NO: 4 comprising one, two, three, four, or five amino acid substitutions. In some embodiments the substitutions are conservative amino acid substitutions. The CD8 polypeptides described herein may be co-expressed with a T-cell receptor or CAR-T in a T-cell and used in methods of adoptive cell therapy (ACT). The T-cell may be an γδ T-cell or a γδ T-cell.

CD8 is a membrane-anchored glycoprotein that functions as a coreceptor for antigen recognition of the peptide/MHC class I complexes by T cell receptors (TCR) and plays an important role in T cell development in the thymus and T cell activation in the periphery. Functional CD8 is a dimeric protein made of either two a chains (CD8αα) or an α chain and a β chain (CD8αβ), and the surface expression of the β chain may require its association with the coexpressed a chain to form the CD8αβ heterodimer. CD8αα and CD8αβ may be differentially expressed on a variety of lymphocytes. CD8αβ is expressed predominantly on the surface of αβTCR+ T cells and thymocytes, and CD8αα on a subset of αβTCR+, γSTCR+ intestinal intraepithelial lymphocytes, NK cells, dendritic cells, and a small fraction of CD4+ T cells.

For example, the human CD8 gene may express a protein of 235 amino acids. FIG. 1 shows a CD8α protein (CD8α1—SEQ ID NO: 258), which in an aspect is divided into the following domains (starting at the amino terminal and ending at the carboxy terminal of the polypeptide): (1) signal peptide (amino acids −21 to −1), which may be cleaved off in human cells during the transport of the receptor to the cell surface and thus may not constitute part of the mature, active receptor; (2) immunoglobulin (Ig)-like domain (in this embodiment, amino acids 1-115), which may assume a structure, referred to as the immunoglobulin fold, which is similar to those of many other molecules involved in regulating the immune system, the immunoglobulin family of proteins. The crystal structure of the CD8αα receptor in complex with the human MHC molecule HLA-A2 has demonstrated how the Ig domain of CD8αα receptor binds the ligand; (3) membrane proximal region (in this embodiment, amino acids 116-160), which may be an extended linker region allowing the CD8αα receptor to “reach” from the surface of the T-cell over the top of the MHC to the a3 domain of the MHC where it binds. The stalk region may be glycosylated and may be inflexible; (4) transmembrane domain (in this embodiment, amino acids 161-188), which may anchor the CD8αα receptor in the cell membrane and is therefore not part of the soluble recombinant protein; and (5) cytoplasmic domain (in this embodiment, amino acids 189-214), which can mediate a signaling function in T-cells through its association with p56lck, which may be involved in the T cell activation cascade of phosphorylation events. CD8α1 (SEQ ID NO: 258) may be encoded by SEQ ID NO: 434.

CD8α sequences may generally have a sufficient portion of the immunoglobulin domain to be able to bind to MHC. Generally, CD8α molecules may contain all or a substantial part of immunoglobulin domain of CD8α, e.g., SEQ ID NO: 258, but in an aspect 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 molecules of the present disclosure may be dimers (e.g., CD8αα or CD8αβ), and CD8α monomer may be included within the scope of the present disclosure. In an aspect, CD8α of the present disclosure may comprise CD8α1 (SEQ ID NO: 258) and CD8α2 (SEQ ID NO: 259). In an aspect, the present disclosure may comprise CD8α1 (SEQ ID NO: 258) encoded by SEQ ID NO: 434.

CD8α and β subunits 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 about 20 amino acids. CD8α and β chains have two and one N-linked glycosylation sites, respectively, in the Ig-like domains where they share <20% identity in their amino acid sequences. The CD8β stalk region is 10-13 amino acids shorter than the CD8α stalk and is highly glycosylated with O-linked carbohydrates. These carbohydrates on the β, but not the α, stalk region appear to be quite heterogeneous due to complex sialylations, which may be differentially regulated during the developmental stages of thymocytes and upon activation of T cells. Glycan adducts have been shown to play regulatory roles in the functions of glycoproteins and in immune responses. Glycans proximal to transmembrane domains can affect the orientation of adjacent motifs. The unique biochemical properties of the CD8β chain stalk region may present a plausible candidate for modulating the coreceptor function.

The CD8α polypeptide may be modified by replacing CD8α stalk region with a CD8β stalk region to generate a modified CD8α polypeptide. In some embodiments the modified CD8α polypeptides described herein may have a CD8β stalk region 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: 2. The modified CD8α 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. 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. Modified CD8 polypeptides described herein may have a cytoplasmic tail 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: 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 comprise one or more signal peptide comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 directly or indirectly fused to the N-terminus or directly or indirectly fused to the C-terminus of mCD8α polypeptide. The CD8 polypeptides described herein may have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

T-cells may express membrane-bound IL-15, the CD8 polypeptides described herein, or any combination thereof. As a non-limiting example, a T-cell may co-express a T-cell Receptor (TCR) and an IL-15/IL-15Rα fusion polypeptide. As another non-limiting example, a T-cell may co-express a T-cell Receptor (TCR) and a modified CD8 polypeptide described herein. As another non-limiting example, a T-cell may co-express a T-cell Receptor (TCR), an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide described herein. T-cells may also express a chimeric antigen receptor (CAR), CAR-analogues, or CAR derivatives. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

The T-cell may be an αβ T cell, a γδ T cell, a natural killer T cell, or a combination thereof if in a population. The T cell may be a CD4+ T cell, CD8+ T cell, or a CD4+/CD8+ T cell. In some embodiments a cell may comprise an αβ T cell, a γδ T cell, a natural killer T cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof. Specifically, the T cell may be a γδ1 T cell or a γδ2 T cell.

A T cell may be an αβ T cell and may express a CD8 polypeptide described herein. A T cell may be an αβ T cell and may express a CD8 polypeptide described herein, for example, a modified CD8α polypeptide or a CD8α polypeptide with a CD8β stalk region, e.g., m1CD8α in Constructs #11 and #12 (FIG. 4) and CD8α* (FIG. 55B). A T cell may be an αβ T cell and may express one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A T cell may be a γδ T cell and may express a CD8 polypeptide described herein and/or a membrane-bound IL-15 as described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified. In some embodiments a T cell may be a γδ T cell and may express a CD8 polypeptide described herein, for example, a modified CD8α polypeptide or a modified CD8α polypeptide with a CD8β stalk region, e.g., m1CD8α in Constructs #11 and #12 (FIG. 4) and CD8α* (FIG. 55B). A T cell may be a γδ T cell and may express one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A T cell or cells comprising, or comprising nucleic acid(s) 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, an IL-15 polypeptide, an IL-15Rα polypeptide, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A T cell or cells comprising, or comprising nucleic acid(s) encoding, one or any combination of a TCR comprising an α chain and a β chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. A T cell or cells comprising, or comprising nucleic acid(s) encoding, one or any combination of a TCR comprising a γ chain and a 8 chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. A T cell or cells comprising, or comprising nucleic acid(s) encoding, one or any combination of a CAR, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A T cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising an α chain and a β chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments a T cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. A T cell or cells comprising, or comprising nucleic acid(s) encoding, a CAR, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A T cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising an α chain and a β chain and/or a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) may be provided. A T cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain and a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) may be provided. A T cell or cells comprising, or comprising nucleic acid(s) encoding, a CAR and a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A T cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising an α chain and a β chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments a T cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising a γ chain and a 8 chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide may be provided. A T cell or cells comprising, or comprising nucleic acid(s) encoding a CAR, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

Natural Killer (NK) Cells

Natural Killer (NK) cells may also be engineered and used in adoptive cell therapy (ACT). See, e.g., Morton L T, et al., “T cell receptor engineering of primary NK cells to therapeutically target tumors and tumor immune evasion”, J Immunother Cancer, Mar. 14, 2022; 10:e003715, which is incorporated by reference herein in its entirety. In some embodiments engineered NK cells are provided.

NK cells may express membrane-bound IL-15, the CD8 polypeptides described herein, or any combination thereof. As a non-limiting example, a NK cell may co-express a T-cell Receptor (TCR) and an IL-15/IL-15Rα fusion polypeptide. As another non-limiting example, a NK cell may co-express a T-cell Receptor (TCR) and a modified CD8 polypeptide described herein. As another non-limiting example, a NK cell may co-express a T-cell Receptor (TCR), an IL-15/IL-15Rα fusion polypeptide, and a CD8 polypeptide described herein. NK cells may also express a chimeric antigen receptor (CAR), CAR-analogues, or CAR derivatives. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

The NK cell may express a CD8 polypeptide described herein. A NK cell may express a CD8 polypeptide described herein, for example, a modified CD8α polypeptide or a CD8α polypeptide with a CD8β stalk region, e.g., m1CD8α in Constructs #11 and #12 (FIG. 4) and CD8α* (FIG. 55B). A NK cell may express one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, an IL-15/IL-15Rα fusion polypeptide, a CD8 polypeptide, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A NK cell or cells comprising, or comprising nucleic acid(s) 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, an IL-15 polypeptide, an IL-15Rα polypeptide, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A NK cell or cells comprising, or comprising nucleic acid(s) encoding, one or any combination of a TCR comprising an α chain and a β chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. A NK cell or cells comprising, or comprising nucleic acid(s) encoding, one or any combination of a TCR comprising a γ chain and a δ chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. A NK cell or cells comprising, or comprising nucleic acid(s) encoding, one or any combination of a CAR, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A NK cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising an α chain and a β chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments a NK cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising a γ chain and a 8 chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. A NK cell or cells comprising, or comprising nucleic acid(s) encoding, a CAR, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A NK cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising an α chain and a β chain and/or a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) may be provided. A NK cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain and a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) may be provided. A NK cell or cells comprising, or comprising nucleic acid(s) encoding, a CAR and a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A NK cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising an α chain and a β chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and/or a CD8 polypeptide may be provided. In some embodiments a NK cell or cells comprising, or comprising nucleic acid(s) encoding, a TCR comprising a γ chain and a δ chain, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide may be provided. A NK cell or cells comprising, or comprising nucleic acid(s) encoding a CAR, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide may be provided. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

Table 1 shows examples of the peptides to which TCRs bind when the peptide is in a complex with an MHC molecule. (MHC molecules in humans may be referred to as HLA, human leukocyte-antigens).

T-Cell Receptor and Peptides

TCR name
Peptide (SEQ ID NO:)

Tumor Associated Antigens (TAA)

Tumor associated antigen (TAA) peptides may be used with the IL-15/IL-15Rα fusion polypeptides and/or CD8 polypeptides constructs, methods and embodiments described herein. For example, the T-cell receptors (TCRs) described herein may specifically bind to the TAA peptide when bound to a human leukocyte antigen (HLA). This is also known as a major histocompatibility complex (MHC) molecule. The MHC-molecules of the human are also designated as human leukocyte-antigens (HLA).

Tumor associated antigen (TAA) peptides that may be used with the IL-15/IL-15Rα fusion polypeptides and/or CD8 polypeptides described herein include, but are not limited to, those listed in Table 3 and those TAA peptides described in U.S. Patent Application Publication No. 2016/0187351; U.S. Patent Application Publication No. 2017/0165335; U.S. Patent Application Publication No. 2017/0035807; U.S. Patent Application Publication No. 2016/0280759; U.S. Patent Application Publication No. 2016/0287687; U.S. Patent Application Publication No. 2016/0346371; U.S. Patent Application Publication No. 2016/0368965; U.S. Patent Application Publication No. 2017/0022251; U.S. Patent Application Publication No. 2017/0002055; U.S. Patent Application Publication No. 2017/0029486; U.S. Patent Application Publication No. 2017/0037089; U.S. Patent Application Publication No. 2017/0136108; U.S. Patent Application Publication No. 2017/0101473; U.S. Patent Application Publication No. 2017/0096461; U.S. Patent Application Publication No. 2017/0165337; U.S. Patent Application Publication No. 2017/0189505; U.S. Patent Application Publication No. 2017/0173132; U.S. Patent Application Publication No. 2017/0296640; U.S. Patent Application Publication No. 2017/0253633; U.S. Patent Application Publication No. 2017/0260249; U.S. Patent Application Publication No. 2018/0051080; U.S. Patent Application Publication No. 2018/0164315; U.S. Patent Application Publication No. 2018/0291082; U.S. Patent Application Publication No. 2018/0291083; U.S. Patent Application Publication No. 2019/0255110; U.S. Pat. Nos. 9,717,774; 9,895,415; U.S. Patent Application Publication No. 2019/0247433; U.S. Patent Application Publication No. 2019/0292520; U.S. Patent Application Publication No. 2020/0085930; U.S. Pat. Nos. 10,336,809; 10,131,703; 10,081,664; 10,081,664; 10,093,715; 10,583,573; and U.S. Patent Application Publication No. 2020/00085930; the contents of each of these publications, sequences, and sequence listings described therein are herein incorporated by reference in their entireties. The Tumor associated antigen (TAA) peptides described herein may be bound to an HLA (MHC molecule). The Tumor associated antigen (TAA) peptides bound to an HLA may be recognized by a TCR described herein, optionally co-expressed with CD8 polypeptides described herein.

Methods for the activation, transduction, and/or expansion of T cells, e.g., tumor-infiltrating lymphocytes, CD8+ T cells, CD4+ T cells, and T cells, that may be used for transgene expression are described herein. T cells may be activated, transduced, and expanded, while depleting α- and/or β-TCR positive cells. The T-cell may be a αβ T cell, γδ T cell, or a natural killer T cell.

Methods for the ex vivo expansion of a population of engineered γδ T-cells for adoptive transfer therapy are described herein. Engineered γδ T cells of the disclosure may be expanded ex vivo. Engineered T cells described herein can be expanded in vitro without activation by APCs, or without co-culture with APCs, and aminophosphates. Methods for transducing T cells are described in U.S. Patent Application No. 2019/0175650, published on Jun. 13, 2019, the contents of which are incorporated by reference in their entirety. Other methods for transduction and culturing of T-cells may be used.

T cells, including γδ T cells, may be isolated from a complex sample that is cultured in vitro. In some embodiments whole PBMC population, without prior depletion of specific cell populations, such as monocytes, αβ T-cells, B-cells, and NK cells, can be activated and expanded. In some embodiments enriched T cell populations can be generated prior to their specific activation and expansion. In some embodiments activation and expansion of γδ T cells may be performed with or without the presence of native or engineered antigen presenting cells (APCs). In some embodiments, isolation and expansion of T cells from tumor specimens can be performed using immobilized T cell mitogens, including antibodies specific to γδ TCR, and other γδ TCR activating agents, including lectins. In some 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 to γδ TCR, and other γδ TCR activating agents, including lectins. T cells, including γδ T cells, may be isolated from leukapheresis of a subject, for example, a human subject. In some embodiments γδ T cells are not isolated from peripheral blood mononuclear cells (PBMC). The T cells may be isolated using anti-CD3 and anti-CD28 antibodies, optionally with recombinant human Interleukin-2 (rhIL-2), e.g., between about 50 and 150 U/mL rhIL-2.

The isolated T cells can rapidly expand in response to contact with one or more antigens. Some γδ T cells, such as Vγ9Vδ2+ T cells, can rapidly expand in vitro in response to contact with some antigens, like prenyl-pyrophosphates, alkyl amines, and metabolites or microbial extracts during tissue culture. Stimulated T-cells can exhibit numerous antigen-presentation, co-stimulation, and adhesion molecules that can facilitate the isolation of T-cells from a complex sample. T cells within 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 of time. Stimulation of T cells with a suitable antigen can expand T cell population in vitro.

Activation and expansion of γδ T cells can be performed using activation and co-stimulatory agents described herein to trigger specific γδ T cell proliferation and persistence populations. In some embodiments activation and expansion of γδ T-cells from different cultures can achieve distinct clonal or mixed polyclonal population subsets. In some embodiments different agonist agents can be used to identify agents that provide specific γδ activating signals. In some embodiments agents that provide specific γδ activating signals can be different monoclonal antibodies (MAbs) directed against the γδ TCRs. In some embodiments companion co-stimulatory agents to assist in triggering specific γδ T cell proliferation without induction of cell energy and apoptosis can be used. These co-stimulatory agents can include ligands binding 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 co-stimulatory agents can be antibodies specific to unique epitopes on CD2 and CD3 molecules. CD2 and CD3 can have different conformation structures when expressed on αβ or γδ T-cells. In some embodiments specific antibodies to CD3 and CD2 can lead to distinct activation of γδ T cells.

The ability of γδ T cells to recognize a broad spectrum of antigens can be enhanced by genetic engineering of the γδ T cells. The γδ T cells can be engineered to provide a universal allogeneic therapy that recognizes an antigen of choice in vivo. Genetic engineering of the γδ T-cells may comprise stably integrating a construct expressing a tumor recognition moiety, such as αβ TCR, γδ TCR, chimeric antigen receptor (CAR), which combines both antigen-binding and T-cell activating functions into a single receptor, an antigen binding fragment thereof, or a lymphocyte activation domain into the genome of the isolated γδ T-cell(s), a cytokine (for example, 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 ex vivo and in vivo. Genetic engineering of the isolated γδ T-cell may also include deleting or disrupting gene expression from one or more endogenous genes in the genome of the isolated γδ T-cells, such as the MHC locus (loci).

Engineered (or transduced) T cells, including γδ T cells, can be expanded ex vivo without stimulation by an antigen presenting cell or aminobisphosphonate. Antigen reactive engineered T cells of the present disclosure may be expanded ex vivo and in vivo. In some embodiments an active population of engineered T cells may be expanded ex vivo without antigen stimulation by an antigen presenting cell, an antigenic peptide, a non-peptide molecule, or a small molecule compound, such as an aminobisphosphonate but using certain antibodies, cytokines, mitogens, or fusion proteins, such as IL-17 Fc fusion, MICA Fc fusion, and CD70 Fc fusion. Examples of antibodies that can be used in the expansion of a γδ T-cell population include anti-CD3, anti-CD27, anti-CD30, anti-CD70, anti-OX40, anti-NKG2D, or anti-CD2 antibodies, examples of cytokines may comprise IL-2, IL-15, IL-12, IL-21, IL-18, IL-9, IL-7, and/or IL-33, and examples of mitogens may comprise CD70 the ligand for human CD27, phytohaemagglutinin (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 Lectin (VGA) or another suitable mitogen capable of stimulating T-cell proliferation.

A population of engineered T cells, including γδ T cells, can be expanded in less than about 60 days, less than about 48 days, less than about 36 days, less than about 24 days, less than about 12 days, or less than about 6 days. In some embodiments a population of engineered T cells can be expanded from about 7 days to about 49 days, about 7 days to about 42 days, from about 7 days to about 35 days, from about 7 days to about 28 days, from about 7 days to about 21 days, or from about 7 days to about 14 days. The T-cells may be expanded for between about 1 and about 21 days. For example, the T-cells may be expanded for about 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.

In some embodiments the same methodology may be used to isolate, activate, and expand αβ T cells.

In some embodiments the same methodology may be used to isolate, activate, and expand γδ T cells.

Vectors

Engineered cells may be generated using various methods, including those recognized in the literature. For example, a polynucleotide encoding an expression cassette that comprises a tumor recognition, or another type of recognition moiety, can be stably introduced into the T-cell by a transposon/transposase system or a viral-based gene transfer system, such as a lentiviral or a retroviral system, or another suitable method, such as transfection, electroporation, transduction, lipofection, calcium phosphate (CaPO4), nanoengineered substances, such as Ormosil, viral delivery methods, including adenoviruses, retroviruses, lentiviruses, adeno-associated viruses, or another suitable method. A number of viral methods have been used for human gene therapy, such as the methods described in WO 1993/020221, the content of which is incorporated herein in its entirety. Non-limiting examples of viral methods that can be used to engineer cells may comprise γ-retroviral, adenoviral, lentiviral, herpes simplex virus, vaccinia virus, pox virus, or adeno-virus associated viral methods. A cell may comprise an αβ T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a CD4+ T cell, CD8+ T cell, a CD4+/CD8+ cell, or any combination thereof. Specifically, the cell may comprise a γδ1 T cell and/or a γδ2 T cell.

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

A single lentiviral cassette can be used to create a single lentiviral vector, expressing at least four individual monomer proteins of two distinct dimers from a single multi-cistronic mRNA so as to co-express the dimers on the cell surface. For example, the integration of a single copy of the lentiviral vector was sufficient to transform T cells to co-express TCRαβ and CD8αβ, optionally αβ T cells or γδ T cells.

Vectors may comprise a multi-cistronic cassette within a single vector capable of expressing more than one, more than two, more than three, more than four genes, more than five genes, or more than six genes, in which the polypeptides encoded by these genes may interact with one another or may form dimers. The dimers may be homodimers, e.g., two identical proteins forming a dimer, or heterodimers, e.g., two structurally different proteins forming a dimer.

Additionally, multiple vectors may be used to transfect cells with the constructs and sequences described herein. One or more vectors may comprise any combination of TCR transgene(s), IL-15/IL-15Rα fusion polypeptide transgene(s), and CD8 transgene(s) in any order. As a non-limiting example, a first vector may comprise a transgene encoding a TCR, a second vector may comprise a transgene encoding an IL-15/IL-15Rα fusion polypeptide, and a third vector may comprise a transgene encoding a CD8 α polypeptide described herein, and the vectors may be transfected into cells either simultaneously or sequentially in any order, using recognized methods. As another non-limiting example, a single vector may encode two transgenes in any order, or a single vector may encode three or more transgenes in any order. As another non-limiting example, a cell line that is stably transfected with one or more transgene(s) may then be transfected with one or more other transgene(s).

One or more vector may comprise a nucleic acid encoding a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide). One or more vector may comprise a nucleic acid encoding a CD8 polypeptide. One or more vector may comprise a nucleic acid encoding a CD8α polypeptide. One or more vector may comprise a nucleic acid encoding a CD8β polypeptide.

One or more vector may comprise a nucleic acid encoding a T cell receptor (TCR) comprising an α chain and a β chain. One or more vector may comprise a nucleic acid encoding a T cell receptor (TCR) comprising an γ chain and a δ chain. One or more vector may comprise a nucleic acid encoding a chimeric antigen receptor (CAR).

More than one vector may comprise a nucleic acid or nucleic acids encoding one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A single vector may comprise a nucleic acid or nucleic acids encoding one or any combination of an IL-15 polypeptide, an IL-15Rα polypeptide, a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), a CD8 polypeptide, a TCR comprising an α chain and a β chain, a TCR comprising an γ chain and a δ chain, and/or a CAR. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

As used herein, the term “cistron” refers to a section of a nucleic acid molecule that specifies the formation of one polypeptide chain, i.e. coding for one polypeptide chain. For example, “mono-cistron” refers to one section of a nucleic acid molecule that specifies the formation of one polypeptide chain, i.e. coding for one polypeptide chain; “bi-cistron” refers to two sections of a nucleic acid molecule that specify the formation of two polypeptide chains, i.e. coding for two polypeptide chains; “tri-cistron” refers to three sections of a nucleic acid molecule that specify the formation of three polypeptide chains, i.e. coding for three polypeptide chains; etc.; “multicistron” refers two or more sections of a nucleic acid molecule that specify the formation of two or more polypeptide chains, i.e. coding for two or more polypeptide chains.

As used herein, the term “arranged in tandem” refers to the arrangement of the genes contiguously, one following or behind the other, in a single file on a nucleic acid sequence. The genes are ligated together contiguously on a nucleic acid sequence, with the coding strands (sense strands) of each gene ligated together on a nucleic acid sequence.

A transgene may further include one or more multicistronic element(s) and the multicistronic element(s) may be positioned, as non-limiting examples, between any, some, or each of a nucleic acid encoding a TCRα or a portion thereof, a nucleic acid encoding a TCRβ or a portion thereof, a nucleic acid encoding a CD8α or a portion thereof, a nucleic acid encoding a CD8β or a portion thereof, and/or a nucleic acid encoding a IL-15/IL-15Rα fusion polypeptide or a portion thereof. The multicistronic element(s) may be positioned, as non-limiting examples, between any two nucleic acid sequences encoding of TCRα, TCRβ, CD8α, CD8β, and/or a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide), and these coding sequences may be in any order. The multicistronic element(s) may include a sequence encoding a ribosome skip element selected from among a T2A, a P2A, a E2A or a F2A or an internal ribosome entry site (IRES).

As used herein, the term “self-cleaving 2A peptide” refers to relatively short peptides (of the order of 20 amino acids long, depending on the virus of origin) acting co-translationally, by preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping to the next codon, and the nascent peptide cleaving between the Gly and Pro. After cleavage, the short 2A peptide remains directly or indirectly fused to the C-terminus of the ‘upstream’ protein, while the proline is added to the N-terminus of the ‘downstream’ protein. 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 content of which including 2A nucleic acid and amino acid sequences are incorporated herein by reference in their entireties). By adding one or more linker sequences (such as, but not limited to, GSG, LE, SGSG (SEQ ID NO: 266), or the linkers set forth in SEQ ID NO: 383, 385, 387, 389, 393, 396-432) before the self-cleaving 2A sequence, this may enable efficient synthesis of biologically active proteins, e.g., TCRs.

As used herein, the term “internal ribosome entry site (IRES)” refers to a nucleotide sequence located in a messenger RNA (mRNA) sequence, which can initiate translation without relying on the 5′ cap structure. IRES is usually located in the 5′ untranslated region (5′UTR) but may also be located in other positions of the mRNA. In some embodiments IRES may be selected from IRES from viruses, IRES from cellular mRNAs, in particular IRES from picornavirus, such as polio, EMCV and FMDV, flavivirus, such as hepatitis C virus (HCV), pestivirus, such as classical swine fever virus (CSFV), retrovirus, such as murine leukemia virus (MLV), lentivirus, such as simian immunodeficiency virus (SIV), and insect RNA virus, such as cricket paralysis virus (CRPV), and IRES from cellular mRNAs, e.g. translation initiation factors, such as eIF4G, and DAP5, transcription factors, such as c-Myc, and NF-κB-repressing factor (NRF), growth factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), platelet-derived growth factor B (PDGF-B), homeotic genes, such as antennapedia, survival proteins, such as X-linked inhibitor of apoptosis (XIAP), and Apaf-1, and other cellular mRNA, such as BiP.

Constructs and vectors described herein may be used with the methodology described in U.S. Patent Application Publication No. 2019/0175650, published on Jun. 13, 2019, the contents of which are incorporated by reference in their entirety.

In some embodiments a 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) (such as, but not limited to wild type WPRE, such as but not limited to SEQ ID NO: 264, or a mutated 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: 437), variant(s) thereof, or any combination thereof.

In some embodiments a vector may further comprise one or more promoter. In some embodiments the promoter(s) may be selected from cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, myelin basic protein (MBP) promoter, glial fibrillary acidic protein (GFAP) promoter, modified MoMuLV LTR comprising myeloproliferative sarcoma virus enhancer (MNDU3), Ubiqitin C promoter, EF-1 alpha promoter, Murine Stem Cell Virus (MSCV) promoter, the promoter from CD69, nuclear factor of activated T-cells (NFAT) promoter, IL-2 promoter, minimal IL-2 promoter, or a combination thereof.

In some embodiments a vector may comprise one or more Kozak sequence. In some embodiments, the Kozak sequence may initiate, increase, or facilitate translation, or a combination thereof. In some embodiments, the Kozak sequence may be GCCACC. In some embodiments, the Kozak sequence may be ACCATGG. In some embodiments, the Kozak sequence may be GCCNCCATGG. where N is a purine (A or G) (SEQ ID NO: 382).

In some embodiments a vector may comprise one or more Factor Xa sites.

In some embodiments a vector may comprise one or more enhancer. In some embodiments the enhancer may comprise Conserved Non-Coding Sequence (CNS) 0, CNS 1, CNS2, CNS 3, CNS 4, or portions or any combination thereof.

In some embodiments a vector may be a viral vector or a non-viral vector.

In some embodiments a vector may be pseudotyped with an envelope protein of a virus selected from the native feline endogenous virus (RD114), a chimeric version of RD114 (RD114TR), gibbon ape leukemia virus (GALV), a chimeric version of 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 retroviral envelope glycoprotein (BaEV), lymphocytic choriomeningitis virus (LCMV), or a combination thereof.

Non-viral vectors may also be used with the sequences, constructs, and cells described herein.

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 guns), microinjection, or any combination thereof. Various methods of transfecting cells are known in the art. See, e.g., Sambrook & Russell (Eds.) Molecular Cloning: A Laboratory Manual (3rd Ed.) Volumes 1-3 (2001) Cold Spring Harbor Laboratory Press; Ramamoorth & Narvekar “Non Viral Vectors in Gene Therapy—An Overview.” J Clin Diagn Res. (2015) 9(1): GE01-GE06.

Gene Editing

Compositions

Compositions may comprise a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or the CD8 polypeptides described herein. Further, compositions described herein may comprise a T-cell and/or a natural killer cell expressing a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or CD8 polypeptides described herein and/or a TCR as described herein. The compositions described herein may comprise a T-cell and/or a natural killer cell expressing a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or CD8 polypeptides described herein and a T-cell receptor (TCR), optionally a TCR that specifically binds one of the TAA described herein complexed with an antigen presenting protein, e.g., MHC, referred to as HLA in humans, for human leukocyte antigen. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

To facilitate administration, the T cells and/or natural killer cells described herein can be made into a pharmaceutical composition or made into an implant appropriate for administration in vivo, with pharmaceutically acceptable carriers or diluents. The means of making such a composition or an implant are described in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980).

The T cells and/or natural killer cells described herein can be formulated into a preparation in semisolid or liquid form, such as a capsule, solution, infusion, or injection. Means known in the art can be utilized to prevent or minimize release and absorption of the composition until it reaches the target tissue or organ, or to ensure timed-release of the composition. Desirably, however, a pharmaceutically acceptable form is employed that does not hinder the cells from expressing the CARs or TCRs. Thus, desirably the T cells and/or natural killer cells described herein can be made into a pharmaceutical composition comprising a carrier. The T cells and/or natural killer cells described herein can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. The carrier and composition can be sterile. Carriers include, for example, a balanced salt solution, such as Hanks' balanced salt solution, or normal saline. The formulation should suit the mode of administration. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, as well as any combination thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, that do not deleteriously react with the T-cells and/or natural killer cells. The cells may be αβ T cells, γδ T cells, and/or natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or CD8 polypeptides described herein, optionally a TCR described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A composition of the present disclosure can be provided in unit dosage form wherein each dosage unit, e.g., an injection, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents.

The compositions described herein may be a pharmaceutical composition. Pharmaceutical composition described herein may further comprise an adjuvant selected from the group consisting of colony-stimulating factors, including but not limited to Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, interferon-alpha, or a combination thereof.

Pharmaceutical compositions described herein may comprise an adjuvant selected from the group consisting of colony-stimulating factors, e.g., Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod and resiquimod.

Other examples for useful adjuvants include, but are not limited to, chemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such as Poly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, immune checkpoint inhibitors including ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system (e.g. anti-CD40, anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may act therapeutically and/or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present disclosure can readily be determined by the skilled artisan without undue experimentation.

CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly it enhances dendritic cell maturation and differentiation, resulting in enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) generation, 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 alum or incomplete Freund's adjuvant (IFA) that normally promote a TH2 bias. CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak. They also accelerate the immune response and enable the antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG in some experiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response. A CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany). In some embodiments dSLIM may be a preferred component of a pharmaceutical composition described herein. Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Methods of Treatment and Preparation

Engineered T cells and/or engineered natural killer cells may express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or CD8 polypeptide(s) described herein. Further, engineered T cells and/or engineered natural killer cells may express a TCR described herein. The TCR expressed by the engineered T cells and/or engineered natural killer cells may recognize a TAA bound to an HLA as described herein. Engineered T cells and/or engineered natural killer cells of the present disclosure can be used to treat a subject in need of treatment for a condition, for example, a cancer described herein. The cells may be αβ T cells, γδ T cells, and/or natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide, and optionally a TCR described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

A method of treating a condition (e.g., ailment) in a subject with T cells and/or natural killer cells described herein may comprise administering to the subject a therapeutically effective amount of engineered T cells and/or engineered natural killer cells described herein, optionally γδ T cells. T cells and/or natural killer cells described herein may be administered at various regimens (e.g., timing, concentration, dosage, spacing between treatment, and/or formulation). A subject can also be preconditioned with, for example, chemotherapy, radiation, or a combination of both, prior to receiving engineered T cells and/or engineered natural killer cells of the present disclosure. A population of engineered T cells and/or engineered natural killer cells may also be frozen or cryopreserved prior to being administered to a subject. A population of engineered T cells and/or engineered natural killer cells can include two or more cells that express identical, different, or a combination of identical and different tumor recognition moieties. For instance, a population of engineered T-cells and/or engineered natural killer cells can include several distinct engineered T cells and/or engineered natural killer cells that are 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 cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide described herein, and optionally a TCR described herein.

T cells and/or natural killer cells described herein, including αβ T-cells and γδ T cells, may be used to treat various conditions. The cells may be αβ T cells, γδ T cells, and/or natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide, and optionally a TCR described herein. T cells and/or natural killer cells described herein may be used to treat a cancer, including solid tumors and hematologic malignancies. Non-limiting examples of cancers 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, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer.

The T cells and/or natural killer cells described herein may be used to treat an infectious disease. The T cells and/or natural killer cells described herein may be used to treat an infectious disease, an infectious disease may be caused a virus. The T cells and/or natural killer cells described herein may be used to treat an immune disease, such as an autoimmune disease. The cells may be αβ T cells, γδ T cells, and/or natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide, and optionally a TCR described herein.

Treatment with T cells and/or natural killer cells described herein, optionally γδ T cells, may be provided to the subject before, during, and after the clinical onset of the condition. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. A treatment can include administering to a subject a pharmaceutical composition comprising engineered T cells described herein. The cells may be αβ T cells, γδ T cells, and/or natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide, and optionally a TCR described herein.

In some embodiments administration of engineered T cells and/or engineered natural killer cells of the present disclosure to a subject may modulate the activity of endogenous lymphocytes in a subject's body. In some embodiments administration of engineered T cells and/or engineered natural killer cells to a subject may provide an antigen to an endogenous T-cell and may boost an immune response. In some embodiments the memory T cell may be a CD4+ T-cell. In some embodiments the memory T cell may be a CD8+ T-cell. In some embodiments administration of engineered T cells and/or engineered natural killer cells of the present disclosure to a subject may activate the cytotoxicity of another immune cell. In some embodiments the other immune cell may be a CD8+ T-cell. In some embodiments the other immune cell may be a Natural Killer T-cell. In some embodiments administration of engineered γδ T-cells and/or engineered natural killer cells of the present disclosure to a subject may suppress a regulatory T-cell. In some embodiments the regulatory T-cell may be a FOX3+ Treg cell. In some embodiments the regulatory T-cell may be a FOX3-Treg cell. Non-limiting examples of cells whose activity can be modulated by engineered T cells and/or engineered natural killer cells of the disclosure may comprise: hematopioietic stem cells; B cells; CD4; CD8; red blood cells; white blood cells; dendritic cells, including dendritic antigen presenting cells; leukocytes; macrophages; memory B cells; memory T-cells; monocytes; natural killer cells; neutrophil granulocytes; T-helper cells; and T-killer cells. The cells may be αβ T cells, γδ T cells, and/or natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide, and optionally a TCR described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

During most bone marrow transplants, a combination of cyclophosphamide with total body irradiation may be conventionally employed to prevent rejection of the hematopietic stem cells (HSC) in the transplant by the subject's immune system. In some embodiments incubation of donor bone marrow with interleukin-2 (IL-2) ex vivo may be performed to enhance the generation of killer lymphocytes in the donor marrow. Interleukin-2 (IL-2) is a cytokine that may be necessary for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies of the adoptive transfer of γδ T-cells into humans may require the co-administration of γδ T-cells and interleukin-2. However, both low- and high-dosages of IL-2 can have highly toxic side effects. IL-2 toxicity can manifest in multiple organs/systems, most significantly the heart, lungs, kidneys, and central nervous system. In some embodiments the disclosure provides a method for administrating engineered T cells and/or engineered natural killer cells to a subject without the co-administration of a native cytokine or modified versions thereof, such as IL-2, IL-15, IL-12, IL-21. In some embodiments engineered T cells and/or engineered natural killer cells can be administered to a subject without co-administration with IL-2. In some embodiments engineered T cells and/or engineered natural killer cells may be administered to a subject during a procedure, such as a bone marrow transplant without the co-administration of IL-2.

In some embodiments the methods may further comprise administering a chemotherapy agent. The dosage of the chemotherapy 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 T-cells and/or natural killer cells. The chemotherapy agent may be cyclophosphamide, fludarabine, or a combination thereof. The chemotherapy agent may comprise dosing at about 400-600 mg/m2/day of cyclophosphamide. The chemotherapy agent may comprise dosing at about 10-30 mg/m2/day of fludarabine.

In some embodiments the methods may further comprise pre-treatment of the patient with low-dose radiation prior to administration of the composition comprising T-cells and/or natural killer cells. The low dose radiation may comprise about 1.4 Gy for about 1-6 days, such as about 5 days, prior to administration of the composition comprising T-cells.

In some embodiments the patient may be HLA-A*02.

In some embodiments the patient may be HLA-A*06.

In some embodiments the methods may further comprise administering an anti-PD1 antibody. The anti-PD1 antibody may be a humanized antibody. The anti-PD1 antibody may be pembrolizumab. The dosage of the anti-PD1 antibody may be about 200 mg. The anti-PD1 antibody may be administered every 3 weeks following T-cell administration.

In some embodiments the dosage of T-cells and/or natural killer cells may be between about 0.8-1.2×109 T cells and/or natural killer cells. The dosage of the T cells and/or natural killer cells may be about 0.5×108 to about 10×109 T cells and/or natural killer cells. The dosage of T-cells and/or natural killer cells may be about 1.2-3×109 T cells and/or natural killer cells, about 3-6×109 T cells and/or natural killer cells, about 10×109 T cells and/or natural killer cells, about 5×109 T cells and/or natural killer cells, about 0.1×109 T cells and/or natural killer cells, about 1×108 T cells and/or natural killer cells, about 5×108 T cells and/or natural killer cells, about 1.2-6×109 T cells and/or natural killer cells, about 1-6×109 T cells and/or natural killer cells, or about 1-8×109 T cells and/or natural killer cells.

In some embodiments the T cells and/or natural killer cells may be administered in 3 doses. The T-cell and/or natural killer cell doses may escalate with each dose. The T-cells and/or natural killer cells may be administered by intravenous infusion.

In some embodiments the membrane-bound IL-15 and/or CD8 sequences described herein and associated products and compositions may be used autologous or allogenic methods of adoptive cellular therapy. In some embodiments, membrane-bound IL-15 sequences, CD8 sequences, T cells and/or natural killer cells thereof, and compositions may be used in, for example, methods described in U.S. Patent Application Publication 2019/0175650; U.S. Patent Application Publication 2019/0216852; U.S. Patent Application Publication 2019/024743; and U.S. Provisional Patent Application 62/980,844, each of which is incorporated by reference in its entirety.

The disclosure also provides for a population of modified T cells and/or modified natural killer cells that express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or present an exogenous CD8 polypeptide described herein and/or a T cell receptor wherein the population of modified T cells and/or natural killer cells is activated and expanded with a combination of IL-2 and IL-15. In some embodiments, the population of modified T cells and/or natural killer cells is expanded and/or activated with a combination of IL-2, IL-15, and zoledronate. In some embodiments, the population of modified T cells and/or natural killer cells is activated with a combination of IL-2, IL-15, and zoledronate while expanded with a combination of IL-2, IL-15, and without zoledronate. The disclosure further provides for use of other interleukins during activation and/or expansion, such as IL-12, IL-18, IL-21, and any combination thereof.

In an aspect, IL-21, a histone deacetylase inhibitor (HDACi), or any combination thereof may be utilized in the field of cancer treatment, with methods described herein, and/or with ACT processes described herein. In some embodiments the present disclosure provides methods for re-programming 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, phenylbutyrate, valproic acid, vorinostat (suberanilohydroxamic acid), belinostat, panobinostat, dacinostat, entinostat, tacedinaline, and mocetinostat.

Compositions comprising engineered T cells and/or engineered natural killer cells described herein may be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, pharmaceutical compositions can 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. An engineered T-cell and/or engineered natural killer cell can also be administered to lessen a likelihood of developing, contracting, or worsening a condition. Effective amounts of a population of engineered T-cells and/or natural killer cells for therapeutic use can vary based on the severity and course of the disease or condition, previous therapy, the subject's health status, weight, and/or response to the drugs, and/or the judgment of the treating physician. The cells may be αβ T cells, γδ T cells, and/or natural killer cells engineered to express a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptide described herein and optionally a TCR described herein. In some embodiments, a CD8 polypeptide may comprise a CD8α chain and/or a CD8β chain, and the CD8α chain and/or CD8β chain may independently be modified or unmodified.

Methods of Administration

One or multiple engineered T cell populations and/or natural killer cell populations described herein may be administered to a subject in any order or simultaneously. If simultaneously, the multiple engineered T cells and/or engineered natural killer cells can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, subcutaneous injections or pills. Engineered T-cells and/or engineered natural killer cells can be packed together or separately, in a single package or in a plurality of packages. One or all of the engineered T cells and/or engineered natural killer cells can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some embodiments engineered T cells and/or engineered natural killer cells can expand within a subject's body, in vivo, after administration to a subject. Engineered T cells and/or engineered natural killer cells can be frozen to provide cells for multiple treatments with the same cell preparation. Engineered T cells and/or engineered natural killer cells of the present disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit may comprise instructions (e.g., written instructions) on the use of engineered T cells and/or engineered natural killer cells and compositions comprising the same.

A method of treating a cancer may comprise administering to a subject a therapeutically-effective amount of engineered T cells and/or engineered natural killer cells, in which the administration treats the cancer. In some embodiments, the therapeutically-effective amount of engineered γδ T cells and/or engineered natural killer cells may be administered for at least about 10 seconds, 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 the engineered T cells and/or engineered natural killer cells may be administered for at least one week. In some embodiments the therapeutically-effective amount of engineered T cells and/or engineered natural killer cells may be administered for at least about two weeks.

Engineered T-cells and/or engineered natural killer cells expressing a membrane-bound IL-15 (e.g., an IL-15/IL-15Rα fusion polypeptide) and/or a CD8 polypeptides described herein, optionally αβ T cells and/or γδ T cells, may be present in a composition in an amount of at least about 1×103 cells/ml, at least about 2×103 cells/ml, at least about 3×103 cells/ml, at least about 4×103 cells/ml, at least about 5×103 cells/ml, at least about 6×103 cells/ml, at least about 7×103 cells/ml, at least about 8×103 cells/ml, at least about 9×103 cells/ml, at least about 1×104 cells/ml, at least about 2×104 cells/ml, at least about 3×104 cells/ml, at least about 4×104 cells/ml, at least about 5×104 cells/ml, at least about 6×104 cells/ml, at least about 7×104 cells/ml, at least about 8×104 cells/ml, at least about 9×104 cells/ml, at least about 1×105 cells/ml, at least about 2×105 cells/ml, at least about 3×105 cells/ml, at least about 4×105 cells/ml, at least about 5×105 cells/ml, at least about 6×105 cells/ml, at least about 7×105 cells/ml, at least about 8×105 cells/ml, at least about 9×105 cells/ml, at least about 1×106 cells/ml, at least about 2×106 cells/ml, at least about 3×106 cells/ml, at least about 4×106 cells/ml, at least about 5×106 cells/ml, at least about 6×106 cells/ml, at least about 7×106 cells/ml, at least about 8×106 cells/ml, at least about 9×106 cells/ml, at least about 1×107 cells/ml, at least about 2×107 cells/ml, at least about 3×107 cells/ml, at least about 4×107 cells/ml, at least about 5×107 cells/ml, at least about 6×107 cells/ml, at least about 7×107 cells/ml, at least about 8×107 cells/ml, at least about 9×107 cells/ml, at least about 1×108 cells/ml, at least about 2×108 cells/ml, at least about 3×108 cells/ml, at least about 4×108 cells/ml, at least about 5×108 cells/ml, at least about 6×108 cells/ml, at least about 7×108 cells/ml, at least about 8×108 cells/ml, at least about 9×108 cells/ml, at least about 1×109 cells/ml, or more, from about 1×103 cells/ml to about at least about 1×108 cells/ml, from about 1×105 cells/ml to about at least about 1×108 cells/ml, or from about 1×106 cells/ml to about at least about 1×108 cells/ml.

T cells, natural killer (NK) cells, and pharmaceutical compositions described herein may be used in therapy, in particular in a method of treating cancer. The present disclosure therefore also provides the use of the T cells, natural killer (NK) cells, and pharmaceutical compositions described herein in the therapy, in particular in a method of treating cancer. Further, 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, in particular 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, urinary bladder cancer, kidney cancer, leukemia, ovarian cancer, esophageal cancer, brain cancer, gastric cancer, and prostate cancer. The features and aspects described in connection with the methods of treating, preparing and administering above are also applicable to the uses described herein, mutatis mutandis.

Sequences

The sequences described herein may comprise about 80%, about 85%, about 90%, about 85%, about 96%, about 97%, about 98%, or about 99%, or about 100% identity to the sequence of any of SEQ ID NO: 1-97, 256-266, 293, 294, or 305-436. The sequences described herein may comprise at least about 80%, at least about 85%, at least about 90%, at least about 85%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the sequence of any of SEQ ID NO: 1-97, 256-266, or 305-436. A sequence “at least 85% identical to a reference sequence” is a sequence having, on its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the entire length of the reference sequence.

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

Percentage of identity may be calculated using a global pairwise alignment (e.g., the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The «needle» program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (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 percentage of identity between two polypeptides, in accordance with the present disclosure, is calculated using the EMBOSS: needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

Proteins comprising or consisting of an amino acid sequence “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”, or similar recitations, to a reference sequence may comprise mutations such as 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, the protein consisting of an amino acid sequence 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 derived from another species than the reference sequence.

Amino acid substitutions may be conservative or non-conservative. In some embodiments, substitutions may be conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties.

Conservative substitutions may comprise those, which are described by Dayhoff in “The Atlas of Protein Sequence and Structure. Vol. 5”, Natl. Biomedical Research, the contents of which are incorporated by reference in their entirety. For example, In some embodiments amino acids, which belong to one of the following groups, can be exchanged for one another, thus, constituting a conservative exchange: 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); and Group 6: aspartic acid (D), glutamic acid (E). In some embodiments a conservative amino acid substitution may be selected from the following of T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and/or T→S.

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

TABLE A

Conservative Amino Acid substitution

Conservative Amino Acid Substitutions

Amino Acid
Substitutions (others are known in the art)

Conservative substitutions in the polypeptides described herein may be those shown in Table B under the heading of “conservative substitutions.” If such substitutions result in a change in biological activity, then more substantial changes, denominated “exemplary substitutions” in Table B, may be introduced and the products screened if needed.

TABLE B

Amino Acid substitution

Amino Acid Substitutions

Original Residue

occurring amino
Conservative

Pro (P)
Ala
Ala

Thr (T)
Ser
Ser

Nucleic acids comprising or consisting of a nucleic acid sequence “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”, or similar recitations, to a reference sequence may comprise mutations such as 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. Due, for example, to codon degeneracy, mutations or substitutions to a reference nucleic acid sequence may result in a mutated nucleic acid sequence that encodes protein identical to the protein encoded by the reference sequence. Mutated nucleic acid sequences that encode a protein having a different sequence from the protein encoded by the reference sequence are also contemplated. Mutated nucleic acid sequences encoding conservative amino acid mutations are contemplated. In the case of substitutions, the nucleic acid sequence 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 derived from another species than the reference sequence.

Unless otherwise indicated, all terms used herein have the same meaning as they would to one skilled in the art.

It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific embodiments of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.

All references cited in this specification are herein incorporated by reference as though 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 such reference by virtue of prior invention. Additional information regarding CD8 polypeptides, TCR polypeptides, and further information, may be found in U.S. patent application Ser. No. 17/563,599, filed Dec. 28, 2021, entitled “CD8 POLYPEPTIDES, COMPOSITIONS, AND METHODS OF USING THEREOF”, which is incorporated by reference herein in its entirety.

Unless otherwise specified herein, ranges of values set forth herein are intended to operate as a scheme for referring to each separate value falling within the range individually, including but not limited to the endpoints of the ranges, and each separate value of each range set forth herein is hereby incorporated into the specification as if it were individually recited.

This specification may include references to “one embodiment”, “an embodiment”, “embodiments”, “one 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, and may refer to more than one embodiment or aspect. Various embodiments and aspects may be combined in any manner consistent with this disclosure.

“Activation” as used herein refers broadly to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are proliferating.

“Antibodies” as used herein refer broadly to antibodies or immunoglobulins of any isotype, fragments of antibodies, which retain specific binding to antigen, including, but not limited to, Fab, Fab′, Fab′-SH, (Fab′)2 Fv, scFv, divalent scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins including 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.

“Antigen” or “Antigenic,” as used herein, refers broadly to a peptide or a portion of a peptide capable of being bound by an antibody which is additionally capable of inducing an animal to produce an antibody capable of binding to an epitope of that antigen. An antigen may have one epitope or have more than one epitope. The specific reaction referred to herein indicates that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

“Chimeric antigen receptor” or “CAR” or “CARs” as used herein refers broadly to genetically modified receptors, which graft an antigen specificity onto cells, for example T cells, NK cells, macrophages, and stem cells. CARs 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 (CSDs), and an intracellular activating domain (IAD). In certain some embodiments, the CSD is optional. In some embodiments, the CAR is a bispecific CAR, which is specific to two different antigens or epitopes. After the ASTR binds specifically to a target antigen, the IAD activates intracellular signaling. For example, the IAD can redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of antibodies. The non-MHC-restricted antigen recognition gives T cells expressing the CAR the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

“Cytotoxic T lymphocyte” (CTL) as used herein refers broadly to a T lymphocyte that expresses CD8 on the surface thereof (e.g., a CD8+ T cell). Such cells may be “memory” T cells (TM cells) that are antigen-experienced.

“Effective amount”, “therapeutically effective amount”, or “efficacious amount” as used herein refers broadly to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

“Genetically modified” as used herein refers broadly to methods to introduce exogenous nucleic acids into a cell, whether or not the exogenous nucleic acids are integrated into the genome of the cell. “Genetically modified cell” as used herein refers broadly to cells that contain exogenous nucleic acids whether or not the exogenous nucleic acids are integrated into the genome of the cell.

“Immune cells” as used herein refers broadly to white blood cells (leukocytes) derived from hematopoietic stem cells (HSC) produced in the bone marrow “Immune cells” include, without limitation, lymphocytes (T cells, B cells, natural killer (NK) (CD3-CD56+) cells) and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). “T cells” include all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T-regulatory cells (Treg) and gamma-delta T cells, and NK T cells (CD3+ and CD56+). A skilled artisan will understand T cells and/or NK cells, as used throughout the disclosure, can include only T cells, only NK cells, or both T cells and NK cells. In certain illustrative embodiments and aspects provided herein, T cells are activated and transduced. Furthermore, T cells are provided in certain illustrative composition embodiments and aspects provided herein. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, NK-T cells, γδ T cells, and neutrophils, which are cells capable of mediating cytotoxicity responses.

“Peripheral blood mononuclear cells” or “PBMCs” as used herein refers broadly to any peripheral blood cell having a round nucleus. PBMCs include lymphocytes, such as T cells, B cells, and NK cells, and monocytes.

“Polynucleotide” and “nucleic acid”, as used interchangeably herein, refer broadly to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

“T cell” or “T lymphocyte,” as used herein, refer broadly to thymocytes, naïve T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. Illustrative populations of T cells suitable for use in particular embodiments include, but are not limited to, helper T cells (HTL; CD4+ T cell), a cytotoxic T cell (CTL; CD8+ T cell), CD4+CD8+ T cell, CD4−CD8− T cell, natural killer T cell, T cells expressing αβ TCR (αβ T cells), T cells expressing γδ TCR (γδ T cells), or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular 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 and if desired, can be further isolated by positive or negative selection techniques.

In the present disclosure, the term “homologous” refers to the degree of identity between sequences of two amino acid sequences, e.g., peptide or polypeptide sequences. The aforementioned “homology” is determined by comparing two sequences aligned under optimal conditions over the sequences to be compared. Such a sequence homology can be calculated by creating an alignment using, for example, the ClustalW algorithm. Commonly available sequence analysis software, more specifically, Vector NTI, GENETYX or other tools are provided by public databases.

The terms “sequence homology” or “sequence identity” are used interchangeably herein. For the purpose of this disclosure, in order to determine the percentage of sequence homology or sequence identity of two amino acid sequences or of two nucleotide sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences, gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full-length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 5, about 10, about 20, about 50, about 100 or more nucleotides or amino acids. The sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the identity between two sequences (Kruskal, J. B. (1983) An overview of sequence comparison. In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, Addison Wesley). The percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mal. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose 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 sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentage of sequence identity between a query sequence and a sequence of the present disclosure is calculated as follows: Number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The identity can be obtained from NEEDLE by using the NOBRIEF option and is labelled in the output of the program as “longest-identity”. The nucleotide and amino acid sequences of the present disclosure can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mal. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, word length=12 to obtain nucleotide sequences homologous to polynucleotides of the present disclosure. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to 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.

“T-cell receptor (TCR)” as used herein refers broadly to a protein receptor on T cells that is composed of a heterodimer of an alpha (α) and beta (β) chain, although in some cells the TCR consists of gamma and delta (γ/δ) chains. The TCR may be modified on any cell comprising a TCR, including a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, or a gamma delta T cell.

The TCR is generally found on the surface of T lymphocytes (or T cells) that is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. It is a heterodimer consisting of an alpha and beta chain in 95% of T cells, while 5% of T cells have TCRs consisting of gamma and delta chains. Engagement of the TCR with antigen and MHC results in activation of its 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 composed of four distinct chains (CD3-γ, CD3δ, and two times CD3ε) in mammals, that associate with molecules known as the T-cell receptor (TCR) and the ζ-chain to generate an activation signal in T lymphocytes. The TCR, ζ-chain, and CD3 molecules together comprise the TCR complex. The CD3-γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The transmembrane region of the CD3 chains is negatively charged, a characteristic that allows these chains to associate with the positively charged TCR chains (TCRα and TCRβ). The intracellular tails of the CD3 molecules contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM for short, which is essential for the signaling capacity of the TCR.

“Treatment,” “treating,” and the like, as used herein refer broadly to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, e.g., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease.

The ability of dendritic cells (DC) to activate and expand antigen-specific CD8+ T cells may depend on the DC maturation stage and that DCs may need to receive a “licensing” signal, associated with IL-12 production, in order to elicit cytolytic immune response. In particular, the provision of signals through CD40 Ligand (CD40L)-CD40 interactions on CD4+ T cells and DCs, respectively, may be considered important for the DC licensing and induction of cytotoxic CD8+ T cells. DC licensing may result in the upregulation of co-stimulatory molecules, increased survival and better cross-presenting capabilities of DCs. This process may be mediated via CD40/CD40L interaction [S. R. Bennet et al., “Help for cytotoxic T-cell responses is mediated by CD40 signalling,” Nature 393(6684):478-480 (1998); S. P. Schoenberger et al., “T-cell help for cytotoxic T-cell help is mediated by CD40-CD40L interactions,” Nature 393(6684):480-483 (1998)], but CD40/CD40L-independent mechanisms also exist (CD70, LTBR). In addition, a direct interaction between CD40L expressed on DCs and CD40 on expressed on CD8+ T-cells has also been suggested, 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)].

Exemplary Nucleic Acid and Amino Acid Sequences

Nucleic Acid
Amino Acid

Construct
(SEQ ID
SEQ ID

Acid
Acid
Acid
Amino Acid
Nucleic Acid

“IgESP” refers to a signal protein derived from IgE.

In some embodiments, construct G/G′ as comprised in SEQ ID NO: 454 may be preferred. T cells transduced with said construct may express an IL-15-L-IL15Rα fusion polypeptide having an amino acid sequence of SEQ ID NO: 329. Additionally, T cells preferably express a TCR alpha chain variable domain having the amino acid sequence of SEQ ID NO: 455 as comprised in SEQ ID NO: 15, and a TCR beta chain variable domain having the amino acid sequence of SEQ ID NO: 456.

The inventors found that the various CD8 elements in the vector lead to a surprising increase in expression and activity. For example, despite the observation that Construct #10 has lower viral titers than Constructs #9b, #11, and #12 (FIG. 5A), T cells transduced with Construct #10 expressing CD8αβ heterodimer and TCR at the lowest viral volumetric concentration, e.g., 1.25 μl/106 cells, generated higher CD8+CD4+ TCR+ cells (56.7%, FIG. 9B) than that of transduced with Construct #9b expressing CD8α and TCR (42.3%, FIG. 9A), Construct #11 expressing CD8αCD8βstalk with CD8α transmembrane and intracellular domain and TCR (51.6%, FIG. 9C), and Construct #12 expressing CD8αCD8βstalk with Neural Cell Adhesion Molecule 1 (NCAM1) transmembrane and intracellular domain and TCR (14.9%, FIG. 9D).

A vector may comprise any one or more of nucleic acid sequences of SEQ ID NO: 72, 73, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356-366, 433-436, or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 310 with or without a nucleic acid encoding a linker therebetween; SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 312 with or without a nucleic acid encoding a linker therebetween; SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 314 with or without a nucleic acid encoding a linker therebetween; or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of 316 with or without a nucleic acid encoding a linker therebetween. A linker may be as described herein. Optionally SEQ ID NO: 368 may be directly or indirectly fused to a 5′ end of SEQ ID NO: 308.

A T-cell and/or natural killer cell or any combination thereof may be transduced to express any one or more of the nucleic acid of SEQ ID NO: 72, 73, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356-366, or 433-436; or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 310 with or without a nucleic acid encoding a linker therebetween; SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 312 with or without a nucleic acid encoding a linker therebetween; SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 314 with or without a nucleic acid encoding a linker therebetween; or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 316 with or without a nucleic acid encoding a linker therebetween. A linker may be as described herein. Optionally SEQ ID NO: 368 may be directly or indirectly fused to a 5′ end of SEQ ID NO: 308.

A vector may comprise any one or more of nucleic acid sequences of SEQ ID NO: 72, 72, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 318, 320, 322, 324, 326, 328, 330, 332, 334, 338, 340, 342, 344, 346, 348, 350, 352, 354, 357-365, or 433-436; or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 312 with or without a nucleic acid encoding a linker therebetween; SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 314 with or without a nucleic acid encoding a linker therebetween; or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 316 with or without a nucleic acid encoding a linker therebetween. A linker may be as described herein. Optionally SEQ ID NO: 368 may be directly or indirectly fused to a 5′ end of SEQ ID NO: 308.

A T-cell and/or natural killer cell or any combination thereof may be transduced to express any one or more of the nucleic acid of SEQ ID NO: 72, 73, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 295, 297, 299, 301, 318, 320, 322, 324, 326, 328, 330, 332, 334, 338, 340, 342, 344, 346, 348, 350, 352, 354, 357-365, or 433-436, or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 312 with or without a nucleic acid encoding a linker therebetween; SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 314 with or without a nucleic acid encoding a linker therebetween; or SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 316 with or without a nucleic acid encoding a linker therebetween. A linker may be as described herein. Optionally SEQ ID NO: 368 may be directly or indirectly fused to the 5′ end of SEQ ID NO: 308.

Full Transgene Constructs

Nucleotide sequence

According to the first aspect of the present disclosure, a vector may comprise any one of the nucleic acid sequences of SEQ ID NOs: 454, 451, 448, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 450, 452 or 453, or a nucleic acid 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 any one of those sequences. Specifically, a vector may comprise any one of nucleic acid sequences according to SEQ ID NOs. 454, 451 or 448, or a nucleic acid 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 any of those sequences. More specifically, a vector may comprise a nucleic acid sequence according to SEQ ID NO. 454, or a nucleic acid 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 thereto.

According to the first aspect of the present disclosure, a T-cell and/or natural killer cell or any combination thereof may be transduced to express any one or more of the nucleic acid sequences of SEQ ID NOs: 454, 451, 448, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 449, 450, 452 or 453, or a nucleic acid 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 any one of those sequences. Specifically, a T-cell and/or natural killer cell or any combination thereof may be transduced to express any one or more of the nucleic acid sequences of SEQ ID NOs 454, 451, 448, or a nucleic acid 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 any one of those sequences. More specifically, a T-cell and/or natural killer cell or any combination thereof may be transduced to express a nucleic acid sequence of SEQ ID NO: 454, or a nucleic acid 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 thereto. In some embodiments, the T cell is an αβ T cell or a γδ T cell, specifically a γδ1 or a γδ2 T cell.

A membrane-bound IL-15 may comprise an IL-15 amino acid sequence selected from Table 2C linked directly or indirectly to an IL-15Rα amino acid sequence selected from Table 2D. A membrane-bound IL-15 may be encoded by an IL-15 nucleic acid sequence selected from Table 2C linked directly or indirectly to an IL-15Rα nucleic acid sequence selected from Table 2D. A signal peptide may be operatively coupled to the IL-15 or IL-15Rα. The signal peptide may be derived from an IgE. A signal peptide derived from IgE may comprise SEQ ID NO: 367 and/or may be encoded by SEQ ID NO: 368.

However, In some embodiments nucleic acids, vectors, and/or T cells and/or natural killer cells do not comprise and/or are not transduced to express (i) SEQ ID NO: 336, 356, or 366, (ii) any sequence having about 80%, about 85%, about 90%, or about 95% or more sequence identity to SEQ ID NO: 336, 356, or 366, (iii) SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 310 with a nucleic acid encoding a linker therebetween; or (iv) any sequence having about 80%, about 85%, about 90%, or about 95% or more sequence identity to SEQ ID NO: 308 directly or indirectly fused to the 5′ end of SEQ ID NO: 310 with a nucleic acid sequence encoding a linker therebetween.

Several of the elements of the constructs in Table 2 are described in Table 3.

Representative Protein and Nucleic Acid (DNA) Sequences

SEQ ID

NO:
Description
Sequence

transmembrane

domain

cytoplasmic tail

peptide

alpha chain
TNFTCSFPSSNFYALHWYRKETAKSPEALFVMTLNGDEKK

chain nucleic acid
ggtttcacctggactgcgtgtcctctatcctgaatgtgga

chain nucleic acid
tggtggccaagcacacagacgccggcgtgatccagtcccc

peptide

peptide

305
Full Wild Type
MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFS

306
Full Wild Type
MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVE

Acid Sequence

Acid Sequence
AAGTGTTTCCTGCTGGAACTCCAAGTCATCAGCCTCGAAT

or
HQPPGVYPQGHSDTTVAVAGCVFLLISVLLLSGLSRQTPP

Amino Acid

Sequence

or
CCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCC

Sequence
L

or
CCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCC

deletion of Exon
AAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNW

Amino Acid

Sequence

deletion of Exon
CATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCC

Acid Sequence
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTSKESGSV

Acid Sequence
CGAGTCAGATGTGCATCCGAGCTGCAAGGTCACCGCGATG

Acid Sequence
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEGKSSG

Acid Sequence
CGAGTCAGATGTGCATCCGAGCTGCAAGGTCACCGCGATG

Acid Sequence
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTSKESGSV

Acid Sequence
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEGKSSG

Acid Sequence
TESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSG

Acid Sequence
CGAGTCAGATGTGCATCCGAGCTGCAAGGTCACCGCGATG

Acid Sequence
VENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS

Acid Sequence
GAAGAAGATTGAAGATCTGATCCAGTCCATGCACATTGAC

Acid Sequence
VENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS

Acid Sequence
GAAGAAGATTGAAGATCTGATCCAGTCCATGCACATTGAC

Acid Sequence
VENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQS

Acid Sequence
GAAGAAGATTGAAGATCTGATCCAGTCCATGCACATTGAC

Peptide Amino

Acid Sequence

Acid Sequence

Peptide Amino

Acid Sequence

Peptide Amino

Acid Sequence

Amino Acid

Sequence

Transmembrane

Domain Amino

Acid Sequence

Domain Nucleic

Acid Sequence

Transmembrane

Domain Amino

Acid Sequence

Domain Nucleic
G

Acid Sequence

Transmembrane

Domain Amino

Acid Sequence 1

Domain Nucleic

Acid Sequence 1

Transmembrane

Domain Amino

Acid Sequence 2

Domain Nucleic

Acid Sequence 2

Nucleic Acid

Sequence

Acid Sequence
TGAAGCT

382
Kozak Sequence
GCCNCCATGG where N is a purine (A or G)

Acid Sequence

Sequence

Acid Sequence

Nucleic Acid
CC

Sequence

Acid Sequence

Acid Sequence

Sequence

Acid Sequence

Sequence

Acid Sequence

Acid Sequence
GGCAGC

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence
KEAAAKA

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

Acid Sequence

436
Full Wild Type
ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCC

Acid Sequence
CGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCA

Linker Amino
LE

Acid Sequence

alpha variable
AKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGS

beta variable
LELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPS

The constructs in Table 2A and in Table 2B each may be assemblages of the individual components described in Table 3. The inventors found that the combination, order, and inclusion of transcription enhancers from Table 3 as described in Table 2A provided unexpected improvements in transfection efficiency, expression levels, and induction of cytotoxic T-cell activities, e.g., IL-12 secretion, IFN-γ secretion, TNF-α secretion, granzyme A secretion, MIP-1α secretion, IP-10 secretion, granzyme B secretion, and any combination thereof.

Tumor Associated Antigens (TAA)

In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T cells bearing specific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or -associated antigens, and to be used in a therapy, particular prerequisites must be fulfilled. The antigen should be expressed mainly by tumor cells and not, or in comparably small amounts, by normal healthy tissues. In some embodiments the peptide may be over-presented by tumor cells as compared to normal healthy tissues. It is furthermore desirable that the respective antigen is not only present in a type of tumor, but also in high concentrations (e.g., copy numbers of the respective peptide per cell). Tumor-specific and tumor-associated antigens are often derived from proteins directly involved in transformation of a normal cell to a tumor cell due to their function, e.g., in cell cycle control or suppression of apoptosis. Additionally, downstream targets of the proteins directly causative for a transformation may be up-regulated and thus may be indirectly tumor-associated. Such indirect tumor-associated antigens may also be targets of a vaccination approach. Singh-Jasuja et al. Cancer Immunol. Immunother. 53 (2004): 187-195. Epitopes are present in the amino acid sequence of the antigen, making the peptide an “immunogenic peptide”, and being derived from a tumor associated antigen, leads to a T-cell-response, both in vitro and in vivo.

Any peptide able to bind an MHC molecule may function as a T-cell epitope. For the induction of a T-cell-response, the TAA must be presented a T cell having a corresponding TCR and the host must not have immunological tolerance for this particular epitope. Exemplary Tumor Associated Antigens (TAA) that may be used with the CD8 polypeptides described herein are disclosed herein.

TAA Peptide sequences

ID
Amino Acid

Different from CD8α polypeptide, e.g., CD8α1 (SEQ ID NO: 258) and CD8α2 (SEQ ID NO: 259), a modified CD8α polypeptide, e.g., m1CD8α (SEQ ID NO: 7) and m2CD8α (SEQ ID NO: 262), may contain additional regions, such as sequence stretches from a CD8β polypeptide. In some embodiments SEQ ID NO: 2 or variants thereof are used with a CD8α polypeptide. In other embodiments, a portion of a CD8α polypeptide, e.g., SEQ ID NO: 260, is removed or not included in modified CD8 polypeptides described herein. FIG. 2 shows a sequence alignment between CD8α1 (SEQ ID NO: 258) and m1CD8α (SEQ ID NO: 7). FIG. 3 shows a sequence alignment between CD8α2 (SEQ ID NO: 259) and m2CD8α (SEQ ID NO: 262), in which the cysteine substitution is indicated by an arrow. The stalk regions are shown within the boxes. CD8α polypeptide CD8α1 (SEQ ID NO: 258) may be encoded by SEQ ID NO: 434. Modified CD8α polypeptide m1CD8α (SEQ ID NO: 7) may be encoded by SEQ ID NO: 435.

Modified CD8 expressing cells showed improved functionality in terms of cytotoxicity and cytokine response as compared to original CD8 expressing T cells transduced with the TCR.

Membrane-bound IL-15 may comprise, for example, an IL-15/IL-15Rα fusion polypeptide and/or an IL-15Rα/IL-15 fusion polypeptide. One or more linkers may be disposed between IL-15 and IL-15Rα or between IL-15Rα and IL-15. An exemplary IL-15/IL-15Rα fusion polypeptide comprising one or more linker is depicted in FIG. 67A. An exemplary IL-15Rα/IL-15 fusion polypeptide comprising one or more linker is depicted in FIG. 67B. The IL-15 polypeptide in FIGS. 67A and 67B may be immature wild type, immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptide in FIGS. 67A and 67B may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments the IL-15 polypeptide in FIG. 67A and FIG. 67B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 67A and FIG. 67B is mature and may or may not be mutated. In some embodiments the IL-15 polypeptide in FIG. 67A and FIG. 67B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. FIG. 67A and FIG. 67B is mature and mutated. Although a linker is depicted in FIG. 67A and FIG. 67B, a linker is optional and a mbIL-15 polypeptides not comprising a linker are also contemplated.

An IL-15/IL-15Rα fusion polypeptide and/or an IL-15Rα/IL-15 fusion polypeptide may also comprise one or more signal peptide, such as, but not limited to, a signal peptide derived from IgE, such as the signal peptide of SEQ ID NO: 367, encoded by SEQ ID NO: 368. An exemplary IL-15/IL-15Rα fusion polypeptide comprising one or more linker and at least one signal peptide is depicted in FIG. 68A. An exemplary 15Rα/IL-15 fusion polypeptide comprising at least one linker and at least one signal peptide is depicted in FIG. 68B. The IL-15 polypeptide in FIGS. 68A and 68B may be immature wild type, immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptide in FIGS. 68A and 68B may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments the IL-15 polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated. In some embodiments the IL-15 polypeptide in FIG. 68A and FIG. 68B is mature and may or may not be mutated, and the IL-15Rα polypeptide in FIG. 68A and FIG. 68B is mature and mutated. Although a linker is depicted in FIG. 68A and FIG. 68B, a linker is optional and a mbIL-15 polypeptides not comprising a linker are also contemplated.

An IL-15/IL-15Rα fusion polypeptide may comprise or consist of appropriate amino acid sequences identified herein. An IL-15/IL-15Rα fusion polypeptide may be encoded by one or more nucleic acids comprising or consisting of appropriate nucleic acid sequences identified herein.

Lentiviral Viral Vectors

The lentiviral vectors used herein contain several elements that enhance vector function, including a central polypurine tract (cPPT) for improved replication and nuclear import, a promoter from the murine stem cell virus (MSCV) (SEQ ID NO: 263), which lessens vector silencing in some cell types, a woodchuck hepatitis virus posttranscriptional responsive element (WPRE) (SEQ ID NO: 264) for improved transcriptional termination, and the backbone was a deleted 3′-LTR self-inactivating (SIN) vector design that improves safety, sustained gene expression and anti-silencing properties. Yang et al. Gene Therapy (2008) 15, 1411-1423.

In some embodiments vectors, constructs, or sequences described herein comprise mutated forms of WPRE. In some embodiments sequences or vectors described herein comprise mutations 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 with the same construct containing SEQ ID NO: 257 as WPREmut2, with the difference between Construct #9 and Construct #9b being the titer consistent with Table 4. In some embodiments WPRE mutants comprise at most one mutation, at most two mutations, at most three mutations, at least four mutations, or at most five mutations. In some embodiments vectors, constructs, or sequences described herein do not comprise WPRE. In an aspect, WPRE sequences described in U.S. 2021/0285011, the content of which is incorporated by reference in its entirety, may be used together with vectors, sequences, or constructs described herein.

In some embodiments vectors, constructs, or sequences described herein do not include an X protein promoter. The WPRE mutants described herein do not express an X protein. WPRE promotes accumulation of mRNA, theorized to promote export of mRNA from nucleosome to cytoplasm to promote translation of the transgene mRNA.

To generate lentiviral vectors co-expressing TCRαβ and mCD8α and/or CD8β, a nucleotide encoding furin-linker (GSG or SGSG (SEQ ID NO: 266))-2A peptide may be positioned between TCRα chain and TCRβ chain, between mCD8α chain and CD8β chain, between a TCR chain and a CD8 chain, and/or between a CD8 or TCR chain and a membrane-bound IL-15 to enable 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).

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

Lentiviral vectors may be pseudotyped with RD114TR (for example, SEQ ID NO: 97), which is a chimeric glycoprotein comprising an extracellular and transmembrane domain of feline endogenous virus (RD114) directly or indirectly fused to cytoplasmic tail (TR) of murine leukemia virus. Other viral envelop proteins, such as VSV-G env, MLV 4070A env, RD114 env, chimeric envelope protein RD114pro, baculovirus GP64 env, or GALV env, or derivatives thereof, may also be used. RD114TR variants comprising at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98%, at least about 99%, or about 100% identity to SEQ ID NO: 97 also provided for.

As another example, FIG. 70 depicts exemplary vectors that are provided In some embodiments. For example, Constructs K-U depicted in FIG. 70 are provided In some embodiments. The TCRs in FIG. 70 may be, for example, TCRβ directly or indirectly fused to TCRα with or without a linker and/or other elements therebetween or TCRα directly or indirectly fused to TCRβ with or without a linker and/or other elements therebetween. The IL-15 polypeptides in FIG. 70 may be immature wild type, immature mutated, mature wild type, or mature mutated. The IL-15Rα polypeptides in FIG. 70 may be immature wild type, immature mutated, mature wild type, or mature mutated. In some embodiments the IL-15 polypeptides in FIG. 70 are mature and may or may not be mutated, and the IL-15Rα polypeptides in FIG. 70 are mature and may or may not be mutated. In some embodiments the IL-15 polypeptides in FIG. 70 are mature and may or may not be mutated, and the IL-15Rα polypeptides in FIG. 70 are mature and mutated. The CD8α, CD8β, and TCR polypeptides in FIG. 70 may independently be as described herein and/or may independently by modified or unmodified. In some embodiments CD8α may comprise or consist of CD8α1 (SEQ ID NO: 258, which may be encoded by SEQ ID NO: 434). In some embodiments CD8α may comprise or consist of m1CD8α (SEQ ID NO: 7, which may be encoded by SEQ ID NO: 435). In some embodiments CD8β may comprise or consist of CD8β1 (SEQ ID NO: 8, which may be encoded by SEQ ID NO: 433). In some embodiments, constructs express an IL-15 polypeptide fused to a WPRE element, a linker and a CD25 or CD28 transmembrane domain as defined herein.

In some embodiments, the nucleic acid encoding mbIL-15 in any of Constructs K-U may be selected from nucleic acid sequences encoding (i) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 311 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (ii) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 313 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; or (iii) SEQ ID NO: 307 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to an N terminus of SEQ ID NO: 315 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (iv) any of SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, 337, 339, 341, 343, 345, 347, 349, 351, or 353; or (vi) a sequence 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 any of SEQ ID NO: 317, 319, 321, 323, 325, 327, 329, 331, 333, 337, 339, 341, 343, 345, 347, 349, 351, or 353. In some embodiments, a sequence encoding a signal peptide may be directly or indirectly fused to the 5′ end of a nucleic acid encoding any of SEQ ID NO: 307, 317, 319, 321, 323, 325, 327, 329, 331, or 333 or a sequence 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, 317, 319, 321, 323, 325, 327, 329, 331, or 333. In some embodiments the signal peptide may be derived from an IgE polypeptide. In some embodiments the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the nucleic acid encoding mbIL-15 in any of Constructs K-U may be selected from nucleic acid sequences encoding (i) any of SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, 337, 341, 345, 347, 349, 351, or 353 or (ii) a sequence 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 any of SEQ ID NO: 317, 321, 325, 327, 329, 331, 333, 337, 341, 345, 347, 349, 351, or 353. In some embodiments, a sequence encoding a signal peptide may be directly or indirectly fused to the 5′ end of a nucleic acid encoding any of SEQ ID NO: 317, 321, 325, 327, 329, 331, or 333, or a sequence 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: 317, 321, 325, 327, 329, 331, or 333. In some embodiments the signal peptide may be derived from an IgE polypeptide. In some embodiments the signal peptide derived from an IgE polypeptide may comprise SEQ ID NO: 367 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the nucleic acid encoding mbIL-15 in any of Constructs K-U may be selected from nucleic acid sequences comprising or consisting of (i) SEQ ID NO: 308 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to 5′ end of SEQ ID NO: 312 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (ii) SEQ ID NO: 308 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to 5′ end of SEQ ID NO: 314 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; or (iii) SEQ ID NO: 308 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, directly or indirectly fused to 5′ end of SEQ ID NO: 316 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto, with or without a linker therebetween; (iv) any of SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, 334, 338, 340, 342, 344, 346, 348, 350, 352, or 354; or (vi) a sequence 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 any of SEQ ID NO: 318, 320, 322, 324, 326, 328, 330, 332, 334, 338, 340, 342, 344, 346, 348, 350, 352, or 354. In some embodiments, a sequence encoding a signal peptide may be directly or indirectly fused to the 5′ end of a nucleic acid encoding any of SEQ ID NO: 308, 318, 320, 322, 324, 326, 328, 330, 332, or 334 or a sequence 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, 318, 320, 322, 324, 326, 328, 330, 332, or 334. In some embodiments the signal peptide may be derived from an IgE polypeptide. In some embodiments the nucleic acid encoding the signal peptide derived from an IgE polypeptide may comprise or consist of SEQ ID NO: 368 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

In some embodiments, the nucleic acid encoding mbIL-15 in any of Constructs K-U may be selected from nucleic acid sequences comprising or consisting of (i) any of SEQ ID NO: 318, 322, 326, 328, 330, 332, 334, 338, 342, 346, 348, 350, 352, or 354 or (ii) a sequence 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 any of SEQ ID NO: 318, 322, 326, 328, 330, 332, 334, 338, 342, 346, 348, 350, 352, or 354. In some embodiments, a sequence encoding a signal peptide may be directly or indirectly fused to the 5′ end of a nucleic acid encoding any of SEQ ID NO: 318, 322, 326, 328, 330, 332, or 334, or a sequence 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: 318, 322, 326, 328, 330, 332, or 334. In some embodiments the signal peptide may be derived from an IgE polypeptide. In some embodiments the nucleic acid encoding the signal peptide derived from an IgE polypeptide may comprise or consist of SEQ ID NO: 368 or a sequence at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical thereto.

Further exemplary constructs (Constructs #13-#19 and #21-#25) are described in Table 2 above. In particular, Constructs #13, #14, and #16 are 4-in-1 constructs co-expressing TCR, CD8α, and CD8β3 with various combinations of signal peptides (SEQ ID NO: 6 [WT CD8α signal peptide]; SEQ ID NO: 293 [WT CD8 signal peptide]; and SEQ ID NO: 294 [S19 signal peptide]) and differing element order. Constructs #15 and #17 are 4-in-1 constructs co-expressing TCR, CD8α, and CD8β5. Construct #15 comprises the WT CD8α signal peptide (SEQ ID NO: 6) and WT CD8β signal peptide (SEQ ID NO: 293), whereas Construct #17 comprises the S19 signal peptide (SEQ ID NO: 294) at the N-terminal end of both CD8α and CD8β5. Construct #21 is a 4-in-1 constructs co-expressing TCR, CD8α, and CD8β2 comprising WT CD8α signal peptide (SEQ ID NO: 6) and WT CD8β signal peptide (SEQ ID NO: 293). Construct #18 is a variant of Construct #10 in which the WT signal peptides for CD8α and CD8β1 (SEQ ID NOs: 6 and 293, respectively) were replaced with 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) was replaced with the S19 signal peptide (SEQ ID NO: 294). Construct #22 is a variant of Construct #11 in which the CD4 transmembrane and intracellular domains are directly or indirectly fused to the C-terminus of the CD8β stalk sequence in place of the CD8α transmembrane and intracellular domains. Construct #25 is a variant of Construct #22 in which the CD8β stalk sequence (SEQ ID NO: 2) is replaced with the CD8α stalk sequence (SEQ ID NO: 260).

Further constructs within the scope of the present disclosure CD8CD8 are listed in Table 2E).

Viral Titers

Constructs

For construct 12, NCAMfu refers to NCAMFusion protein expressing modified CD8α extracellular and Neural cell adhesion molecule 1 (CD56) intracellular domain.

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

T Cell Manufacturing

Activation

FIG. 6 shows that, on Day +0, PBMCs (about 9×108 cells) obtained from two donors (Donor #1 and Donor #2) were thawed and rested. Cells were activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the presence of serum. Activation markers, e.g., CD25, CD69, and human low density lipoprotein receptor (H-LDL-R) are in CD8+ and CD4+ cells, were subsequently measured. FIG. 7A shows that % CD3+CD8+CD25+ cells, % CD3+CD8+CD69+ cells, and % CD3+CD8+H-LDL-R+ cells increase after activation (Post-A) as compared with that before activation (Pre-A). Similarly, FIG. 7B shows that % CD3+CD4+CD25+ cells, % CD3+CD4+CD69+ cells, and % CD3+CD4+H-LDL-R+ cells increase after activation (Post-A) as compared with that before activation (Pre-A). These results support the activation of PBMCs.

Transduction

FIG. 6 shows that, on Day +1, activated PBMCs were transduced with viral vectors, e.g., Constructs #1, #2, #8, #9, #10, #11, and #12, in G-Rex® 6 well plates at about 5×106 cells/well in the absence of serum. The amounts of virus used for transduction are shown in Table 6.

Expansion

FIG. 6 shows that, on Day +2, transduced PBMCs were expanded in the presence of serum. On Day +6, cells were harvested for subsequent analysis, e.g., FACS-Dextramer and vector copy number (VCN) and were cryopreserved. FIGS. 8A and 8B show fold expansion on Day +6 of transduced T cell products obtained from Donor #1 and donor #2, respectively. Viabilities of cells is greater than 90% on Day +6.

Characterization of T Cell Products

FIG. 18 shows % Tet+ of CD3+ cells from Donor #1 (upper panel) and Donor #2 (lower panel) transduced with Construct #1, #2, #8 (TCR), #9, #10, #11, or #12 at 1.25 μl, 2.5 μl, or 5 μl per 1×106 cells. These results show higher frequencies of CD3+Tet+ in cells transduced with Construct #9 or #11 than that transduced with Construct #10 or #12. It appears more % Tet+CD3+ cells in cells transduced with Construct #10 (WPREmut2) than that transduced with Construct #2 (wild type WPRE) at 5 μl per 1×106 cells. FACS analysis was gated on live singlets, followed by CD3+, followed by CD3+, and followed by Tet+.

In sum, these results show (1) higher % CD8+CD4+ cells obtained by transducing cells with vectors expressing CD8α and TCR with wild type WPRE (Construct #1) and WPREmut2 (Construct #9) than that transduced with Construct #10, #11 or #12; (2) % CD8+CD4+Tet+ cells was comparable among cells transduced with different constructs; (3) dose dependent increase in % tetramer, e.g., 5 μl per 1×106 cells showed better results than 1.25 μl and 2.5 μl per 1×106 cells; (4) % CD8+ cells comparable among cells transduced with different constructs; (5) higher frequencies of CD8+Tet+ in cells transduced with Construct #9, #11, or #12 than that transduced with Construct #10; (6) higher frequencies of CD3+Tet+ in cells transduced with Construct #9 or #11 than that transduced with Construct #10 or #12; (7) higher VCN in cells transduced with Construct #11 or #12 than that transduced with Construct #9 or #10; and (8) higher CD3+tet+/VCN in cells transduced with Construct #9 than that transduced with Construct #10, #11, or #12.

T cell products transduced with viral vector expressing a transgenic TCR and modified CD8 co-receptor showed superior cytotoxicity and increased cytokine production against target positive cell lines.

Tumor Death Assay

FIG. 20A-C depicts data showing that constructs (#10, #11, & #12) are comparable to TCR-only in mediating cytotoxicity against target positive cells lines expressing antigen at different levels (UACC257 at 1081 copies per cell and A375 at 50 copies per cell).

Tumor Cell Line
Antigen Positivity

Construct #9 loses tumor control over time against the low target antigen expressing A375 cell line.

IFNγ secretion was measured in UACC257 and A375 cells lines. IFNγ secretion in response in UACC257 cell line was comparable among constructs. However, in the A375 cell line, Construct #10 showed higher IFNγ secretion than other constructs. IFNγ quantified in the supernatants from Incucyte plates. FIG. 21A-B.

FIG. 22 depicts an exemplary experiment design to assess 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.

IFNγ secretion in response to A375 increases in the presence of immature DC (iDCs). In the tri-cocultures with iDCs, IFNγ secretion is higher in Construct #10 compared to the other constructs. However, comparing Construct #9 with Construct #11 expressing wild type and modified CD8 coreceptor sequences respectively, T cells transduced with #11 induced stronger cytokine response measured as IFNγ quantified in the culture supernatants of three-way cocultures using donor D600115, E:T:iDC::1:1/10:1/4. FIG. 23A-B.

IFNγ secretion in response to A375 increases in the presence of iDCs. In the tri-cocultures with iDCs, IFNγ secretion was higher in Construct #10 compared to the other constructs. IFNγ quantified in the supernatants from DC cocultures D150081, E:T:iDC::1:1/10:1/4. FIG. 24A-B

IFNγ secretion in response to UACC257 increases in the presence of iDCs. In the tri-cocultures with iDCs, IFNγ secretion is higher in Construct #10 compared to the other constructs. However, comparing Construct #9 with Construct #11 expressing wild type and modified CD8 coreceptor sequences respectively, T cells transduced with Construct #11 induced stronger cytokine response measured as IFNγ quantified in the culture supernatants of three-way cocultures using donor D600115, E:T:iDC::1:1/10:1/4. FIG. 25A-B. These results demonstrate that T cell products co-expressing a transgenic TCR and CD8 co-receptor (αβ heterodimer or modified CD8α homodimer) are able to license DCs in the microenvironment through antigen cross presentation and therefore hold the potential to mount a stronger anti-tumor response and modulate the tumor microenvironment.

Viral Titers

T Cell Manufacturing

Activation

FIG. 26 shows that, on Day +0, PBMCs obtained from two HLA-A02+ donors (Donor #1 and Donor #2) were thawed and rested. Cells were activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the absence of serum. Activation markers, e.g., CD25, CD69, and human low density lipoprotein receptor (H-LDL-R) are in CD8+ and CD4+ cells, were subsequently measured. FIG. 27A shows that % CD3+CD8+CD25+ cells, % CD3+CD8+CD69+ cells, and % CD3+CD8+H-LDL-R+ cells increase after activation (Post-A) as compared with that before activation (Pre-A). Similarly, FIG. 27B shows that % CD3+CD4+CD25+ cells, % CD3+CD4+CD69+ cells, and % CD3+CD4+H-LDL-R+ cells increase after activation (Post-A) as compared with that before activation (Pre-A). These results support the activation of PBMCs.

Transduction

FIG. 26 shows that, on Day +1, activated PBMCs were transduced with viral vectors, e.g., Constructs #8, #10, #10n, #11, #11n, and #13-#21, in G-Rex® 24-well plates at about 2×106 cells/well in the absence of serum. The amounts of virus used for transduction are shown in Table 8.

Expansion

FIG. 26 shows that, on Day +2, transduced PBMCs were expanded in the absence of serum. On Day +6, cells were harvested for subsequent analysis, e.g., FACS-Tetramer and vector copy number (VCN) and were cryopreserved. FIG. 28 shows fold expansion on Day +6 of transduced T cell products. Viabilities of cells is greater than 90% on Day +6.

Characterization of T Cell Products

In sum, these results show (1) viral vectors with CD8β1, CD8β3 and CD8β5 isoforms had good transducing titers; (2) all constructs were capable of successful manufacturing (e.g., high viability, fold expansions in the range of 6-12); (3) frequencies of CD3+tet+ among CD8β isoforms: CD8β1 (Construct #10) was greater than CD8β3 (Construct #16) and CD8β5 (Constructs #15 and #17), with Construct #21 showing the lowest values; (4) frequency of CD3+tet+ in Constructs #11 and #19 (m1CD8α (SEQ ID NO: 7)) showed the highest values; and (5) saturation in % CD3+tet+, % CD8+tet+ and % CD4+CD8+tet+ observed at 10 μl/e6. Optimal vector dose ranges between 3.3-10 μl/e6 for all constructs.

T Cell Manufacturing

FIG. 37 shows that, on Day +0, PBMCs obtained from four HLA-A02+donors were thawed and rested. Cells were activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the absence of serum. On Day +1, activated PBMCs were transduced with viral vectors, e.g., Constructs #8, #10n, #11n, and #13-#19, in G-Rex® 6-well plates at about 7×106 cells/well in the absence of serum. The amounts of virus used for transduction are shown in Table 9.

Expansion

FIG. 37 shows that, on Day +2, transduced PBMCs were expanded in the absence of serum. On Day +7, cells were harvested for subsequent analysis, e.g., FACS-Tetramer and vector copy number (VCN) and were cryopreserved. Fold expansion on Day +7 was comparable for all constructs (approximately 30-fold expansion). Viabilities of cells is greater than 90% on Day +7.

Characterization of T Cell Products

Similar to results described in Example 6, comparable frequencies of CD8+CD4+ cells were obtained by transduction with Construct #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells. Construct #8 (TCR only) serves as negative control. FIG. 38 shows % Tet of CD8+CD4+ cells transduced with Construct #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells. Similar to results described in Example 6, these results show that there was a trend towards higher frequencies of CD4+CD8+tet+ in CD8β1 isoforms (Construct #10n) compared to CD8β3 isoforms (Constructs #13, #14, #16) and CD8β5 isoforms (Constructs #15 and #17). FACS analysis was gated on live singlets, followed by CD3+, followed by CD4+CD8+, and followed by Tet+.

Similar to results described in Example 6, results show no difference in the CD8 frequencies (% CD8+CD4− of CD3+) in cells transduced with Construct #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells among the constructs (data not shown). Comparable frequencies of CD8+Tet+ (of CD3+) in cells transduced with Construct #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells (data not shown). FACS analysis was gated on live singlets, followed by CD3+, followed by CD8+CD4−, and followed by Tet+.

FIG. 41 shows % Tet+ of CD3+ cells transduced with Construct #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells. These results show slightly higher frequencies of CD3+Tet+ in cells transduced with Construct #10 (CD8β1) compared to those transduced with CD8β3 (Constructs #13, #14, and #16) and CD8β5 (Construct #15). FACS analysis was gated on live singlets, followed by CD3+, and followed by Tet+. Slightly higher total CD3+tet+ cell counts were observed in PBMC transduced with Construct #10 CD8β1) compared to those transduced with CD8β3 (Constructs #13, #14, and #16) and CD8β5 (Construct #15) (data not shown).

FIG. 42 shows vector copy number (VCN) of cells transduced with Construct #10n, #11n, #13-#19 at 2.5 μl or 5.0 μl per 1×106 cells. These results show vector copies per cell remained below 5 in PBMC product derived using each individual construct at vector dose of 2.5 μl or 5.0 μl per 1×106 cells.

FIG. 43 shows the % T cell subsets in cells transduced with Construct #10, #11, #13, and #15 for each donor. Construct #8 (TCR only) and non-transduced cells were used as controls. These results show that TCR-only condition has slightly more naïve cells compared to the other constructs, consistent with lower fold-expansion. FIG. 44A and FIG. 44B shows % T cell subsets in cells transduced with Construct #10, #11, #13, and #15 for each donor. Construct #8 (TCR only) and non-transduced cells were used as controls. FACS analysis was gated on CD4+CD8+ for FIG. 44A and on CD4-CD8+TCR+ for FIG. 44B. These results show donor-to-donor variability between frequencies of T cell memory subsets but little difference in the frequencies of Tnaive and Tcm between constructs.

In sum, these results show (1) viability and fold expansions were comparable among all constructs at day 7; (2) slightly higher frequency of CD3+tet+ observed in CD8β1 (Construct #10) compared to CD8β3 (Constructs #13, #14, and #16) and CD8β5 (Constructs #15 and #17); (3) vector copies per cell <5 for majority of the constructs at 2.5-5 μl/106 dose; and (4) donor-to-donor variability between frequencies of T cell memory subsets but generally, Construct #10 has less naïve but more Tcm cells than the other β isoform constructs.

FIGS. 45A and 45B depicts data showing that Constructs #13 and #10 are comparable to TCR-only in mediating cytotoxicity against UACC257 target positive cells lines expressing high levels of antigen (1081 copies per cell). Construct #15 was also effective but slower in killing compared to Constructs #13 and #10. The effector:target ratio used to generate these results was 4:1. Similar results were obtained with a 2:1 effector:target ratio (data not shown).

IFNγ secretion was measured in the UACC257 cells line. FIG. 46 shows IFNγ secretion in response in UACC257 cell line was higher with Construct #13 compared to Construct #10. IFNγ quantified in the supernatants from Incucyte plates. The effector:target ratio used to generate these results was 4:1. Similar results were obtained with a 2:1 effector:target ratio (data not shown).

Expression of various cytokines was measured in UACC257 cells co-cultured at a 4:1 E:T ratio with PBMC transduced with Constructs #10, #11, #13, and #15. FIG. 48A-48G show increased expression of IFNγ, IL-2, and TNFα with 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, 4:1 E:T. FIG. 49A-49G show increased expression of IFNγ, IL-2, MIP-1β, and TNFα with 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, 4:1 E:T. FIG. 50A-50G show increased expression of IL-2 and TNFα with CD3+TCR+ cells transduced with construct #10 (WT signal peptide, CD8β1) compared to other constructs. MIP-1β expression is highest in Construct #11 (similar results when gated on CD4+CD8+ cells). FACS analysis was gated on CD3+TCR+ cells against UACC257, 4:1 E:T.

Expression of various cytokines was measured in A375 cells co-cultured at a 4:1 E:T ratio with PBMC transduced with Constructs #10, #11, #13, and #15. FIG. 51A-51C show results from FACS analysis gated on CD4+CD8+ cells against A375, 4:1 E:T. FIG. 52A-52C show results from FACS analysis gated on CD4-CD8+ cells against A375, 4:1 E:T. FIG. 53A-53C show results from FACS analysis gated on CD3+TCR+ cells against A375, 4:1 E:T. Overall, results were more variable when cells are co-cultured with A375+RFP, but similar trends are observed compared to activation by UACC257+RFP.

T Cell Manufacturing

FIG. 54 shows that, on Day +0, PBMCs obtained from three HLA-A02+ donors were thawed and rested. Cells were activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the absence of serum. On Day +1, activated PBMCs were transduced with viral vectors, e.g., Constructs #8, #10n, #11n, #13, #16, #18, and #19 in G-Rex® 100 cell culture vessels at about 5×107 cells/vessel in the absence of serum. The amounts of virus used for transduction are shown in Table 10.

Expansion

FIG. 54 shows that, on Day +2, transduced PBMCs were expanded in the absence of serum. On Day +7, cells were harvested for subsequent analysis, e.g., FACS-Tetramer and vector copy number (VCN) and were cryopreserved. Fold expansion on Day +7 was comparable for all constructs (approximately 30-fold expansion). Viabilities of cells is greater than 90% on Day +7.

Characterization of T Cell Products

Tumor death assays and cytokine expression in the presence and absence of autologous immature dendritic cells was also measured.

The results were consistent with the prior examples and are summarized in Table 11.

TCR only

Construct
Construct
Construct
Construct
Construct

mean ± SD

killing with UACC

DC Licensing by CD4 Cells Expressing Constructs of the Present Disclosure

FIG. 59 shows a scheme of determining the levels of cytokine secretion by dendritic cells (DC) in the presence of PBMCs transduced with constructs of the present disclosure and in the presence of target cells, e.g., UACC257 cells. Briefly, Day 0, PBMCs (n=3) were thawed and rested, followed by monocyte isolation and autologous immature DCs (iDC) generation in the presence of IL-4 and GM-CSF; Day 2 and Day 4-5, DC were fed in the presence of IL-4 and GM-CSF; Day 6, iDC (+DC) were co-cultured with PBMC transduced with Construct #13, #16, #10n, #18, #11n, or #19 (Effector) and UACC257 cells (Target) at a ratio of Effector:Target:iDC=1:1/10:1/4 or without iDC (−DC), PBMCs transduced with TCR only, PBMCs without transduction (NT), PBMCs treated with iDC and LPS, and iDC only serve as controls; and Day 7 (after co-culturing for 24 hours), supernatants from the co-cultures were harvested, followed by cytokine profiling including, e.g., IL-12, IL-6, and TNF-α, using Multiplex.

Increased secretion of pro-inflammatory cytokines in tri-cocultures of autologous immature dendritic cells, UACC257 tumor cell line, and CD4+ T cell product expressing CD8αβ heterodimer and TCR (Construct #10) compared with that expressing CD8α* homodimer, in which the stalk region is replaced with CD8β stalk region, and TCR (Construct #11).

To determine the ability of CD4+ T cells expressing Constructs #10 or #11 to license DC, bulk PBMCs were transduced with Constructs #10 or #11, followed by selection of CD8+ and CD4+ cells from the product. Tri-cocultures of PBMCs, CD8+CD4-selected-product, or CD4+CD8+ selected-product with UACC257 tumor cell line in the presence or absence of autologous immature dendritic cells (iDCs) for 24 h followed by cytokine quantification of IL-12, TNF-α and IL-6 using Multiplex; iDCs alone or with LPS as controls, N=4-7, mean±SD, P values based on 2way ANOVA.

In the presence of immature dendritic cells (iDCs) and UACC257 cells, CD4+ T cells expressing Construct #10 (CD4+CD8+ T cells) performed better by inducing higher levels of IL-12 (FIG. 56), TNF-α (FIG. 57), and IL-6 (FIG. 58) secreted by dendritic cells (DC) than CD4+ T cells expressing Construct #11. On the other hand, the levels of IL-12, TNF-α, and IL-6 were comparable between CD8+ T cells expressing Constructs #10 and #11 (CD8+CD4-T cells). These results suggest that CD4+ T cells expressing CD8αβ heterodimer and TCR (Construct #10) may be a better product than CD4+ T cells expressing CD8α* homodimer and TCR (Construct #11) in DC licensing. The negative controls include the cytokine levels obtained (1) in the absence of iDCs (−iDCs), (2) in the presence of non-transduced T cells (NT)+UACC257 cells, and (3) in the presence of T cells transduced with TCR only (TCR)+UACC257 cells. The positive control includes the cytokine levels obtained from iDCs treated with lipopolysaccharide (LPS), which can activate DC.

Assessment of DC Maturation and Cytokine Secretion by PBMC Products in Response to UACC257 Targets

FIG. 60 shows IL-12 secretion levels induced by co-culturing PBMCs transduced with constructs of the present disclosure in the presence or absence of iDC and target cells, e.g., UACC257 cells. For example, IL-12 secretion was increased by co-culturing PBMCs transduced with Constructs #10 and 13 in the presence of iDC (+DC) and UACC257, as compared with that by co-culturing PBMCs transduced with TCR only. Increase of IL-12 secretion suggests (1) polarization towards Th1 cell-mediated immunity including TNF-α production (see, FIG. 61), (2) T cell proliferation, (3) IFN-γ production, and (4) cytolytic activity of cytotoxic T lymphocytes (CTLs).

FIG. 61 shows TNF-α secretion levels induced by co-culturing PBMCs transduced with constructs of the present disclosure in the presence or absence of iDC and target cells, e.g., UACC257 cells. For example, TNF-α secretion was increased by co-culturing PBMCs transduced with Constructs #10 and 13 in the presence of iDC (+DC) and UACC257, as compared with that by co-culturing PBMCs transduced with TCR only.

The increased IL-6 secretion (in addition to IL-12, TNF-α) may signify dendritic cell maturation, which may be augmented by CD40-CD40L interactions between CD4+ T cells and DCs. DC maturation and subsequent cytokine secretion may aid in modulation of the proinflammatory environment.

FIG. 62 shows IL-6 secretion levels induced by co-culturing PBMCs transduced with constructs of the present disclosure in the presence or absence of iDC and target cells, e.g., UACC257 cells. For example, IL-6 secretion was increased by co-culturing PBMCs transduced with Constructs #10 and 13 in the presence of iDC (+DC) and UACC257, as compared with that by co-culturing PBMCs transduced with TCR only.

These results show that PBMC products containing CD4+ T cells co-expressing transgenic TCR and CD8 co-receptor (CD8αβ heterodimer or CD8α homodimer) may license DCs in the microenvironment through antigen cross presentation to modulate the tumor microenvironment by, e.g., increasing IL-12, IL-6, and TNF-α secretion.

Table 12 shows comparison between constructs based on manufacturability and functionality.

Construct
Construct
Construct
Construct

expression

mean ± SD

number

of killing with

with UACC257

(16 h with

and IL-6

Table 13 shows construct comparison and ranking (the smaller the number the better).

Construct

Construct

*Time delay here refers to any delay from, for example, GMP Vector manufacturing or any delay due to incomplete data set, which may add delay in implementation of constructs in clinical trials.

In sum, while manufacturability in terms of, e.g., viability, fold expansion, transgene expression, and vector copy number, may be equally good, as ranked 1, among cells transduced with Construct #10, #11, #13, or #19, functionality in terms of, e.g., cell killing, cytokine secretion, DC licensing, and 3D spheroid forming ability, of cells transduced with Construct #10 and #13 may be better, as ranked 1, than those transduced with Construct #11 and #19, as ranked 1-3.

To determine the efficacy of T cells transduced with constructs of the present disclosure, e.g., Constructs #10 and #11, against target cells, EC50s were determined based on the levels of IFNγ produced by the transduced cells in the presence of PRAME peptide-pulsed T2 cells.

For example, to compare EC50s of CD4+ selected T cells transduced with Construct #10 (CD8αβ-TCR), Construct #11 (m1CD8α-TCR), or Construct #8 (TCR only), CD4+ selected products (TCR+ normalized) were co-cultured with PRAME peptide-pulsed T2 cells at defined concentrations at E:T ratio of 1:1 for 24 h. IFNγ levels were quantified in the supernatants after 24 h. FIGS. 63A-63C show IFNγ levels produced by the transduced CD4+ selected T cells obtained from Donor #1, #2, and #3, respectively. In general, CD4+selected T cells transduced with Construct #10 were more sensitive to PRAME antigen as compared with that transduced with Construct #11 (m1CD8α TCR+CD4 T cells), as indicated by lower EC50 values (ng/ml) of CD4+ selected T cells transduced with Construct #10 than that transduced with Construct #11 (FIG. 63D). No response was observed among TCR+CD4+ cells (FIGS. 63A-63D). These results suggest that CD8αβ heterodimer may impart increased avidity to CD8αβ TCR+CD4+ T cells as compared to m1CD8α homodimer, leading to better efficacy against target cells.

Similar experiments were performed using PBMC obtained from Donor #1, #3, and #4. Briefly, PBMC products (TCR+non-normalized) were co-cultured with PRAME peptide-pulsed T2 cells at defined concentrations at E:T ratio of 1:1 for 24 h. IFNγ levels were quantified in the supernatants after 24 h. FIGS. 64A-64C show IFNγ levels produced by the transduced PBMC obtained from Donor #4, #1, and #3, respectively. Donor-to-donor variability was observed in the EC50 values. For example, while Donor #3 (FIGS. 64C and 64D) shows lower EC50 of PBMC transduced with Construct #10 as compared with that transduced with TCR only, Donors #1 (FIG. 64B) and #4 (FIG. 64A) show comparable EC50s between Construct #10 and TCR only (FIG. 64D). Thus, the increased avidity and efficacy observed in CD4+ selected T cell products expressing TCR and CD8αβ heterodimer as compared with that expressing TCR only may be obtained but to lesser extent when using PBMC products.

To compare EC50s of different T cell products obtained from the same donor, PBMC products, CD8+ selected products, and CD4+ selected products obtained from a single donor were co-cultured with PRAME peptide-pulsed T2 cells (TCR+ normalized) at defined concentrations at E:T ratio of 1:1 for 24 h. IFNγ levels were quantified in the supernatants after 24 h. FIGS. 65A-65C show that IFNγ levels produced by PBMC products (FIG. 65A), CD8+ selected products (FIG. 65B), and CD4+ selected products (FIG. 65C), respectively. Consistently, EC50 of CD4+ selected T cells transduced with Construct #10 was lower than that transduced with Construct #11 or TCR only (FIG. 65C), while EC50s of the transduced PBMC and CD8+ selected T cells were comparable between Construct #10 and TCR only transduction. Thus, the increased avidity and efficacy observed in CD4+selected T cell products expressing TCR and CD8αβ heterodimer as compared with that expressing TCR and m1CD8α homodimer or with that expressing TCR only may be obtained but to lesser extent when using PBMC products or CD8+ selected T cell products.

T Cell Manufacturing

Activation: Similar to the procedure shown in FIG. 6, on Day +0, PBMCs (about 300 million to 1 billion cells per donor) obtained from donors are thawed and rested. Cells are activated in bags (AC290) coated with anti-CD3 and anti-CD28 antibodies in the presence of serum.

Transduction: Similar to the procedure shown in FIG. 6, on Day +1, activated PBMCs are transduced with viral vectors, e.g., (i) TCR only (ii) TCR and membrane bound IL-15, (iii) CD8βα.TCR, (iv) CD8βα.TCR and membrane bound IL-15, (v) m1CD8α.TCR, or (vi) m1CD8α.TCR and membrane bound IL-15, in G-Rex® 6 well plates at about 5×106 cells/well in the absence of serum. One vector encoding multiple polypeptides may be used to transduce T cells, or multiple vectors may be used. As non-limiting examples, (i) to obtain cells expressing TCR only, Construct #8 may be transduced into cells; (ii) to obtain cells expressing TCR and membrane-bound IL-15, Construct #8 and a vector comprising a nucleic acid encoding membrane-bound IL-15 may be transduced into cells, or Construct O or Construct U may be transduced into cells; (iii) to obtain cells expressing CD8βα.TCR, Construct #10 may be transduced into cells; (iv) to obtain cells expressing CD8βα.TCR and membrane-bound IL-15, Construct #10 and a vector comprising a nucleic acid encoding membrane-bound IL-15 may be transduced into cells, or Construct L may be transduced into cells; (v) to obtain cells expressing m1CD8α.TCR, Construct #11 may be transduced into cells; and/or (vi) to obtain cells expressing m1CD8α.TCR and membrane-bound IL-15, Construct #10 and a vector comprising a nucleic acid encoding membrane-bound IL-15 may be transduced into cells, or Construct M may be transduced into cells.

Vector copy number in cells is determined, and other cell characterization is performed.

Expansion: Similar to the procedure shown in FIG. 6, on Day +2, transduced PBMCs are expanded in the presence of serum. On Day +6 or Day +7, cells are harvested for subsequent analysis, e.g., FACS-Dextramer and vector copy number (VCN) and are cryopreserved.

Characterization of Cell Products:

Cell fold expansion and/or viability of transduced and non-transduced cells are determined. Percent of transduced cells expressing each polypeptide of interest is determined. Cells are characterized through phenotyping (flow-based) and through functional studies. For phenotyping, tetramer, intracellular marker, Tmem, and/or ICS panels may be run to assess different markers of interest. Marker expression may be assessed, as non-limiting examples, in the following populations: CD3+ TCR+, CD8+ TCR+, CD8+, CD4+CD8+, or CD4+CD8+ TCR+. Activation, tetramer frequency and CD4/CD8 frequencies, memory subsets, exhaustion status, and effector molecule expression (via ICS and/or intracellular staining) may be assessed. For cells transduced to express mbIL-15, the following populations may be assessed: CD3+ TCR+mbIL-15+, CD8+ TCR+mbIL-15+, CD8+mbIL-15+, CD4+CD8+mbIL-15+, and/or CD4+CD8+ TCR+mbIL-15+. Additional assays, such as cell trace proliferation assays and/or cell death and apoptosis assays may be performed. Probing for IL-15/IL-15Rα fusion polypeptide may be performed using an antibody against IL-15Rα.

Serial Killing Assays

Transduced and non-transduced cells are cocultured with tumor cells. For example, the following tumor cell lines may be used: UACC257 (high antigen density of the antigen PRAME (preferentially expressed antigen in melanoma)), A375 (low antigen density of the antigen PRAME), or MCF7 (negative for the antigen PRAME). Cells are cocultured for up to 21 days in an IncuCyte and are imaged about every 2 hours. Effector (T cell product) to target (tumor cell line) ratio (E/T) is as follows: about 4:1 E/T for UACC257 (about 40,000 effectors to about 10,000 tumor cells), about 8:1 E/T for A375 (about 80,000 effectors to about 10,000 tumor cells), or about 4:1 E/T for MCF7 (about 40,000 effectors to about 10,000 tumor cells). Effector numbers are normalized to TCR positivity to account for the variability in transduction efficiency between cellular products. Prior to co-culture setup, the tumor cells are seeded onto 96-well IncuCyte ImageLock plates and allowed to attach for about 1-4 hours before effector cells are added. Tumor cell-only wells are included as controls for each serial killing IncuCyte assay performed. Effectors and tumor cells are allowed to coculture for 3-4 days before an add-back is performed in which about 10,000 fresh tumor cells are added to the wells (referred to as a tumor challenge or stimulation). The number of tumor challenges may vary between experiments but typically, 3-6 tumor challenges are performed. 16-24 hours after coculture is initiated and after every subsequent add-back, about 50-100 μl of supernatant from the wells is harvested for use in IFNγ ELISA or Luminex assays. Data acquisition and processing is performed by the Incucyte® S3 Live-Cell Analysis Instrument with values graphed using Prism/GraphPad statistical software.

T Cell Phenotype

Prior to the coculture setup (time 0) for the serial killing IncuCyte assays, a fraction (about 1-2e6 cells per condition) of cellular products are stained for surfaces markers indicative of T cell activation and exhaustion and assessed for expression by flow cytometry. The panel includes a live-dead stain and assesses the expression of 12 different surfaces molecules: CD8, CD3, CD4, engineered TCR, TIM-3, TIGIT, 4-1BB, 2B4, CD39, PD-1, CD69, and LAG3. Upon the completion of the serial killing IncuCyte assay, cells are harvested and stained with the same panel, allowing for the comparison of ICI marker expression pre- and post-antigen exposure. Data analysis is performed using FlowJo and graphed using Prism/GraphPad statistical software.

16-24 hours after coculture is initiated for the serial killing IncuCyte assay and after every subsequent add-back of tumor cells, about 50-100 μl of supernatant from the wells is harvested for use in cytokine detection assays. Supernatants are stored at about −80° C. until use. For interferon γ (IFNγ) ELISAs, supernatants are thawed and diluted with assay buffer. The dilutions are dependent on the tumor cell line used for the coculture and the time point the supernatant was collected. Typically, the following dilutions are used: Against UACC257, 1:20 for post-stimulation #1-3 and 1:10 for post-stimulation #4-6; against A375, 1:5 for post-stimulation #1-3 and 1:2 for post-stimulation #4-6; against MCF7, 1:5 for post-stimulation #1-3. IFNγ ELISAs are conducted with the human IFNγ Quantikine ELISA kit from R&D Systems following the manufacturer's protocol with plates are read at 450 nm wavelength using the Synergy 2 microplate reader. Data analysis is performed using Prism/GraphPad statistical software.

T Cell Manufacturing

TCR-transduced products co-expressing TCR specific for PRAME-004 and mbIL15 were generated using a standard manufacturing process. Briefly, donor peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor leukaphereses and cryopreserved. PBMCs are later thawed in TexMACS medium supplemented with 5% by volume human AB serum (“Complete TexMACS”), washed, resuspended in Complete TexMACS, and treated with benzonase nuclease for a short duration. Cells are then rested in a cell stack. Following rest, PBMC are counted, concentration-adjusted, and added to tissue culture bags coated with immobilized anti-CD3 and anti-CD28 antibodies for activation. Cells are activated overnight at 37° C.

Following activation, cells are removed from the activation bags, washed, and counted. They are then added to G-Rex vessels containing a transduction master mix. For transduced cells, lentiviral supernatant was added at 2.5 μL per million activated PBMC. For non-transduced (NT) cells, no lentivirus was added.

Flow cytometry was used to get transgene frequencies with analysis performed using FlowJo software. Harvest metrics including TCR frequency, mbIL15+TCR+ DP frequency, fold expansion, and total TCR+ cells are shows in FIG. 71A-D. All constructs were expressed.

T cell products were previously generated using the manufacturing described in Example 21 were thawed, washed, and resuspended in Complete TexMACS and treated with benzonase nuclease (25 U/mL) for 15 minutes. Cells are then rested overnight in Complete TexMACS within a Grex vessel at 37° C. (no exogenous cytokines are added for overnight rest).

The next day, tumor lines are harvested using 0.05% trypsin, washed, and counted. Red fluorescent protein (RFP)-labeled tumor cells were plated at 10,000 per well in a flat-bottomed 96-well ImageLock plate in 100 μL of Complete TexMACS. Plates were placed in an incubator at 37° C. until effector T cells were ready for plating.

Overnight-rested effector T cells were removed from the incubator and counted. Depending on the intended effector-to-target (E:T) ratio, a certain number of effectors cells were added in 100 μL to their respective well on the 96-well plate. Effector numbers were normalized with respect to T cell receptor (TCR)-positive cells with the total number of T cells added adjusted to account for the transduction efficiency. Typical E:T ratios include, but are not limited to, 10:1, 8:1, 5:1, 4:1, 3:1, or 1:1 depending on the target cells used and the question(s) being investigated.

Effector/target co-culture plates were placed into the IncuCyte S3 imager at 37° C. and 5% CO2 and imaged every 4 hours for the duration of the assay (typically ˜3 to 12 days).

Supernatant, if needed for cytokine analysis, was collected between 16 and 24 hours after the initiation of co-culture, and the plate replenished with fresh Complete TexMACS. Harvested supernatant was frozen down at −80° C. for use in downstream IFNγ ELISAs.

In assays including multiple tumor challenges, co-culture plates were removed 3-4 days following the last tumor cell stimulation and 50 μL of supernatant was removed using a micropipette. Complete TexMACS medium containing the same number of tumor target cells as at assay initiation was added to bring each well to full volume. If a given condition did not require the addition of tumor cells, they were provided with fresh medium. Cells were placed back in the IncuCyte until the next tumor cell stimulation timepoint.

Data was exported from the IncuCyte S3 software into Microsoft Excel and GraphPad Prism for further analysis. Fold tumor growth (RFP+ cell count) was normalized to 0 hr timepoint.

Results are shown in FIGS. 72-75 and 83. All products were expressed with the majority of mbIL15-containing products showing increased killing and cytokine production upon repeated antigen stimulation compared to TCR only (“TCR”).

Cell Phenotyping

Flow cytometry was performed on overnight-rested T cell products produced as described in Example 21 before or after antigen stimulation (through co-culture with tumor cells). For the “post-antigen” stimulation analysis, co-culture wells from the IncuCyte cytotoxicity assay described in Example 22 were harvested and used after the IncuCyte assay concluded. Product was stained with antibodies against memory and exhaustion markers. Flow analysis was performed using FlowJo software.

Results are shown in FIG. 76-77. Constructs show comparable memory subset distribution pre-antigen exposure with a predominant shift to Tem after antigen exposure. Exhaustion marker frequencies are similar across all constructs prior to antigen exposure.

Cell Death & Apoptosis Assay

Overnight-rested effector T cell product was co-cultured with antigen (PRAME)-positive tumor cells lines as described in the IncuCyte assay method of Example 22 except in a 24-well rather than a 96-well tissue culture plate. After co-culture setup, plates were incubated at 37° C. and 5% CO2 with re-stimulations occurring every 2-3 days. A total of four stimulations were performed. Wells were harvested after ˜9-10 days in culture and the cell mixture analyzed by flow cytometry for dead and apoptotic cells. Flow analysis was performed using FlowJo software.

Results are shown in FIG. 78. All mbIL15-containing constructs exhibited improved survival compared to TCR only (“TCR”) or non-transduced (NT”) products.

Proliferation

T cell product was thawed and rested as in the IncuCyte cytotoxicity assay described in Example 22. Tumor cells were similarly plated as in the IncuCyte cytotoxicity assay but in 1 mL per well in a 24-well rather than a 96-well tissue culture plate.

On the day of co-culture, effector T cells were counted, washed, and resuspended in PBS containing a CellTrace Violet proliferation dye at 1:1000 dilution (1 μL dye per mL PBS) and incubated for 20 minutes at 37° C.

After labeling incubation, Complete TexMACS with 5% human AB serum was added in excess to bind remaining free dye and incubated for another 5 minutes at 37° C.

Labeled effector T cells were then washed, counted, and resuspended in Complete TexMACS and added in 1 mL per well to previously prepared tumor targets for a total of 2 mL per well. E:T ratios varied but mirrored the IncuCyte cytotoxicity assays as described in Example 22 to ensure comparability. Co-cultured tumor target and effector T cells were incubated for ˜6 days at 37° C. after which point they were harvested, washed, and stained with a panel consisting of a TCR-specific tetramer and antibodies against surface antigens.

Proliferation modeling and statistics were generated using the Proliferation Modeling feature of FlowJo.

Results are shown in FIG. 79.

Persistence Assay

Overnight-rested effector T cell product was cultured in a Grex 24-well vessel at a concentration of 1.0e6 cells/ml either in the absence of any exogenous IL-7 & IL-15 addition or in the presence of IL-7 & IL-15 for up to 31 days. Every 3-4 days cells were counted using a cellometer and a 50% fresh medium change was performed. Complete TexMACS was used for the entire duration of the assay. After 31 days, cells were counted and then used in a IncuCyte cytotoxicity assay against antigen-positive tumor cell lines through one stimulation.

IncuCyte data was exported from the IncuCyte S3 software into Microsoft Excel and GraphPad Prism for further analysis. Fold tumor growth (RFP+ cell count) was normalized to 0 hr timepoint.

Results are shown in FIGS. 80 and 81. All mbIL15-containing products persisted through 31 days in culture in the absence of cytokine addition and no antigen stimulation. Cytolytic activity was retained in all products containing mbIL15 against antigen positive tumor cell lines.

Long-Term Cytotoxicity Assay and Cytokine Assay

Effector T cells were prepared as described in Example 22. Overnight-rested effector T cells were co-cultured with antigen (PRAME)-positive tumor cell line UACC257 at an effector:target ratio of 3:1 and as described in the IncuCyte assay method of Example 22. After co-culture setup, plates were incubated at 37° C. and 5% CO2 with re-stimulations occurring every 3-4 days. A total of 13 re-stimulations were performed. On day 28, effector T cells were harvested and added to a new, UACC257 cell line seeded plate. Wells were harvested after ˜44 days in culture and analyzed as described in Example 22. Results are shown in FIG. 82A-F.